Disease-Related Hepatotoxicity

#### **Chapter 5**

## COVID-19 Outcomes and Liver Disease

*Umar Hayat, Hafiz Zubair, Muhammad Farhan, Ahmad Haris and Ali Siddiqui*

#### **Abstract**

The novel severe acute respiratory syndrome coronavirus (SARS CoV-2) is the cause of coronavirus disease (COVID-19), a pandemic that represents a global health challenge. COVID-19 is usually a self-limiting disease; however, it is associated with a significant (3–7%) mortality rate. The excessive production of pro-inflammatory cytokines because of SARS-CoV-2 infection is mainly associated with high mortality due to multiple organ failure. The global burden of chronic liver disease (CLD) is vast. Approximately 122 million people worldwide have cirrhosis, 10 million living with decompensated cirrhosis. The preexisting chronic liver disease is associated with inflammation and immune dysfunction that might predispose to poor clinical outcomes in COVID-19, such as disease severity, rate of ICU admission, and mortality. The overlapping risk factors for SARS CoV-2 and chronic liver diseases such as obesity, advanced age, diabetes, and metabolic dysregulation are the major causes of these poor outcomes. Furthermore, progressive liver disease is associated with immune dysregulation, contributing to more severe COVID-19. This book chapter will explain the natural history and pathogenesis of COVID-19 in CLD patients along with the likely underlying SARS CoV-2-related liver injury mechanisms.

**Keywords:** SARS CoV-2, COVID-19, chronic liver disease, cirrhosis, hepatocellular carcinoma, COVID-19 clinical outcomes

#### **1. Introduction**

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-20) is a novel member of the coronavirus family first reported in Wuhan, China [1]. It causes COVID-19, which has infected millions of people worldwide, representing a global challenge. COVID-19 is generally a self-limiting disease presenting with flu-like symptoms but can also be deadly with a 0.7–5.8% fatality rate [2]. However, the disease severity and fatality vary by geographic areas and country, related to distinct population and disease demographics [2]. Mild COVID-19 cases may present with dry cough, fever, fatigue, dyspnea, and diarrhea. In contrast, severe cases may give a complex picture of acute hypoxia, respiratory distress syndrome (RDS), encephalopathy, and multiple organ failure [3]. Patients with advanced age and comorbidities such as hypertension, diabetes mellitus, obesity, chronic lung disease, chronic liver disease, cardiovascular disease, and

cancer are at the greater risk of having severe illness and fatality due to COVID-19 [4]. Previously healthy patients with severe and critical COVID-19 also experience some liver injury, mainly presenting with deranged liver enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), and hypo-functioning of the liver in the form of hypoalbuminemia [3, 5–8].

COVID-19 leads to host immune dysregulation and cytokine storm by producing inflammatory markers [3]. This cytokine storm has been implicated in causing lung and liver injury and multiorgan failure (**Figure 2**). COVID-19 patients have been studied to have an elevated level of cytokines such as interleukin (IL)-1B, interleukin-6, tumor necrosis factor (TNF) interferon-gamma (INF-γ), interferon gammainduced protein 10, macrophage inflammatory proteins (1alpha, 1beta), and vascular endothelial growth factor (VEGF) [3]. Although COVID-19 patients exhibit a highly variable immune response, the interleukin-6 level has been associated with COVID-19 severity and mortality [9].

The world is also dealing with another ongoing obesity pandemic due to sedentary lifestyles and food habitus [10]. This pandemic has led to various diseases such as diabetes mellitus, insulin resistance, and chronic liver disease (CLD) [10, 11]. CLD is prevalent worldwide and imposes a significant burden on healthcare costs and services. The most common causes of CLD include nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), viral hepatitis B and C. CLD can further progress to fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC) as an end-stage liver disease [10, 11]. Hepatocytes constitute a significant source of many proteins involved in both the body's innate and adaptive immune responses [12]. The liver plays a vital role in regulating immune homeostasis by two fundamental mechanisms. First, it prevents the systemic spread of dietary and microbial antigens from the gut; second, it produces the soluble molecules essential for effective body immune responses to the foreign antigens [12]. Thus, any liver injury can compromise the synthesis of proteins involved in the immune responses resulting in a compromised body immune surveillance against antigens [12]. It is categorized as an immune dysregulation in both CLD and liver cirrhosis.

The impairment of the liver's homeostasis in CLD leads to specific molecular patterns from the damaged hepatocytes, which may prompt the circulating immune cells to activate and induce an inflammatory response by releasing pro-inflammatory cytokines (interleukins and tumor necrosis factor) in the serum [13]. Furthermore, this immune dysregulation process emanates the possibility of increased infection susceptibility. Margot et al. have demonstrated that patients with CLD and cirrhosis are at a higher risk of morbidity and mortality due to COVID-19 infection [14]. However, the mechanisms of COVID-19-induced liver injury are multifactorial and are not fully understood [15, 16]. Cytokine storm hypothesis suggests that immune dysregulation because of SARS CoV-2 infections plays a vital role in liver pathophysiology in COVID-19 [15, 16].

This chapter aims to discuss the COVID-19 implications on healthy liver and CLD. The effect of COVID-19 on clinical outcomes in patients with cirrhosis and hepatocellular carcinoma will also be reviewed and discussed.

#### **2. Pathophysiology of liver injury in COVID-9**

SARS CoV-2 virus has two major binding sites. The spike glycoprotein (S) is essential for viral entry into the host cell, and the inner nucleocapsid phosphoprotein (N) interacts with the host RNA [17]. There are two possible mechanisms of liver injury in COVID-19 infection.

#### **2.1 Viral immunological injury and systemic inflammatory response**

One mechanism suggests that the SARS CoV-2 virus infects the target cells by binding to the angiotensin-converting enzymes 2 receptors on cell surfaces and replicates further inside to infect other cells [17]. These receptors are present on the bile duct epithelial cells, liver parenchymal cells, and alveolar type 2 cells in the lungs [18]. Some studies have suggested that the virus does not directly infect the hepatocytes, but it enters the portal circulation and, by reaching the liver, induces the Kupffer cells to activate immune systems, and thus produces inflammatory changes [19]. These inflammatory changes are the primary source of liver injury in SARS CoV-2 infection [19]. As a result of this inflammation, the liver enzymes (AST, ALT) were reported to be elevated >2 times the upper limit of normal in 14–53% of COVID-19 cases [20].

On the other hand, gamma-glutamyl transpeptidase (GGT) has been found to be elevated in 24% of the COVID-19 hospitalized patients suggesting a biliary epithelial cell injury [20]. Higher levels of liver enzymes have been associated with the severity of COVID-19 [21]. Moreover, antiviral drugs used for COVID-19 treatment are associated with liver injury. For instance, remdesivir use in severe COVID-19 patients has also been associated with elevated liver enzymes [22]. **Figure 1** illustrates the etiological factors of liver injury in COVID-19.

#### **2.2 Hypoxic injury and cytokine storm**

Hypoxia and cytokine storm following SARS CoV-2 infection can also affect the liver and are associated with multiorgan failure in some patients with severe COVID-19 (**Figure 2**) [23]. Hypoxia also causes Kupffer cells to produce more cytokines and triggers the recruitment and activation of other polymorphonuclear leukocytes to produce

#### **Figure 1.**

*Etiology of liver injury in COVID-19. Abbreviations: SIRS: Systemic inflammatory response syndrome; TNF-α: Tumor necrosis factor-alpha; IL18: Interleukin-18.*

#### **Figure 2.**

*Pathophysiology of SARS CoV-2 infection. A cytokine storm may occur following SARS CoV-2 infection, which can cause ineffective pathogen recognition with immune evasion leading to inappropriate inflammatory response or failure to return to the homeostasis mechanism.*

more cytokines. This cytokine storm has also been implicated in thrombocytopenia and disseminated intravascular coagulation (DIC) observed in many COVID-19 patients [24]. Furthermore, it has been associated with liver vascular endotheliitis, complement system activation, and fibrin microthrombi formation in the liver sinusoids leading to hepatic dysfunction [25–28].

In essence, regardless of etiology, aminotransferases elevation is commonly observed in COVID-19 patients, and it appears to mirror disease severity [29]. Both ALT and AST have been observed to be elevated in 93% of hospitalized COVID-19 patients. However, most of the COVID-19 patients have been found to have AST predominant aminotransferase elevations. AST can be higher in non-hepatic injuries such as myositis, but correlations with creatinine kinase (CK) were weak [29].

#### **3. Impact of COVID-19 on non-alcoholic fatty liver disease**

Chronic diseases such as diabetes mellitus, hypertension, and obesity are associated with severe COVID-19 and lousy prognosis [30–33]. Together these conditions are part of the metabolic syndrome that predisposes to non-alcoholic fatty liver disease (NAFLD) [34]. The worldwide prevalence of NAFLD is 20–30% among Western populations and about 5–15% among Asian people. Thus, a large proportion of the population is at a higher risk of developing severe COVID-19 [35]. Shanghai et al. demonstrated that the patients with the fatty liver disease diagnosed on liver CT scan were more likely to have severe COVID-19 than the general population [36]. Elevated liver enzymes AST/ALT >2 times the upper limit are independently associated with the worst clinical COVID-19 outcomes [37–39]. Patients with NAFLD, compared with those without NAFLD, reportedly show a higher risk of liver enzymes elevation throughout the disease course (70% vs. 11.1%), a higher

risk of disease progression (6.6% vs. 44.7%), and a longer viral shedding time (17.5 ± 5.2 days vs. 12.1 ± 4.4 days) [40].

The severity of liver fibrosis in NAFLD is associated with the worst COVID-19 clinical outcomes [41]. Furthermore, the patients with NAFLD who have been diagnosed with hepatic fibrosis on liver CT scan (OR, 4.32; 95% CI, 1.94–9.59) or with intermediate or high fibrosis index (Fib-4) (OR, 5.73; 95% CI, 1.84–17.9) have a significantly higher risk of developing severe COVID-19, regardless of the presence of other comorbidities [41, 42]. Moreover, the need for mechanical ventilation and ICU admission among COVID-19 patients was independently associated with diabetes mellitus, obesity, and FIB-4. FIB-4 is also associated with increased 30-day mortality (OR, 8.4; 95% CI, 2.23–31.7) [43].

It has been proposed that the patients with NAFLD/NASH have a higher expression of genes for ACE2 and TMPRSS2 receptors, which may explain the worse COVID-19 clinical outcomes among these patients. However, further studies are needed to support this hypothesis [44]. Because there is no therapy for NAFLD/ NASH, it has been demonstrated that the patients with NAFLD/NASH are at a higher risk of COVID-19 severity, ICU admission, and mortality.

Similarly, metabolic-associated fatty liver disease (MAFLD) is one of the most common causes of chronic liver disease. It affects approximately 26–39% of the global population [45]. It is also a well-known risk factor for chronic diseases such as cardiovascular disease and diabetes mellitus, resulting in higher morbidity and mortality among these patients [45]. The criteria to diagnose MAFLD are based on hepatic steatosis and three other measures, including the presence of obesity, DM2, metabolic dysregulation [45]. Studies have demonstrated that preexisting MAFLD is linked with severe COVID-19 outcomes such as a high hospitalization rate and disease severity [46]. According to a proposed mechanism of liver injury in COVID-19 patients, the presence of MAFLD could release more pro-inflammatory cytokines to exacerbate the SARS CoV-2-induced inflammatory response [46]. SARS CoV-2 uses angiotensinconverting enzyme 2 (ACE2) receptors for cellular entry. The patients with MAFLD had reported having an increased expression of ACE 2 receptors, thus leading to more severe disease and worst clinical outcomes [47]. Lastly, MAFLD patients have an increased production of reactive oxygen species that further swirls the inflammatory storm responsible for disease severity [48].

#### **4. COVID-19 and alcohol-associated liver disease**

Worldwide alcohol consumption has been increased lately [49]. Social distancing and lockdown situations in the COVID-19 pandemic have further accelerated alcohol abuse, aggravating the alcohol-associated liver injury and chronic liver disease [50]. Alcohol consumption causes approximately 3.3 million annual deaths. CLD and cirrhosis are the main pathologies linked to alcohol consumption [50]. It has been suggested that excessive alcohol consumption may have immune-modulating effects in the human body and may predispose to bacterial and viral infections [51, 52]. Moreover, there has been an unprecedented rise in the listing rate for hepatic transplantation of ALD patients compared with HCV and NASH combined [53].

Patients with alcoholic liver disease (ALD) exhibit more severe liver injury if they have COVID-19 [14]. Therefore, ALD is independently associated with a 1.8-fold increased mortality risk among COVID-19 patients [14]. A recent study has indicated that alcoholic liver damage (OR, 7.05; 95% CI, 6.30–7.88) and alcoholic cirrhosis (OR, 7.00; 95% CI, 6.15–7.97) are significantly associated with the severity of COVID-19 [54]. Another study reported the higher severity of COVID-19 among patients with ALD. They have suggested this increase due to an increased proportion of alcoholic hepatitis among these patients due to a substantial increase in alcohol consumption since the pandemic's beginning [53, 54]. Future studies are needed to explore the mechanism and pathogenesis of how alcohol consumption and ALD are related to the severe COVID-19.

### **5. COVID-19 and liver cirrhosis**

Cirrhosis is the end-stage of chronic liver disease characterized by advanced fibrosis. The liver is an essential part of the reticuloendothelial system and plays a vital role in immune regulation [13, 15]. It is responsible for innate immunity and responds to bacterial and viral infections. SARS CoV-2 binds to the selective ACE2 receptors on the surface of bile duct epithelial cells responsible for liver regeneration and immune response [50]. Thus, cirrhosis impairs this homeostasis response of the reticuloendothelial liver component and causes immune dysfunction leading to severe COVID-19 and a bad prognosis [54]. In severely decompensated liver cirrhosis, the pro-inflammatory state of the liver switches to the immune-deficient state [13].

#### **Figure 3.**

*COVID-19 and hepatic cirrhosis interrelationship. The impact of cirrhosis on SARS CoV-2 infection and vice versa.*

#### *COVID-19 Outcomes and Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.103785*

Patients with cirrhosis are at an increased risk for SARS CoV-2 infection, a higher risk of developing severe disease, and a substantial risk for hepatic decompensation [55]. A large multicenter cohort study has demonstrated that COVID-19 infection was strongly associated with hepatic decompensation, increasing the mortality rate from 26.2% to 63.2% [56]. Moreover, studies have shown that cirrhosis is an independent predictor of overall and 30-day mortality in COVID-19 patients [57–59]. A recent analysis on 745 CLD patients infected with SARS CoV-2 virus in 28 countries indicated that cirrhosis was strongly associated with COVID-19 mortality (OR, 9.32; 95% CI, 4.80–18.08) [14]. Among the total, 150 patients died due to COVID-19, and among those, 123 had cirrhosis. The study also revealed that only 19% of the total deaths were due to cirrhosis-related complications, and for rest of the patients, the cause of death was lung injury [14]. These findings suggest that cirrhosis is a strong driving force for lung injury development in COVID-19 patients. This association is related to the cirrhosis-related immune dysfunction triggered by SARS CoV-2 infection [15]. Thus, the potential mechanism for severe COVID-19 in cirrhosis is the combination of cirrhosis-related immune dysfunction, an overwhelming systemic inflammatory response to SARS CoV-2 infection, and coagulopathy [60]. Lastly, cirrhotic patients have a poor response to Hepatitis B and pneumococcal vaccine, suggesting an inadequate response to SARS CoV-2 vaccination [61, 62]. The impact of cirrhosis on SARS CoV-2 infection and vice versa has been described in **Figure 3**.

#### **6. COVID-19 and hepatocellular carcinoma**

Hepatocellular carcinoma accounts for 6% of all the malignancies globally and is the sixth most common cancer [63]. Patients suffering from any malignancy are more prone to developing SARS CoV-2 infection and are at a higher risk of developing severe COVID-19 clinical outcomes [64]. Since SARS CoV-2 directly affects the liver parenchyma and leads to immune dysfunction, it can be hypothesized that the patients with HCC are more susceptible to the severity of the disease and have worse clinical outcomes than the patients with other cancers [65]. Moreover, cancer patients are more likely to be admitted to ICU and have mechanical ventilation and die (39%) than non-cancer patients (8%) [66]. A retrospective study on 28 cancer patients with two HCC patients has demonstrated that the patients with malignancies had poor outcomes compared with the general population [67]. It is also attributed to their advanced age, different comorbidities, and underlying cirrhosis. Also, these patients were more vulnerable to severe infection because of their compromised immunity resulting from poor nutrition status [67]. Additionally, recent chemotherapy treatment within the last month also increased the risk of COVID-19 severity [66].

AASL recommends restricting physician visits in this pandemic. They have also recommended continuing surveillance imaging for HCC with an acceptable delay of 2 months [68]. However, the management of these patients is becoming more and more challenging. It is expected that the interruption of the surveillance programs in high-risk patients and patients with cirrhosis will result in advanced HCC [65].

#### **7. COVID-19 and viral hepatitis**

Hepatitis B virus (HBV) and Hepatitis C (HCV) constitute two primary sources of chronic liver disease [69]. About 300 million and 70 million people are currently infected with HBV and HCV, respectively, instigating a significant burden to the healthcare system. HBV accounts for approximately 12%, and HCV constitutes about 11% of the underlying causes of chronic liver disease [69, 70]. The susceptibility of the HBV and HCV patients to get infected with SARS CoV-2 remains unclear. Similarly, there is only limited data available to conclude the association of HBV and HCV with the severity of COVID-19 [71]. Some studies have reported that viral hepatitis is not associated with the severity of the COVID-19 [72–74]. However, a small retrospective study has shown that COVID-19 patients with HBV disease had more severe disease e (46.7% vs. 24.1%) and a higher mortality rate (13.3% vs. 2.8%) than those without HBV disease [75]. The overall COVID-19 severity and mortality were found to be higher if the viral hepatitis patients have baseline liver injury and liver fibrosis than those without any liver injury (28.57% vs. 3.30%, P = 0.004) [76].

SARS CoV-2-induced lymphopenia and the use of immunosuppressive drugs such as corticosteroids may increase the risk of severe COVID-19 in patients with active or past HBV infection [76]. A retrospective study demonstrated that immunosuppressive therapy in COVID-19 has a low risk of HBV reactivation in patients with resolved HBV infection [77]. AASLD recommends continuing HBV and HCV treatment in COVID-19 patients if started before acquiring SARS CoV-2 infection [68].

#### **8. COVID-19 and liver transplantation**

Liver transplant patients are immune-compromised, thus vulnerable to SARS CoV-2 infection. It also makes them a potential source of infection dissemination to others, especially healthcare workers, by serving as super spreaders [78]. On the other hand, immunosuppression is considered protective against the severe COVID-19 infection as it suppresses the cytokine storm responsible for inflammatory changes [79]. Surprisingly, an international cohort study with 151 liver transplant recipients who had COVID-19 demonstrated that liver transplantation was not an independent predictor of mortality [80]. However, another study revealed that patients with liver transplants and COVID-19 had a higher mortality risk than those without transplantation (OR, 6.91; 95% CI, 1.68–28.48) [81]. COVID-Hep and SECURE-CIRRHOSIS registries described 159 liver transplant patients in their recent report. Of all, 81% were hospitalized, 30% were admitted to the ICU and required mechanical ventilation, and the overall mortality rate was 19% [82, 83]. The European Liver and Intestine Transplant Association (ELITA) revealed that the older patients with liver transplants had higher mortality [84]. In a systematic review of patients with solid organ transplants (SOT) who had COVID-19, the mortality rate among liver transplant recipients was 37.5% [85]. However, the risk of SARS CoV-2 infection and clinical outcomes of COVID-19 remained unclear among liver transplant patients and need further studies for factual inferences [86].

#### **9. Conclusions**

In essence, preexisting liver disease and liver injury are associated with the COVID-19 severity and mortality. The indicators of liver disease such as elevated liver enzymes, liver steatosis, and fibrosis are considered the prognostic markers of severe COVID-19. Additionally, CLD patients with severe COVID-19 tend to develop *COVID-19 Outcomes and Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.103785*

changes in fibrinolytic and coagulative pathways due to the dysfunctional innate immune response of the body against SARS CoV-2, leading to a lousy prognosis.

Moreover, the current co-occurring worldwide NAFLD/NASH pandemic is particularly relevant in the COVID-19 era as this mortal combination results in worse clinical outcomes. CLD patients should be given special attention for screening and treatment of COVID-19. Furthermore, patients with advanced liver disease and cirrhosis should be vaccinated on a priority basis. Lastly, the COVID-19 pandemic may have significantly delayed diagnosing and treating chronic liver disease and contributed to the significant morbidity and mortality associated with liver disease. Unhealthy behaviors and sedentary lifestyle changes in the pandemic can increase the global burden of liver disease in the future. Thus, the ongoing effect of the COVID-19 pandemic on the liver warrants robust measures and further investigation.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Umar Hayat1 \*, Hafiz Zubair2 , Muhammad Farhan3 , Ahmad Haris4 and Ali Siddiqui5

1 Department of Internal Medicine, University of Kansas, Wichita, Kansas, USA

2 Department of Internal Medicine, Creighton University Medical Center, Omaha, Nebraska, USA

3 Hospitalist Medicine, United Regional Hospital, Wichita Falls, Texas, USA

4 Hospitalist Medicine, Wesley Medical Center, Wichita, Kansas, USA

5 Division of Gastroenterology, Centura Healthcare, Rocky Vista State University, Denver, CO, USA

\*Address all correspondence to: umarhayat216@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies

*Youcai Tang, Xuecui Yin and Yuying Ma*

#### **Abstract**

Non-alcoholic fatty liver disease (NAFLD) is diffuse steatosis of hepatocytes and is the most common type of chronic liver disease. The benign and reversible stage of NAFLD is defined as simple fatty liver, which further progresses to non-alcoholic steatohepatitis (NASH), liver fibrosis, and even liver cancer. It is believed that in the future, NASH would be one of the primary reasons for advanced liver failure and the need for liver transplantation. NAFLD is considered to be closely related to genetics, environment, metabolic diseases, such as obesity and hyperlipidemia. From the macro-level of NAFLD understanding, this chapter systematically analyzes the research progress on the etiology, pathogenesis, diagnosis, treatment, and development trends of NAFLD.

**Keywords:** non-alcoholic fatty liver disease, metabolic dysfunction-associated fatty liver disease, insulin resistance, type 2 diabetes mellitus, metabolic syndrome, gut flora, drug

#### **1. Introduction**

Non-alcoholic fatty liver disease (NAFLD) is a general term for a series of liver diseases ranging from hepatic steatosis alone (fatty liver) to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Of these, hepatic steatosis alone (fatty liver) is known as NAFLD, and the occurrence of inflammation and liver cell damage is called NASH. Without effective intervention, the NASH may progress to cirrhosis. In the absence of alcohol or a small amount of alcohol, there is steatosis in more than 5% of liver cells, often combined with IR, metabolic syndrome (MetS), or type 2 diabetes mellitus (T2DM), and genetic variants of PNPLA3 or TM6SF2. The mechanisms are not fully understood but are involved in hepatic lipid accumulation, imbalance in energy metabolism, and inflammatory responses from various cell types. Lipid toxins, mitochondrial function, cytokines, and adipocytokines play major roles in a process of the disease. People with NAFLD often have insulin resistance, and a large number of T2DM patients develop NAFLD and its inflammatory complication NASH. The high incidence of NASH in patients with T2DM further leads to widely recognized complications such as cirrhosis and

HCC. There are no clear clinical criteria for the diagnosis of NAFLD due to the naming of an exclusive diagnosis and the emphasis on alcohol consumption, and ignoring the metabolic causes and heterogeneity of NAFLD. Therefore, in March 2020, an expert consensus from an international team consisting of 30 experts in 22 countries recommended changing the name of NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) [1]. MAFLD is based on histological (liver biopsy), imaging, and blood biomarker to show the evidence of liver fat accumulation (hepatic steatosis), with one of the following three conditions: overweight/obesity, T2DM, and metabolic dysfunction. The prevalence of MAFLD is up to 25%, which poses a serious threat to human health and imposes a huge economic burden on society, and so far in the United States and the European Union, no drugs have been approved to treat this disease. Under the absence of proven and effective therapies, we must combine the etiology of NAFLD and its underlying pathological risk factors to explore therapeutic strategies.

#### **2. Epidemiology**

At present, the pathogenesis and potential pathological risk factors of NAFLD have not been concluded. The definition of NAFLD is also disputed, and these uncertainties prevent the large-scale diagnostic screening of NAFLD. However, the incidence of NAFLD is increasing year by year, and the age of onset is also decreasing through the Healthy People Census and related research reports. With the rapid change of lifestyle, the incidence of NAFLD is increasing year by year, and it has developed into a major global public health crisis. According to statistics, the prevalence of NAFLD is about 25% globally. The prevalence of NAFLD is approximately 24% in the North American general population, 32% in South America, 23.7% in the EU, and 27.4% in Asia [2]. In the past 10 years, the cases of fatty liver in China have jumped from 18% to 29.2%, and middle-aged men have become a high-risk population [3]. The incidence of NAFLD has increased with a rise in obesity, T2DM, and MetS, and according to 2016 statistics, the NAFLD patients in China are predicted to rise from 246 million to 315 million in 2030. Thus, if not controlled, the NAFLD will be one of the leading cause of cirrhosis requiring liver transplantation during the next decade. While the incline in the prevalence of NASH is from 2% to 3%, NASH has been recognized as the main cause of HCC and one of the indications for liver transplantation (LT) in the United States.

#### **3. Etiology**

Based on the pathogenesis, NAFLD can be divided into two types: primary and secondary [4]. Insulin resistance is related to genetic susceptibility, excessive weight gain, and overweight caused by excess nutrition, MetS-related fatty liver such as obesity, diabetes, hyperlipidemia, and cryptogenic fatty liver are all the primary causes. NAFLD caused by malnutrition, total parenteral nutrition, rapid weight loss after bariatric surgery, drug/environmental, industrial poisoning, etc. belong to the category of secondary group.

However, the new definition of MAFLD points out that hepatic steatosis is secondary, and should avoid using the terms "primary" and "secondary" fatty liver to describe. The previous dichotomous classification (simple fatty liver and NAFLD) was replaced by activity and fibrosis to better describe the process of MAFLD [1].

#### **4. Risk factors**

NAFLD is closely related to environmental and genetic risk factors, such as obesity, T2DM, MetS, lifestyle, genetic factors, and so on. It should be noted that lifestyle changes are strongly associated with the incidence of NAFLD.

#### **4.1 Obesity**

Obesity is recognized as an independent risk factor for NAFLD. The World Health Organization (WHO) defines normal as body mass index (BMI) 18.5 < BMI < 24.9, while it is defined as 18.5 < BMI < 23.9 in China. BMI has been the most useful population-level measurement for defining overweight and obesity, with equal or over 25 being overweight and equal or over 30 being obese. And the measurement applies to all adults of all ages. The Report on Nutrition and Chronic Disease Status of Chinese Residents (2020), which conducted a field investigation of more than 600,000 among nearly 600 million people in 31 provinces (autonomous regions and municipalities) across the country, found that more than half of the adult residents were overweight or obese. The overweight and obesity rates of children and adolescents aged 6–17 years old and under the age of 6 were 19% and 10.4%, respectively.

However, BMI neither reflects the distribution of body composition and fat, nor distinguishes between visceral fat and subcutaneous fat. For example, because muscle density is greater than fat, BMI will overestimate the degree of obesity in people with high muscle mass and underestimate the degree of obesity in people with high-fat contents. Therefore, although within the same BMI range, great differences exist in cardiovascular risk and mortality among individuals. Some overweight and obese people have normal metabolism and do not develop T2DM or dyslipidemia, and other metabolic diseases, which are known as metabolically healthy obesity [5]. On the contrary, part of the populations with normal weight has a variety of cardiovascular risk factors, which are prone to metabolic diseases such as T2DM, high blood pressure (HBP), and dyslipidemia.

Metabolic abnormalities are closely related to adipose tissue, mainly manifested as increased abdominal visceral fat [6]. Abdominal visceral fat is the deep adipose tissue wrapped by fascia, accounting for about 20% of the total fat mass in men and 5–8% in women. Compared with subcutaneous fat (SAT), abdominal visceral fat is more closely related to endothelial dysfunction. Glucose transporter-4 is highly expressed in abdominal visceral adipocytes, enhancing the rate of glucose uptake [7]. In addition, abdominal visceral fat is rich in β1, β2 adrenergic receptors, and unique β3 adrenergic receptors required for fat metabolism, so fats are broken down rapidly, producing more free fatty acids (FFA) and glycerol [8, 9]. FFA directly enters the liver through the portal vein, and excessive FFA deposition leads to the inhibition of hepatic glucose utilization, resulting in hepatic IR [10]. The increased oxidation of FFA in peripheral muscles will reduce the oxidative utilization of glucose in peripheral tissues, resulting in IR in peripheral tissues. The release of FFA into the blood will synthesize TG, resulting in TG deposition in many non-adipose tissues and organs.

Because of genetic background, lifestyle, and other reasons, Asian people show the characteristics of a thin body, less muscle content, and easy accumulation of abdominal fat. Under the same weight, they are more likely to develop a cardiovascular disease such as IR and glucose and lipid metabolism disorders than Caucasians. IR is the pathogenesis and core link of the normal-weight metabolic obesity [11]. Insulin can lower blood sugar mainly by inhibiting hepatic glucose production, stimulating

the uptake of glucose by visceral tissues (such as the liver), and promoting the utilization of glucose by peripheral tissues (skeletal muscle, fat). IR refers to the decreased sensitivity of the target organs of insulin action (mainly liver, muscle, and adipose tissue) to the insulin action [12].

#### **4.2 Type 2 diabetes mellitus**

T2DM is characterized by relative insulin deficiency caused by pancreatic β-cell dysfunction and IR in target organs [13]. Globally, obesity, sedentary lifestyles, and aging populations have led to a marked increase in the incidence and prevalence of T2DM in recent years. As the sixth leading cause of disability in 2015, diabetes imposes considerable socioeconomic pressure on the public and significant costs on the global health economy. Long-term high blood glucose, large blood vessels, and micro blood vessels are damaged and endanger the heart, brain, kidneys, peripheral nerves, eyes, feet, and so on. According to the statistics of WHO, there are more than 100 complications related to diabetes. More than half of the deaths from diabetes are caused by cardiovascular and cerebrovascular diseases, and 10% are caused by nephropathy [14]. Amputations due to diabetes are 10–20 times as many as nondiabetic patients with diabetes. The mechanisms of microvascular and macrovascular complications caused by hyperglycemia are endothelial dysfunction, formation of advanced glycation end products, hypercoagulability, increased platelet reactivity, and high expression of sodium-glucose cotransporter-2 (SGLT-2) [15]. In addition, isolated postprandial hyperglycemia is more common in Asian diabetic patients. Unlike obese T2DM insulin resistance mechanisms, Asian non-obese T2DM had higher visceral fat. Although the BMI of Asian T2DM patients is lower than that of European and American T2DM patients, the visceral fat of Asian T2DM patients is higher than that of European and American T2DM patients. It has been studied that higher visceral fat is related to insulin resistance, which may be related to the lipolysis of visceral fat being higher than that of the subcutaneous fat [16]. The decomposed free fatty acids enter the liver through the hepatic portal vein, which increases triglycerides in liver cells and leads to insulin resistance. Defective β-cell function plays a key role in the pathogenesis of T2DM. In the presence of insulin resistance, if β cells can compensate by increasing insulin secretion, the body can maintain normal blood sugar; when the function of β cells cannot compensate for insulin resistance, T2DM occurs. IR results in increased lipolysis and ultimately more free fatty acids entering the liver. Reduced glycogen synthesis and increased gluconeogenesis in the liver are the main features of IR. In diabetic patients, abnormal lipid metabolism will easily lead to fatty liver, which in turn affects blood sugar control, resulting in a vicious circle, overall, fatty liver compromises the ability of hypoglycemic drugs to control blood glucose. IR is not only an important mechanism for the pathogenesis of diabetes but also attracts more and more attention to the central link of the pathogenesis of NAFLD. Previous studies have shown that fatty liver in diabetic patients is more likely to develop NASH, liver fibrosis, and cirrhosis than in non-diabetic patients. People with diabetes have a higher risk of developing fibrosis than non-diabetic individuals [17]. Currently, the histopathological biopsy is the only effective way to determine the presence and severity of NASH [18]. However, due to the limited understanding of NAFLD, NASH diagnosis in T2DM is often missed or diagnosed too late, resulting in the occurrence of end-stage liver diseases and serious consequences caused by metabolic disorders, such as cardiovascular and cerebrovascular diseases. The survival rates of patients also decline, while the medical cost will rise.

*Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

#### **4.3 Metabolic syndrome**

Metabolic syndrome (MetS) may have multiple causes, ranging from a set of unrelated risk factors to the series of risk factors linked by common underlying mechanisms [19]. Previously, MetS is often used as part of an overall risk assessment for cardiovascular disease. The diagnosis is based on abdominal obesity (highly associated with IR), decreased high-density lipoprotein cholesterol (HDL-C), elevated blood pressure, triglycerides, and fasting glucose (IFG or T2DM) [20]. The diagnostic criteria of the Diabetes Society of the Chinese Medical Association for MetS are adopted in China, and those who meet three or more criteria are MetS: a. BMI ≥ 25 kg/m2 ; b. TG ≥ 1.7 mmoL and/or HDLC < 0.9 mmoL (male) or HDLC < 1.0mmo/L (female); c. SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg (1 mmHg = 0.1333 kPa) and/or diagnosed with hypertension and treated; d. FBG ≥ 6.1mmo and/or diagnosed with diabetic patients. NAFLD is considered as a hepatic manifestation of MetS. The liver, as a key organ of systemic metabolism, in turn, affects the risk of MetS and its complications. Increasing pieces of evidence show that the relationship between NAFLD and MetS are bidirectional [21]. These two clinicopathological syndromes share many aspects of their pathophysiology and IR is at the core of both. IR and MetS can exacerbate liver disease. Several cross-sectional studies have indicated that MetS and its components are associated with an increased risk of NAFLD in various populations compared with individuals without MetS.

#### **4.4 Lifestyle**

Rapid urbanization and lifestyle changes are associated with an increased incidence of NAFLD. Urbanization has led to an accelerated pace of life, dietary imbalances, such as irregular diets and high intake of saturated fat, carbohydrates, and trans-fatty acids, which are associated with IR and dyslipidemia. In addition, a sedentary lifestyle is also an important factor in NAFLD [22]. The fast-paced life and convenient transportation in cities make people less and less physically active in their daily and spare time. Age, increased smoking and alcohol consumption, screen time, decreased sleep, education, and stress all amplify the effects of IR and abdominal obesity, further increasing the prevalence of NAFLD.

#### **4.5 Genetic factors**

In addition to IR and MetS, genetic factors also play an important role in the occurrence and development of NAFLD. The human pastatin-like phospholipase domain containing 3 (PNPLA3) gene encodes 481 amino acid proteins called adiponutrin [23]. The exact role of this protein is still unknown, but it is thought to be a membrane-associated protein expressed in liver and adipose tissue, with lipogenic and lipolytic activities. It has been documented that it is located in lipid droplets (LDs) and may play a role in triglyceride hydrolysis. The gene is located in the long arm of chromosome 22. The variant rs738409 is the result of the substitution of cytosine by guanine, encoding isoleucine replaced by methionine at position 148 (I148M) of the protein. Substantial shreds of evidence suggest that this polymorphism is the strongest genetic determinant across the entire NAFLD lineage [23].

According to a study on the association of NAFLD among the medical patients in Uyghur and Beijing, it was found that the genotype frequency of PNPLA3-rs738409CG and GG genotype in NAFLD patients was higher than that in healthy controls, and the

frequency of PNPLA3-rs738409G allele in NAFLD patients was higher than that in healthy controls [24, 25]. At the same time, the univariate logistic regression analysis of the genotype distribution of PNPLA3-rs738409 and NAFLD showed that compared with the PNPLA3-rs738409CC genotype, the GG genotype had a higher risk of NAFLD. Down-regulation of PNPLA3 mutant proteins will have beneficial effects on NAFLD and maybe a new therapeutic target for NAFLD treatment.

A similar situation was found in the transmembrane 6 superfamily member 2 (TM6SF2) gene. TM6SF2 is also present in LDs and mainly expressed in the liver and gut. It is believed as a key regulator of hepatic fat metabolism and secreting triglyceride-rich lipoproteins. The variant, identified as E167K, or rs58542926, is unrelated to NPLA3 variants but associated with susceptibility to NAFLD, and with advanced fibrosis and cirrhosis [26].

#### **4.6 Gut flora**

The influence of gut bacteria on liver homeostasis is based on an anatomical basis between the gastrointestinal tract and the liver, commonly referred to as the "gut-liver axis" [27]. The liver transports bile acids and antibacterial molecules (primary bile acids, IgA, and angiopoietin) to the intestinal lumen via the bile duct to control bacterial overgrowth and maintain intestinal flora balance. Liver products (bile acids) influence gut microbiota composition and barrier integrity. Under normal circumstances, intestinal mucosal epithelial cells, intercellular tight junctions, and biofilm constitute the mechanical barrier of the intestinal tract, which can effectively prevent harmful substances such as bacteria and endotoxins from entering the blood through the intestinal mucosa. Pathologically, microbiota-dysbiotic bacteria and their derivatives translocate to the liver through a disrupted gut barrier, where they cause hepatic inflammatory responses and commensal or metabolite-induced interactions that induce steatosis. In addition, there is increasing evidence that patients with NAFLD also have gut barrier dysfunction or altered gut permeability. Although the causal relationship between NAFLD/NASH co-occurrence and disruption of the gut epithelial barrier is unclear, impaired gut permeability exacerbates NASH [28].

#### **5. Pathophysiology and pathogenesis**

#### **5.1 Theoretical hypothesis of "two-hit" and "multiple hit" in NAFLD**

The pathogenesis of NAFLD is complex and still not fully clarified, and its pathogenesis was initially dominated by the"two-hit" hypothesis [29]. Hepatic steatosis is the first step in the development of NAFLD. A high energy intake from dietary fat, a marginal decrease in fatty acid oxidation, and an increase in hepatic lipid synthesis can all contribute to the abnormal accumulation of lipids in hepatocytes (the first hit). This process is associated with IR, which leads to dysfunction of intracellular triglyceride synthesis and transport. The "second hit" is based on the fact that lipid metabolism dysfunction and mitochondrial dysfunction occur in the liver, triggering inflammation and oxidative stress caused by fatty acid peroxidation mediated by cytokines, inflammatory factors, and endotoxins. These factors can trigger a series of signaling pathways, activate liver Kupffer cells, hepatic stellate cells (HSCs), immune cells, etc., and cause pathological changes in liver tissue such as inflammation, steatosis, and liver fibrosis to form NAFLD.

*Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

In recent years, as the public pays more and more attention to NAFLD, and the research on NAFLD continues to deepen and improve, the complexity of the pathogenesis of NAFLD is far more than the "two-hit" hypothesis, and the "multiple hit" hypothesis has emerged to explain it. The "multiple hit" hypothesis suggests that the progression of NAFLD involves the occurrence of "parallel, multiple" injuries [30]. Oxidative stress, lipid peroxidation, and IR, mitochondrial dysfunction, dysregulation of cytokines, activation of HSCs, and gut-derived bacterial endotoxemia caused by intestinal flora disturbance, as well as dietary habits, environmental factors, and genetic factors are in the occurrence and development of NAFLD play a role at the same time.

#### **5.2 Insulin resistance**

Insulin is a protein hormone secreted by pancreatic islet beta cells stimulated by endogenous or exogenous substances such as glucose and glucagon. The biological action of insulin at the cellular level is initiated by binding to specific receptors on the target cell membrane [31]. Insulin receptors are membrane glycoproteins composed of two separate insulin-binding domains (alpha subunits) and two signaling domains (beta subunits). The binding of insulin to the receptor causes conformational changes in α-subunit, so that adenosine triphosphate (ATP) can bind to the intracellular domain of β-subunit. After binding to ATP, the tyrosine kinase in the β- subunit is activated, which in turn auto-phosphorylates the insulin receptor [32]. Insulin mainly acts on the liver, muscle, and adipose tissue, and controls the metabolism and storage of the three major nutrients, protein, sugar, and fat. Normally, insulin reduces glucose production by reducing hepatic gluconeogenesis and glycogenolysis, accelerates glucose uptake by adipose and skeletal muscle tissue, regulates glucose homeostasis, and prevents the conversion of excess glucose to lipid deposition. Systemic or local IR occurs when the sensitivity and responsiveness of insulin target organs or tissues to endogenous or exogenous insulin are reduced. In a sense, IR is a compensatory response mechanism of the body to excess energy. Eating a lot of carbohydrates can cause our body to store more glycogen, which leads to the continuous release of insulin, the body's sensitivity to insulin slowly decreases over time, until eventually, maybe due to impaired insulin secretion, resistance to peripheral actions of insulin, or both. In IR, on the one hand, insulin cannot effectively promote glycogen synthesis, it specifically reduces hepatic gluconeogenesis and rapidly lowers blood sugar. On the other hand, it is the effect of lipid synthesis in the liver that leads to hyperglycemia and hypertriglyceridemia that greatly affects the metabolic balance of the body. IR in the liver is often associated with T2DM, MetS, and NAFLD [33].

#### **5.3 Lipotoxicity**

Adipose tissues play a central role in body metabolism by regulating fatty acid synthesis, release, and glucose utilization, maintaining the balance of skeletal muscle and liver metabolism. Therefore, fat accumulation is not only associated with obesity but also causes fat-related metabolic disorders, among which obesity-related IR is an important way to affect the body's energy stability. The original concept of lipotoxicity refers to the effect of excess FFA on the secretory function of pancreatic islet B cells under high-fat diet conditions [34]. With the deepening of research, it has been found that excessive lipid deposition in non-adipose tissues such as skeletal muscle, cardiac muscle, and liver can lead to cell dysfunction or cell death. Ectopic

fat deposition leads to metabolic disorders of the corresponding organ, thus expanding the understanding of lipotoxicity. It is generally believed that excess intake of carbohydrates or fat gets stored in subcutaneous fat and visceral fat. When the storage capacity of adipose tissue is exceeded, especially in obese individuals, triglyceride from adipose tissue can be broken down to glycerol and FFA, and FFA can be mobilized by binding to plasma albumin. The FFA level in peripheral blood increases, an imbalance occurs in the uptake and metabolism of fatty acids. The utilization of FFA is hindered, resulting in insufficient lipid oxidation, thereby causing a large number of lipids and their products to accumulate in various tissues and organs. Inadequately oxidized lipids are stored in liver fat droplets in the form of triglycerides. Steatosis of the liver or fatty liver occurs when the accumulation of LDs in hepatocytes exceeds the storage and oxidative capacity of the liver. Steatosis of a large number of hepatocytes can induce liver dysfunction, including lipid accumulation and oxidative stress caused by lipid metabolites, inflammation, apoptosis, and liver fibrosis. This pathological process is called lipotoxicity. The failure of hepatocytes to deal with excess FFA-induced lipotoxicity promotes ER and oxidative stress leading to apoptosis, which is also a major feature of the NAFLD [28].

#### **5.4 Endoplasmic reticulum stress**

The endoplasmic reticulum (ER) is an organelle mainly responsible for physiological functions such as protein and lipid metabolism in eukaryotic cells. The membrane within the cytoplasm forms a series of sheet-like sacs and tubular lumens that communicate with each other to form a conduit system isolated from the cellular matrix. Because the conduit system is close to the inner side of the cytoplasm, it is called the endoplasmic reticulum. The ER is an important organelle related to metabolism. It has a sophisticated and complex control system to participate in intracellular anabolism and catabolism, such as protein synthesis and degradation, glycogen synthesis and decomposition, membrane lipid synthesis and recovery, fat storage, and hormone metabolism (such as production and secretion of insulin, leptin, resistin, etc.), and so on [35]. The ER is also a nutrient sensor in the body. Hyperglycemia, hyperlipidemia, and more inflammatory factors secreted by adipose tissue that accompany obesity are all stress signals of the ER. A long-term high-fat diet will increase blood sugar and fatty acids and induce disorder of glucose and lipid metabolism. Excessive high-sugar and high-fat substances entering cells for anabolism will increase the burden on the ER, increasing unfolded or misfolded proteins. When the accumulation of a large number of unfolded proteins exceeds a certain level, the corresponding unfolded protein response (UPR)-related signaling pathways are activated, resulting in an imbalance of ER function homeostasis. This state of homeostatic imbalance is called ER stress. The URP pathway is highly conserved and mainly mediated by three ER transmembrane proteins: pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor (ATF6) [36]. It is generally believed that these three proteins all have domain located in the lumen of the ER, which can sense the concentration of misfolded proteins in the lumen. Under normal circumstances, ER stress inhibits the synthesis of nascent proteins, promotes the correct folding of unfolded proteins, and accelerates the degradation of misfolded proteins through its associated unfolded protein response (UPR) signaling pathway, thus exerting a protective effect on cells. However, once the UPR is activated excessively or persistently by ER stress, the endoplasmic reticuluminduced apoptosis pathway will be triggered, resulting in apoptosis. ER stress can also inhibit insulin signaling by activating UPR-corresponding kinases, such as IRE1α,

*Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

phosphorylation of JNK, and IkB kinases [37]. In addition, related studies have also shown that FFAs-induced lipotoxicity also promotes ER stress and oxidative stress. CHOP (C/EBP-homologous protein), also known as GADD153 (growth arrest and DNA damage-inducible protein) or DDIT3 (DNA-damage inducible transcription 3). CHOP is considered a proapoptotic marker of ER stress-dependent cell death.

Elevated expression of the ER stress marker CHOP was detected in liver biopsies from patients with NAFLD [38], suggesting that ER stress-induced apoptosis in hepatocytes is likely related to the progression from steatosis to NAFLD in humans.

#### **5.5 Inflammation**

Although the pathogenesis of NAFLD has not been fully elucidated, the inflammatory response runs through the entire pathological process of NAFLD. In NAFLD patients, showing the increase of FFA released into the blood circulation and the decrease of the oxygen content of adipocytes, both act together to induce the activation of hypoxiainducible factor (HIF1) and downstream target genes in adipocytes, and ER stress [39], resulting in cell death and specific inflammatory response. The inflammatory markers tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and C-reactive protein (CRP) in NAFLD patients were significantly higher than those in healthy people [40]. TNF-α is secreted by macrophages and increases with the content of adipose tissues in the body. Highly expressed TNF-α induces phosphorylation and inactivation of insulin receptors in adipose tissues and smooth muscle cells, increases lipolysis to generate FFA, and inhibits adiponectin release. IL-6 is a cytokine produced by adipocytes and immune cells and has a complex regulatory mechanism in the body. The IL-6 production increases with the increased body fat and IR. It acts on the liver, bone marrow, and endothelium, increasing the expression of the acute phase reactant CRP in the liver. Several studies have shown a correlation between high CRP levels and the development of NAFLD as well [41]. Increased production and release of pro-inflammatory factors (TNF-α, IL-6, and CRP) can induce IR in the liver, skeletal muscle, and adipose tissue through insulin-interfering signaling pathways.

Therefore, inflammation and metabolic changes in adipose tissues can also trigger NAFLD.

#### **5.6 Leptin and adiponectin**

Adipokines also play an important role in the process of NAFLD-related liver fibrosis. Leptin is a hormone secreted by adipose tissue that can promote fibrosis [42]. The content of leptin in serum is positively correlated with the content of adipose tissue in the body. Normally, leptin functions primarily as an afferent signal in a feedback loop, acting on neurons in the hypothalamus to regulate feeding and other physiological functions. The researchers found that the level of leptin in the blood circulation increases when the body undergoes an inflammatory response, and many acute-phase factors, such as TNF-α, IL-1, IL-6, and bacterial lipopolysaccharide (LPS) stimulation, can rapidly increase leptin levels [43]. Leptin can also alter insulin action, induce angiogenesis, reduce endothelial NO synthase, and interact with the immune system [44]. In addition, leptin can activate HSCs by activating the JAK/ STAT pathway. HSCs are the main source of extracellular matrix in liver fibrosis [45].

Adiponectin (ADPN) is also a protein hormone mainly secreted by adipocytes. ADPN mainly exists in blood circulation and plays an important role in the regulation of insulin sensitivity and glucose metabolism. ADPN reduces the level of plasma-free

fatty acid (FFAs) by promoting fatty acid oxidation. There are two types of adiponectin receptors, adiponectin receptor 1 (AdipoR1) which is mainly distributed in skeletal muscle, and adiponectin receptor 2 (AdipoR2) which is abundantly expressed in the liver. Studies in mammals have shown that ADPN activates the adenylate-activated protein kinase (AMPK) signaling pathway through AdipoR1 and AdipoR2 [46]. Activated AMPK induces phosphorylation inactivation of acetyl-CoA carboxylase (ACC), thereby promoting fatty acid oxidation. In addition, peroxisome proliferatoractivated receptor alpha (PPAR-α) is a key transcription factor regulating lipid metabolism in animals. As a downstream factor of the AMPK signaling pathway, it is also involved in the effect of ADPN on enhancing fatty acid oxidation [47]. Studies have shown that highly expressed ADPN attenuates the proliferation and migration of HSCs and promotes apoptosis of HSCs by inducing the expression of nitric oxide synthase (iNOS) and messenger RNA (mRNA) in HSCs, which hinders liver fibrosis [48]. In addition, blood ADPN concentrations are significantly reduced in MetS, diabetes, atherosclerosis, and NAFLD, in contrast to other cytokines, making ADPN a possible hallmark of these diseases.

#### **5.7 Hepatic stellate cells**

Hepatic stellate cells (HSCs) are a kind of non-parenchymal cells unique to the liver, accounting for about 8–13% of the total number of liver cells. HSCs have a dual phenotype of quiescence and activation [49]. In normal liver, the cells are quiescent. At this time, the cells act as hepatic fat-storing cells, and the intracellular LDs are abundant. The autofluorescence properties of vitamin-A stored in the LDs under the microscope contribute to the localization of the cells. During the development of NAFLD, multiple factors within the micro-circle promote the activation and transdifferentiation of HSCs into myofibroblasts. Activated HSCs can also massively secrete extracellular matrix (ECM), tissue inhibitors of metalloproteinases (TIMPS), matrix metalloproteinases (MMPs), and α-smooth muscle actin (α-SMA) [50]. The continuous activation of HSCs is a key link in the development and progression of liver fibrosis. On the one hand, HSCs produce 80% of type I collagen in fibrotic tissue, which induces liver remodeling. On the other hand, intra-hepatic sinusoidal pressure is increased by cell contraction. These two types of changes finally laid the pathological basis of NAFLD-related liver fibrosis. Existing studies have found that in the mechanism of liver fibrosis, growth factor signaling has a significant role in the activation of HSCs. Growth factors such as transforming growth factor (TGF)-α, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and other growth factors activate HSCs through signaling, promoting ECM remodeling, leading to collagen formation [51]. The molecular pathways of HSCs activation are complex and involve a variety of signaling pathways. The characteristics of HSCs and their roles in the repair of hepatocyte injury and local immunity in the liver still require more in-depth research.

#### **6. Clinical manifestations**

The onset of NAFLD is insidious, slow onset, and often asymptomatic. A small number of patients may have non-specific symptoms such as fatigue, mild discomfort in the right upper quadrant, dull pain in the liver area, or upper abdominal distension. With the development of the disease, some NAFLD patients may have symptoms such as jaundice, anorexia, nausea, and vomiting, which may be accompanied by hepatomegaly. In the decompensated stage of NAFLD-related liver cirrhosis, the clinical manifestations are similar to those of liver cirrhosis caused by other causes.

#### **7. Diagnosis**

NAFLD represents the liver manifestation of a multi-system disease, with heterogeneity in underlying causes, presentation, course, and outcomes. NAFLD means that the whole body is in a state of metabolic dysfunction.

Liver biopsy is considered to be the gold standard for defining NAFLD and able to distinguish steatosis from NASH. However, it is not recommended routinely because of the increased risk of bleeding and complications. Ultrasound is the most recommended and widely used diagnostic method for the identification of hepatic steatosis due to its sensitivity and non-invasiveness.

Over the past few decades, several expert groups have attempted to develop simple diagnostic criteria for clinical practice to identify NAFLD patients. The latest expert consensus in 2020 clarifies that the diagnosis of MAFLD is mainly based on histology, imaging, or blood biomarker evidence of the presence of fat accumulation in the liver (hepatic steatosis), in addition to one of three criteria (i.e., overweight/obesity, presence of T2DM or evidence of metabolic dysregulation) [1]. The presence of at least two metabolic risk abnormalities may correctly diagnose NAFLD in non-overweight/ obese individuals.

#### **8. Differential diagnosis**

#### **8.1 Alcoholic liver disease**

Before the name of NAFLD was suggested to be changed to MAFLD, the difference between NAFLD and alcoholic liver disease (ALD) is mainly based on the prescribed amount and duration of drinking. Drinking history is a prerequisite for the diagnosis of ALD [52]. If there is no history of drinking, the diagnosis of ALD does not need to be considered. However, if the patient has a history of excessive drinking but the duration is less than 5 years or more than 5 years but the average drinking amount does not exceed the standard, this means that part of the population falls between the two diagnostic criteria when it comes to drinking.

After ethanol enters hepatocytes, it is oxidized by hepatic alcohol dehydrogenase, catalase, and hepatic microsomal alcohol oxidase, and finally forming acetaldehyde. Acetaldehyde has strong lipid peroxidation, and obvious toxic and side effects on hepatocytes, which hinders their metabolism and leads to degeneration and necrosis of hepatocytes. In addition, ethanol can affect the occurrence and development of liver disease by regulating intestinal flora, inflammatory response, and fibrosis [53]. Compared with NAFLD, patients with ALD have obvious liver disease presentation and rapid disease progression, and a higher risk of liver cirrhosis, liver failure, or liver cancer.

At present, a few studies have focused on the differential diagnosis of NAFLD and ALD, and many studies used non-fatty liver patients or healthy people as controls. There are still many problems and unknown factors in the differential diagnosis of NAFLD and alcoholic liver disease. Clinically, ALD is more likely to be diagnosed

when there are obvious clinical manifestations of chronic hepatitis and cirrhosis, especially extrahepatic and neuropsychiatric manifestations. While NAFLD is more likely to be diagnosed when there are mild or even no symptoms. For the patients who drank alcohol, the changes of indicators within 4 weeks after abstinence were helpful for the differential diagnosis of NAFLD and ALD.

#### **8.2 Chronic viral hepatitis**

Viral hepatitis, as an infectious disease, is mainly caused by a variety of hepatitis viruses. There are five recognized types of viral hepatitis, namely hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). All viral hepatitis is contagious, but the route of transmission and the intensity of infection vary. Hepatitis A and E are acute hepatitis, and types B, C, and D, are chronic hepatitis and can develop liver cirrhosis and HCC. Hepatitis D virus can only be transmitted in individuals with the presence of hepatitis B virus, so normal people do not get hepatitis D. Chronic viral hepatitis is an inflammation of the liver caused by the hepatitis virus that lasts for more than 6 months. The hepatitis virus usually causes symptoms after it has severely damaged the liver [54]. Viral hepatitis is an infectious disease with the highest infection rate and the greatest harm to patients in China.

HBV is an enveloped partially double-stranded DNA virus, consisting of an outer lipid envelope embedded with hepatitis B surface antigen (HBsAg) and a nucleocapsid containing hepatitis B core antigen (HBcAg), viral polymerase, and DNA genome. Clinically, it is difficult to distinguish hepatitis B from hepatitis caused by other viral agents, and the diagnosis must be confirmed by laboratory tests. The laboratory tests for hepatitis B surface antigen (HBsAg) are used to diagnose hepatitis B infection. Acute HBV infection is characterized by the presence of hepatitis B surface antigenantibody and immunoglobulin IgM type anti-core antigen-antibody. In the early stage of infection, the serum of patients can also be positive for hepatitis B-e antigen (HBeAg). Chronic infection is characterized by the persistence of HBsAg-antibodies (with or without HBeAg positivity) (>6 months). The persistence of HBsAgantibodies is a primary risk marker for the development of chronic liver disease and progression to HCC. The presence of HBeAg positivity indicates that the blood and body fluids of infected individuals are highly contagious [55].

HCV is a single-stranded RNA virus that can be divided into six genotypes and several subtypes. The genome of HCV encodes a single polyprotein that can be translated and processed into structural and nonstructural proteins. And the nonstructural proteins have key functions in viral replication. During the acute phase of HCV infection, the presence of an HCV-specific CD4-T cells response is associated with the control of viral replication. If the response of the CD4-T cell is sustained and maintained, HCV is permanently eliminated. If the CD4-T cells' response is lost, rebound viral replication or viremia occurs, resulting in a viral persistence [56]. In chronic HCV infection, CD4-T cells are functionally limited due to impaired proliferative capacity, which is caused by HCV core-mediated inhibition of IL-2 secretion.

#### **8.3 Autoimmune liver diseases**

Autoimmune liver diseases (ALDs) refer to a group of non-infectious liver diseases characterized by liver pathological damage and abnormal liver function. Its pathogenesis may be related to autoimmunity, mainly including autoimmune hepatitis (AIH),

#### *Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and any overlapping syndrome of these three diseases. AIH is mainly causing damage to liver cells, while PBC and PSC are mainly damaging the biliary tract. The main damage is related to abnormal autoimmune function. ALDs are chronic diseases with a long natural history and progressive development, which eventually leads to liver cirrhosis and liver failure [57]. China still lacks exact statistics, but the number of clinically detected and reported cases has significantly increased in recent years. At present, it is believed that ALDs are caused by the breakdown of the immune system's immune tolerance to self-antigens, which induces an immune attack on the liver. Genetic susceptibility and environmental factors are the initiating factors, and the pathogenesis may be related to factors such as infection, chemical factors, cytokine networks, and molecular mimicry of self-antigens. However, the specific etiology and pathogenesis are still unclear, and there is currently no single clinical or laboratory index to diagnose ALDs. It is necessary to comprehensively integrate clinical manifestations, laboratory examinations, and liver histological characteristics to exclude other possible causes of chronic hepatitis. Clinically, patients with ALDs lack specificity. Initially, symptoms such as fatigue, pruritus, jaundice, and abdominal pain are often present. Biochemical tests are often abnormal in liver function. The presence of autoantibodies in serum is an important feature for diagnosis and differential diagnosis, such as ANA, SMA, AMAM2, etc., and histopathological examination of the liver is also very important [58].

#### **8.4 Hepatolenticular degeneration**

Hepatolenticular degeneration, also known as Wilson disease (WD), is an autosomal recessive genetic disorder caused by the mutation of the ATPase copper transport β gene ATP7B, resulting in disturbance of copper metabolism in the body [59]. The genetic mutations lead to the defective or loss of ATPase function, resulting in the obstruction of copper excretion in the bile duct, and a large amount of copper accumulates in the brain, liver, kidney, bone, joint, cornea, and other tissues or organs. The carrier frequency and prevalence rate of this disease in the world are 1:100–1:90, and 1:40,000–1:30,000 respectively. Clinically, the clinical manifestations of WD patients are diverse, and the clinical manifestations can be mainly divided into brain type, liver type, mixed type, and other types. The manifestations of cerebral-type patients mainly include Parkinson's syndrome, dyskinesia, oral and mandibular dystonia, and psychiatric symptoms. The main clinical symptoms of liver patients include asymptomatic elevation of transhelicase, hepatomegaly, splenomegaly, hepatitis, fatty liver, cirrhosis, and acute liver failure. Excessive copper will also be deposited in the kidneys, bones and joints, blood, skin, cornea, and other tissues or organs, causing corresponding tissue and organ damage. Since the human body's copper is mainly excreted from the liver in the form of bile, many liver diseases themselves can lead to abnormal copper metabolism indicators in the human body. Therefore, for patients with only liver involvement, the interpretation of auxiliary examination indicators needs to be more cautious, and a comprehensive evaluation should be combined with a variety of examination methods. The new 2021 health guidelines in China remind clinicians to be highly alert the individuals with serum ceruloplasmin <120 mg and children with elevated liver enzymes and 24 h urinary copper ≥40 μg. It is recommended to perform ATP7B gene testing to confirm the diagnosis.

Specific diseases, such as alcoholic liver disease, chronic viral hepatitis, autoimmune liver disease, and Wilson's disease that can lead to fatty liver need to be excluded, as well as drugs (tamoxifen, amiodarone, methotrexate, glucocorticoids, etc.),

total parenteral nutrition, inflammatory bowel disease, hypothyroidism, Cushing's syndrome, lack of β-lipoproteinemia, and congenital IR syndrome-related fatty liver also need to be excluded.

#### **9. Treatment**

Generally, non-alcoholic fatty liver (NAFL) progresses relatively slowly. But when NAFL progresses to NASH without effective intervention, 15–25% of patients can progress to liver cirrhosis or even HCC within 10–15 years. Exploring and eliminating the causes are the fundamental ways to treat this disease. Obese people need to more effectively control their weight, and diabetic patients require effective treatment. People with malnutrition need to adjust to a balanced diet, and so on. The speed of weight loss is a key factor in determining the improvement or deterioration of liver histology.

#### **9.1 Lifestyle**

Because the etiology and pathogenesis of NAFLD are unknown, there is no effective drug therapy for liver disease. None of NASH drugs are currently in Phase III clinical trials, and there are no drugs approved by government regulators to treat NASH.

For obese patients with fatty liver, diet therapy is the basis and key approach. Lifestyle modification is recommended as the primary treatment for NAFLD [60]. For NAFLD patients who are overweight or obese (abdominal obesity), the first optional lifestyle is aimed at weight loss with a range of 8–10%. More than 50% of patients fail to meet the target and require individualized drug treatment. NAFLD patients should adjust their diet, which should be supplemented with high protein, an appropriate amount of fat, and sugar with rationally allocated. The total energy intake should be controlled at about 20–25 kcal per kilogram per day. Meanwhile, patients should strictly control their daily salt intake, avoid foods rich in monosaccharides and disaccharides, such as high-sugar pastries, ice cream, candies, etc.

Exercise is very important in the treatment of NAFLD. It is recommended that patients should take aerobic exercise, such as jogging, brisk walking, swimming, and so on. The specific time and amount of each and gradual exercise need to be personalized. Weight loss is generally controlled at 0.5–1 kg/week because losing weight too quickly is also harmful to the body.

#### **9.2 Obesity management**

Weight loss should be a priority in obese patients and those with MetS. Obesity can be addressed through lifestyle changes such as a low-calorie diet with an adequate intake of fruits and vegetables and increased physical activity. Although medical treatment and bariatric surgery may also be considered, however, the adverse effects cannot be eliminated.

#### **9.3 Pharmacotherapy for patients with T2DM**

NAFLD is an acquired metabolic stress-induced liver injury closely associated with IR and genetic susceptibility. The metabolic disorders in T2DM patients are

#### *Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

similar to NAFLD. Therefore, the glucose metabolism in T2DM patients with NAFLD will further deteriorate, making diabetes difficult to control, and requiring more hypoglycemic drug treatment. Metformin is the preferred treatment for patients with T2DM unless there is a specific contraindication, such as in patients with renal impairment.

Since metformin does not promote insulin secretion, it generally does not cause hypoglycemia when used alone. Animal and in vitro studies have shown that metformin has a protective effect against several T2DM-related cardiovascular diseases, including myocardial infarction, hypertrophic, and diabetic cardiomyopathy, which lead to cardiac insufficiency and the potential progression to heart failure. The molecular mechanisms involved in this protection are multifaceted and function primarily by acting on vascular endothelial cells, cardiomyocytes, and fibroblasts. Since metformin is excreted by the kidney, the accumulation of metformin and lactic acid easily occurs in the body when the kidney functions insufficiently, increasing the risk of acidosis thereby. The doctors generally recommend cessation when the serum creatinine is greater than 150 micromol/liter. In addition, the drug should also be discontinued when there is severe cardiac and liver dysfunction, and the liver and kidney functions should be checked regularly during the medication.

Sulfonylureas, such as gliclazide and glimepiride, act on β cells to stimulate insulin secretion and increase the level of insulin in the body. Some sulfonylurea drugs (such as glimepiride) can enhance the sensitivity of peripheral tissues to insulin, reduce the output of hepatic glycogen, and also have the effect of reducing platelet aggregation, regulating blood lipids and blood viscosity, and improving blood circulation (e.g., gliclazide). Sulfonylureas boost the production of insulin, a hormone that promotes energy storage, which may indirectly contribute to weight gain. Among various sulfonylureas, clinical studies have shown that glipizide controlledrelease tablets and glimepiride have no significant effect on weight gain. Metformin, acarbose, and sodium-glucose cotransporter 2 (SGLT-2) inhibitors also have weight loss effects. For overweight or obese patients, sulfonylureas in combination with these drugs may reduce the risk of weight gain associated with sulfonylureas.

NAFLD patients with diabetes should have effective improvement not only in NASH, but also in NAFLD-related MetS, T2DM, and cardiovascular diseases. In the treatment of NASH, it is necessary to take effective measures to lose 8–10% of body weight, including lifestyle intervention. If the standard is not met, drug treatment can be selected. Patients eligible for bariatric surgery may also be considered.

#### **9.4 Gut flora**

In addition to genetic susceptibility and diet, the gut microbiota influences hepatic carbohydrate, lipid metabolism, and the balance between pro-and anti-inflammatory cytokines in the liver, thereby affecting NAFLD and its progression to NASH. Hyperproliferation of intestinal bacteria can lead to changes in cytokines in the portal vein and liver, so probiotics and antibiotics may help treat this disease. Animal experiments have shown that probiotics can down-regulate TNF-α levels and reduce liver inflammation, but clinical studies are needed to confirm the efficacy. Antibiotics that are not absorbed in the gut may be helpful in the treatment of intestinal bacterial hyperproliferation. Rifaximin, which is rarely absorbed in the gut, is well tolerated and may have certain advantages [61]. However, there is no randomized controlled clinical study to observe the efficacy of antibiotics on NAFLD.

#### **9.5 Potential drugs**

Studies have found that liver fibrosis can be reversed in a series of processes including the occurrence and development of NAFLD. The activation of HSCs to produce collagen is the core link of liver fibrosis. Although great progress has been made in the study of HSCs activation-related genes, few breakthroughs are achieved in the treatment of liver fibrosis, and the search for effective anti-fibrosis drugs is still a research hotspot. By choosing appropriate drugs, the clinical prognosis of NAFLD can be optimal, which has important social and economic significance.

#### *9.5.1 Curcumin*

Turmeric is the dried rhizome of turmeric (*Curcuma longa* L.), which has been used in traditional medicine in China for thousands of years and is widely used in flavoring, dyeing, and pharmaceutical industries. The main active ingredient is a class of diarylheptane compounds derived from ginger plants, which mainly exist in the rhizomes of medicinal plants such as turmeric, tulip, and Curcuma. At present, more than 40 kinds of Curcumin compounds have been isolated from the genus Curcuma, among which Curcumin is the main active substance, and the main chain is unsaturated aliphatic and aromatic groups. Since it was first isolated from plants in 1870 but its molecular structure was determined in 1910, years of research have found that it has a variety of biological functions, such as regulating blood lipids, anti-tumor, anti-virus, and anti-inflammatory effects, and act as antioxidants. Through research on the mechanism and intervention of NAFLD-related hepatic stellate cell activation, it is of great theoretical significance to clarify the potential mechanism of Curcumin to inhibit the occurrence of hepatic fibrosis.

Liver fibrosis is a wound repair response to chronic liver injury (viral infection, alcoholism, cholestasis, etc.), and is a pathological process of excessive extracellular matrix (ECM) production and deposition. Chronic liver injury leads to the accumulation of a large number of inflammatory cells, which release inflammatory factors and growth factors, such as TNF-α and TGF-β1, thereby activating HSCs, which are generated by ECM (especially collagen fibers). Curcumin has received great attention as a dietary supplement for liver protection. Curcumin can inhibit the activities of lipoxygenase and cyclooxygenase-2 (COX-2), inhibit lipid peroxidation, reduce the release of arachidonic acid, especially the inflammatory factors ILs by inhibiting the NF-kB signaling pathway—production of 1β, IL-6, TNF-α. Our previous findings provide new insights into the mechanism of action of curcumin and a therapeutic candidate for the prevention and treatment of hyperleptinemia-induced liver fibrosis in NASH patients with obesity and/or T2DM [62–64]. In recent years, several in vitro and in vivo studies have also shown that curcumin can intervene in the pathological process of liver diseases from multiple links, and has anti-hepatic injury, anti-steatosis, anti-fibrosis, and anti-cancer effects. However, due to the poor water solubility and low bioavailability of curcumin, its clinical application is greatly limited. Therefore, the formulation and structural modification of curcumin as a lead compound are currently hot and crucial research topics.

#### *9.5.2 Vitamin E*

Vitamin E is a fat-soluble vitamin with antioxidant function, which is necessary for the normal growth and reproduction of animals. Studies have found that vitamin

#### *Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies DOI: http://dx.doi.org/10.5772/intechopen.103059*

E has a similar biological activity to a-tocopherol, which can provide a hydrogen ion on the color ring to scavenge free radicals, thereby playing an anti-oxidative stress role. In addition to scavenging reactive oxygen free radicals, vitamin E can also scavenge reactive nitrogen free radicals. Both of them play important roles in the occurrence and development of NAFLD. In vivo experiments in mice found that vitamin E plays an important regulatory role in improving glucose and lipid metabolism, and vitamin E supplementation can significantly improve lipid metabolism in NAFLD mice. Clinical trials have found that vitamin E supplementation can significantly improve liver pathological outcomes in non-diabetic NAFLD patients [65]. However, there was no significant improvement in diabetic patients with NAFLD [66]. Therefore, vitamin E therapy can be considered for non-diabetic NASH patients who have failed lifestyle interventions.

#### *9.5.3 Peroxisome proliferator-activated receptor alpha (PPAR-α) agonist*

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors. PPARs contain three isoforms consisting of PPARα, PPARβ/δ, and PPARγ. Among them, PPARα is abundantly expressed in hepatocytes. PPARα has a key role in regulating fatty acid transport as well as peroxisomal and mitochondrial β-oxidation in the liver. The researchers found that PPARα expression in the human liver was inversely correlated with the severity of NAFLD. Currently, PPARα-agonists have been shown to improve IR and significantly increase energy expenditure. PPARα-agonists improve pathological conditions in a NAFLD mouse model by modulating lipid turnover and energy metabolism in the liver [67].

#### *9.5.4 Farnesoid X receptor agonists*

Farnesoid X Receptor (FXR) is a bile acid receptor, a member of the nuclear receptor superfamily. Studies have found that the nuclear receptor transcription factor FXR can participate in the regulation of various metabolic pathways through the regulation of its corresponding target genes. FXR and retinol X receptor (RXR) bind to the FXR response element in the promoter region of target genes in the form of heterodimers to regulate the transcription of downstream genes. Fibroblast growth factor 21 (FGF21) is an important cytokine downstream of FXR that regulates glucose and lipid metabolism in the body. It can enhance the hydrolysis of adipose tissue, thereby increasing the rate of fatty acid oxidation. Activation of FXR by bile acids can increase the expression and secretion of FGF21, and the increased expression of FGF21 can reduce the content of triglycerides in the liver. Therefore, it can be used as an important drug target for NAFLD [68]. Obeticholic acid is a kind of FXR. In a phase 3 study in the treatment of NAFLD, 25 mg of Obeticholic acid significantly improved fibrosis in NASH patients [69]. Therefore, FXR agonists may also be considered as one of the potential drugs for NAFLD.

#### **10. Future prospects**

Several issues related to NAFLD require further research to clarify. Furthermore, the lack of understanding of the pathogenesis, causality, and genetic factors of NAFLD have hindered the development of new therapeutics. Therefore, further basic and clinical studies are needed to better understand the development of NAFLD from the perspectives of genetic, molecular, and cell signaling, etc. Focusing on the underlying mechanisms may be valuable in identifying new therapeutic targets for metabolic diseases. Lifestyle interventions are the recommended initial therapy for the treatment of NAFLD. To date, there is insufficient evidence to support the use of drugs that primarily target the underlying causes of MetS. Therefore, if lifestyle changes are not sufficient, other measures that target individual risk factors may be needed. Most importantly, improved strategies are needed to achieve and maintain long-term weight loss and increased physical activity. In future research, not only basic medical research will be conducted but also actively innovate and carry out translational medicines. It is believed that with the joint efforts of medicinal chemists and clinical experts, new drugs will be used in the treatment of liver diseases.

### **Acknowledgements**

This work is supported by research grants from the National Natural Science Foundation of China (31471330), National Key R&D Program of China (2020YFC2006100 and 2020YFC2006101), National Key R&D Program of China (2020YFC2009000 and 2020YFC2009006), Henan Provincial Key R&D and Promotion Special Project (212102310033), Zhengzhou University Discipline Key Special Project (XKZDQY202001). Furthermore, we thank Dr. Ihtisham Bukhari (Gastroenterology, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China) for his linguistic assistance.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Youcai Tang1,2,3,4,5\*, Xuecui Yin2 and Yuying Ma<sup>2</sup>

1 Department of Pediatrics, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China

2 Gastroenterology, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China

3 Henan Key Laboratory of Rehabilitation Medicine, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China

4 Henan Joint International Research Laboratory of Chronic Liver Injury, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China

5 Zhengzhou Key Laboratory of Metabolic-dysfunction-associated Fatty Liver Disease, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China

\*Address all correspondence to: tangyoucai@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 7** Hepatocellular Carcinoma

*Rahmat Adetutu Adisa and Lateef Adegboyega Sulaimon*

#### **Abstract**

Over 1 million cases of liver cancer are estimated to occur by 2025, making it a global health challenge. In almost 90% of cases of liver cancer, it is hepatocellular carcinoma (HCC). The main risk factors for HCC development are infection with hepatitis B and C viruses, although nonalcoholic steatohepatitis (NASH) associated with metabolic syndrome or diabetes mellitus is becoming more prevalent in the West. The molecular pathogenesis of nonalcoholic steatohepatitis-associated HCC is unique. A quarter of all HCCs present with mutations that are potentially actionable but have not yet been translated into clinical practice. In the advanced stages of the disease, systemic therapy is expected to be administered 50–60% of the time to HCC patients. In phase III trials, six systemic therapies have been approved (atezolizumab plus bevacizumab, sorafenib, lenvatinib, regorafenib, cabozantinib, and ramucirumab), and new trials are evaluating combination therapies, such as checkpoint inhibitors and tyrosine kinase inhibitors or anti-VEGF therapies. The findings of these clinical trials are expected to alter the landscape of managing HCC at all stages of the disease.

**Keywords:** hepatocellular carcinoma, nonalcoholic steatohepatitis, hepatitis B, hepatitis C, systemic therapies

#### **1. Introduction**

The incidence of liver cancer is growing worldwide [1, 2] and research estimates that millions of people will be affected by the disease annually by 2025 [3]. Hepatocellular carcinoma (HCC) describes the most common type of liver cancer, responsible for nearly 90% of all cases. The most significant risk factor for HCC development is infection with the hepatitis B virus (HBV), accounting for 50% of all cases [4]. With antiviral drugs, patients have achieved sustained virological response (SVR), reducing the risk of hepatitis C virus (HCV) infection substantially [5]. Nevertheless, the risk of HCC for individuals with cirrhosis remains even after HCV clearance. Nonalcoholic steatohepatitis (NASH) is becoming the main cause of HCC in the West, since it is associated with metabolic syndrome and diabetes mellitus [6]. Furthermore, there have also been reports that aristolochic acid and tobacco are potentially pathogenic cofactors for HCC [7].

The incidence of HCC differs depending on the etiology and type of genotoxins, although there is a greater understanding of the pathophysiology and drivers of HCC over the past few years; clinical applications of these insights have yet to emerge. There are actionable mutations of HCC tumors in approximately 25% of cases;

however, most mutations are less than 10%, making proof-of-concept studies difficult [7, 8]. The majority of mutations in HCC remain unsolvable, including those in TERT, TP53, and CTNNB1 [9]. Researchers are also still working on how to establish biomarkers that guide therapy based on molecular and immune classes.

Since the early 2010s, HCC management has vastly improved [8, 10–12]. The mainstay curative treatments in HCC cases have been hepatic resection and liver transplantation. For tumors down-staged beyond Milan criteria, refinements in patient selection have led to improved surgical resection results and outstanding 10-year post-liver transplantation survival rates [10, 13]. In nonsurgical early-stage HCCs, image-guided ablation using radiofrequency remains the gold standard despite advancements in alternative approaches [12]. Following these potentially curative methods, adjuvant therapies to prevent relapse are an unmet medical need, as randomized controlled trials (RCTs) have so far given poor results. The most frequently used and standard treatment for intermediate-stage HCC for the past two decades has been transarterial chemoembolization (TACE) [14]. Transarterial radioembolization (TARE) has been demonstrated to be effective in phase II studies [15], but guidelines have not yet established it as a primary standard of therapy. The arsenal of intermediate therapy is unlikely to improve in the immediate term with more locoregional devices or radiation oncology methods.

There has been a threat to the use of traditional HCC treatments from systemic medicines, such as tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), and monoclonal antibodies. Patients with HCC are predicted to be exposed to systemic therapy 50–60% of the time over their lives, especially in advanced stages of the disease [8]. The development of systemic medicines has progressed dramatically in the last 5 years, with studies showing significant improvements in overall survival and quality of life for patients [8]. As a result of the combination of anti-PDL1 antibody atezolizumab and anti-VEGF antibody bevacizumab, patients with advanced-stage HCC have a quadrupled life expectancy and improved patient-reported outcomes [16]. The most successful single-drug therapies are still sorafenib [17] and lenvatinib [18]. Regorafenib [19], cabozantinib [20], and ramucirumab [21] have similarly shown enhanced survival advantages when switched to single-agent regimens. In 15–20% of responders, single-agent ICIs produce significant therapeutic advantages, although biomarkers have thus far failed to identify this group [22, 23]. Phase III trials are also underway that examine combinations of ICIs with TKIs or PD1/PDL1 axis inhibitors with CTLA4 inhibitors to examine the efficacy of these therapies. The findings of these studies are expected to alter the landscape of managing HCC at all stages of the disease.

#### **2. Epidemiology of HCC**

In 2018, there were 841,080 new cases of liver cancer, making it the sixth most common cancer worldwide and the fourth leading cause of cancer-related death [3]. Despite an increase in HCC incidence and mortality in different parts of Europe and the United States [24], the highest rates are seen in East Asia and Africa. SEER reports that HCC has been the fastest-growing cancer-related cause of death in the United States since the early 2000s. HCC is expected to be the third leading cause of cancerrelated death by 2030 if current trends continue [25].

#### **3. Risk factors of HCC**

Chronic liver disease is responsible for more than 90% of all cases of HCC. All forms of cirrhosis are major risk factors for HCC [10, 11]. Annually, 1–6% of patients with cirrhosis die of HCC. HBV and HCV infection, chronic alcohol consumption, and diabetes- or obesity-related NASH all increase the risk for HCC [26]. Hemochromatosis, antitrypsin deficiency, and cirrhosis from primary biliary cholangitis all represent less common risk factors for HCC. Up to 45% of people with hemochromatosis who develop cirrhosis over their lifetime will develop HCC [27].

#### **4. Hepatitis B viral infection**

The cause of HCC in Asia and Africa is 60% HBV infection, while it is 20% in the West [4]. HBV is a DNA virus that can cause insertional mutagenesis and activate oncogenes by integrating into the host genome [28]. HBV increases the risk of liver cancer even if there is no cirrhosis in most patients with HBV-induced HCC. Due to the high prevalence of endemic HBV in East Asia, males (40 years of age) and females (50 years of age) have a high risk of developing HCC, which necessitates surveillance programs. The incidence of HCC in patients in their early 30s or 40s in Africa is likely due to their exposure to aflatoxin B1, a carcinogen, which increases the risk of developing HCC in combination with HBV [29]. Many Asian countries still do not have universal immunization programs, despite the fact that HBV vaccination programs have reduced HCC incidence in some regions [30].

#### **5. Hepatitis C viral infection**

The most common underlying liver disease in North America, Europe, and Japan is chronic HCV infection [4]. In contrast to HBV, HCV is an RNA virus that does not integrate into the host genome, so those who develop cirrhosis or chronic liver disease with bridging fibrosis are at risk of developing HCC. Direct-acting antiviral (DAA) medications have enabled more and more people to achieve a sustained viral response (SVR), thereby reducing their risk of developing HCC by 50–80% [5]. A number of patients, especially those from minority racial or ethnic groups and those from low-income socioeconomic areas, have not been tested for HCV and thus have no idea of their infection [31]. Additionally, people with HCV-induced cirrhosis remain at risk of developing HCC even after they have achieved sustained virologic response (>2% per year) and, thus, they have to be monitored closely [32, 33].

#### **6. Hepatitis D viral infection**

HBV surface antigens are necessary for HDV to replicate and infect. HDV is an RNA virus. Twenty to forty million people are estimated to be infected with HDV worldwide, and these individuals experience more severe liver disease, notably fibrosis and cirrhosis, than people who have only HBV. Furthermore, several cohort studies have found that co-infection with HDV and HBV may lead to an increased risk of HCC than HBV infection alone. A study reported that patients with acute or

chronic HDV infection were at a significantly higher risk of HCC than those with a sole HBV infection [34].

#### **7. Alcohol**

A fatty liver, cirrhosis, and HCC are all caused by excessive alcohol consumption. Cirrhosis caused by persistent alcohol consumption, also known as NASH, is becoming increasingly common. HCC is associated with alcohol-induced cirrhosis in 15–30% of cases depending on geographic region, with an annual incidence varying between 1% observed in population-based studies and 2–3% recorded in tertiary care referral centers [35]. There is also evidence that chronic alcohol consumption increases the risk of HCC from other causes; for example, several studies suggest that those who drink alcohol and are HBV carriers are more likely to develop HCC [36]. Although alcohol consumption has some similarities with other forms of cirrhosis, particularly NASH, in some pathophysiological processes, there is an indication that alcohol consumption may have different pro-tumorigenic mechanisms in individuals.

#### **8. Nonalcoholic steatohepatitis (NASH)**

Patients with diabetes mellitus or obesity may also develop HCC from NASH, another major factor contributing to cirrhosis. Due to the rising incidence of obesity, NASH has become a leading cause of cirrhosis around the world. Since 2010, the proportion of HCC caused by NASH has risen quickly, now accounting for 15–20% of cases in the Western world [6]. The proportion of metabolic syndrome and NASH attributable to the population is expected to exceed 20% due to the co-occurrence of these two disorders [37]. The incidence of HCC in NASH-associated cirrhosis (1–2% per year) is lower than in virus-related cirrhosis (3–5% per year), but it remains >1.1% per year, demonstrating that surveillance is cost-effective [38]. Several studies have shown that 25–30% of NASH-related HCC occurrences develop without cirrhosis, limiting the relevance of current surveillance programs that primarily target individuals with cirrhosis. However, the National Veterans Affairs Health System has discovered that the incidence of HCC annually is below the cost-effective threshold in people with non-cirrhotic NASH and surveillance should not be performed [38, 39].

#### **9. Other risk factors**

Many sociodemographic factors have been linked to HCC, particularly in individuals with cirrhosis. The risk of HCC increases with age, with those over 70 years of age showing the highest incidence [40]. HCC is also disproportionately male (maleto-female ratio of 2–3:1), which may reflect a clustering of risk factors among men, as well as differences in sex hormones [41]. HCC is more common in racial or ethnic minorities, particularly Hispanics, than in White people, according to studies. This disparity in prevalence could be related in part to the increased prevalence of singlenucleotide polymorphisms in PNPLA3, which are connected to NASH-associated HCC [42]. Smoking has also been linked to an increased risk of HCC in epidemiological studies [43]. Except for studies demonstrating a protective benefit of coffee and aspirin [44], the impact of diet in reducing the incidence of HCC is unknown.

#### **10. Mechanisms/pathophysiology of HCC**

HCC pathophysiology is a multistep process. The early stages of hepatocyte malignant transformation and HCC development are caused by the interaction of several variables. The cellular environment, immune cells, and the severity of chronic liver disease must all be considered, including genetic predisposition, and reciprocal interactions among viral and nonviral risk factors. From the early stages of transformation to invasion and then metastasis, the microenvironment plays an important role in cancer progression.

#### **11. Origin of HCC cell**

HCC's cell of origin is a point of contention. It is possible for liver cancer to originate from liver stem cells, transit-amplifying populations, or mature hepatocytes, just like in any other type of cancer. There is general controversy over whether liver stem cells exist and function. Additionally, mature hepatocytes have a high proliferation capacity after injury, which allows them to survive for long periods of time. Several studies on mouse models reported that HCC is believed to develop from transformed mature hepatocytes; however, other studies suggest HCC may originate from putative stem cells in the liver [45]. Intrahepatic cholangiocarcinomas and tumors with mixed HCC or cholangiocarcinoma form, on the other hand, often appear to emerge from adult hepatocytes, highlighting the principles of metaplasia and cell plasticity (i.e. trans-differentiation). These data back the idea that a tumor's form and epigenetic landscape may not always represent its cell of origin [46, 47].

#### **12. Mutations of cancer-driver genes in HCC**

High-throughput next-generation sequencing has identified cancer-driver genes recurrently changed in HCC with oncogenic or tumor-suppressive properties. In 80% of cases of HCC, driver gene alterations are found in the TERT promoter, chromosome translocations, telomerase activation, and gene amplification [7, 48]. Studies have shown that mutations in AXIN1 (inhibitors of the Wnt pathway), CTNNB1 (encoding-catenin), or APC (inhibitors of the Wnt pathway) inactivation activate the Wnt-β catenin signaling pathway in 30–50% of cases [7, 48]. CCNE1, TP53, ARID1A, RB1, CCNA2, PTEN, RPS6KA3, ARID2, and NFE2L2 are all known to have mutations or genetic changes that affect cell cycle control. AKT-mTOR and MAPK pathways, as well as genes involved in epigenetic regulation and oxidative stress, have been linked to HCC. AKT-mTOR and MAPK pathways, as well as genes involved in epigenetic regulation and oxidative stress, have been linked to HCC. The recurrent overexpression and activation of oncogenic signaling pathways, including receptor tyrosine kinases, are also linked to focal chromosomal amplification of MYC, CCND1, VEGFA, FGF19, and MET [49]. In spite of the fact that cancer-driver gene mutations can occur at random, certain genes seem to be associated with specific molecular HCC subclasses based on transcriptome profiles and histological phenotypes [8, 9, 50]. At least 20–25% of HCC patients have a potentially actionable mutation, according to current standards [7, 8, 51]. In the pathogenesis of HCC, it has been well documented that risk factors cooperate with cancer-driver mutations. In patients with a GSTT1 null mutation, for instance, the harmful effects of aflatoxin B1 are amplified by HBV

infection [52, 53]. In addition, patients who use a lot of alcohol are more likely to have polymorphisms in PNPLA3, TM6SF2, and HSD17B13 [54, 55].

#### **13. Molecular alterations associated with viral infection**

The TERT promoter is the most common locus of HBV-mediated insertional mutagenesis, resulting in overexpression of telomerase, the enzyme responsible for telomere length maintenance [56]. Telomerase activation inhibits the chromosomal erosion that occurs naturally with each cell division as people age. Telomerase activity on the ectopic enhances cell transformation and protects cells against senescence [57]. Other HBV-associated recurrent insertions have been shown to activate potent oncogenes involved in cell cycle control, such as CCNA2 or CCNE1. Replicative stress and complex rearrangements are caused by these oncogenic changes throughout the genome [58]. Adeno-associated virus 2 (AAV2) showed identical insertional oncogenic mutagenesis in a small group of HCC patients, with a shared hot point of the viral insertion inside the TERT promoter, CCNA2, and CCNE1 [59]. These findings show that viral infection activates particular oncogenes, which act as early facilitators of hepatocyte transformation. HCV infection, on the other hand, has no direct carcinogenic effect, and the induction of mutations is driven by the oxidative stress caused by persistent inflammation.

#### **14. Mutational signatures in HCC**

Hepatocytes are subjected to multiple genetic mutations and epigenetic alterations throughout the progression of chronic liver disease and cirrhosis, which are the most common causes of HCC. Several risk factors that cause DNA changes are linked to particular mutational signatures during this process [7, 60]. In exome sequencing analyses of HCC, patients from Asia and Africa who had been exposed to aristolochic acid (A > T mutations in CTG trinucleotide) and aflatoxin B1 (C > A mutations) had mutational signatures 22 and 24, respectively [7, 61]. Mutations of the C > A at dinucleotide sequences in signature 4 were linked with tobacco smoking, while the T > C mutations at TpA dinucleotide in signature 16 were related to alcohol consumption [62]. It remains to be seen whether this discovery can be turned into preventative measures. It is well known that the liver is capable of detoxifying a variety of chemicals that may cause mutations in the hepatocyte genome, leading to the development of cancer.

#### **15. Molecular classes of HCC**

Several studies have created a molecular and immune categorization for HCC based on genomic, epigenomic, histopathological, and immunological analysis [1, 9, 63]. Molecular classes of HCC have been identified based on the principal molecular drivers and pathways involved [9, 63–67] or the tumor's immunology status [8, 68]. The molecular classifications are associated with specific genomic abnormalities, histological signatures, and clinical outcomes. Approximately half of all HCCs are of the proliferation type [49]. The proliferation type is characterized by mutations in TP53 and FGF19 or CCND1 amplification, and it is more common in HBV-associated cancers with poor prognosis. Within the proliferation class, there are two subclasses: proliferation

#### *Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.105473*

progenitor cells and proliferation-Wnt-TGF cells. Twenty-five to thirty percent of HCC are proliferation-progenitor cells, which are characterized by activation of classical cell proliferation pathways, i.e. the expression of progenitor cell markers (such as EPCAM and FTP) is also related to the activation of signaling pathways (PI3K-AKT-mTOR, RAS-MAPK, and MET and IGF signaling cascades [49, 64]. In alcohol- and HCV-related HCC, non-proliferative tumors represent more than half of all cases; these tumors have better outcomes and correspond to TCGA cluster 2 [65]. Within the nonproliferative class, at least two distinct subgroups have been described: one with dominant canonical Wnt signaling and mutations in CTNNB1 [69] and the other with IFN signaling activation [49].

Reports on the classification of HCC based on immune cell status have added to the knowledge of HCC's molecular characteristics [68]. This categorization classifies HCC tumors into four subclasses: immunological-active, immune-exhausted, immune-intermediate, and immune-excluded, and gives additional information based on immune features. Immune cell infiltrations are categorized into two subclasses: immune-active and immune-exhausted. In HCC tumors that are immune-active, helper T (CD4+) and cytotoxic T (CD8+) cells are enriched and ICIs are effective. The depletion of CD8+ cells driven by TGF is prevalent in immuneexhausted tumors. In contrast, immune-excluded tumors lack T cell infiltrates and are characterized by a disproportionate increase in regulatory T cells (Tregs), as well as canonical Wnt signaling and other immune-suppressive pathways. Immune-excluded tumors often develop ICI resistance [70].

**Figure 1.** *The molecular mechanism of HBV-induced HCC.*

Obesity has been related to a higher risk of cancer in a variety of organs [71]. Obesity can cause systemic alterations, such as impaired immune function and endocrine abnormalities, which are common in cancers of many types. According to current research, fatty liver disease is quickly becoming the primary cause of HCC in the Western world [6]. The effects of metabolic and oxidative stress, immune dysfunction, abnormal inflammatory responses, impaired endocrine, and adipokine signaling have all been identified as pathways by which NAFLD or NASH cause HCC (**Figure 1**) [72, 73].

Several classical cell proliferation pathways are activated in HBV-associated HCC tumors, including PI3K-AKT-mTOR, RAS-MAPK, MET, and Wnt-TGF. A high chromosomal instability level and frequent TP53 and AXIN1 mutations are additional features of HBV-induced HCC (**Figure 2**).

Nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis, and hepatitis C virus (HCV) infection promote the development of HCC tumors. Here, the risk factors cause chromosomal instability with frequent mutations in the TERT promoter

**Figure 2.** *The molecular pathogenesis of HCC induced by NASH, HCV, HDV, and alcohol.*

sequence which, in turn, leads to the CTNNB1 mutations and activation of either WNT-β-catenin signaling pathway or IL6-JAK-STAT signaling pathway. The activation of either or both of these signaling pathway promote the proliferation of progenitor cells leading to an inflammatory tumor microenvironment and ultimately to HCC.

#### **16. Oxidative stress and HCC**

Fatty acid overload causes oxidative stress and endoplasmic reticulum (ER) stress in hepatocytes, resulting in pathological inflammation and cell death [72, 74]. HCC was induced in one study in mice following ER stress-induced inflammation via NF-κB and TNF-α signaling pathways [75]. These toxicological processes of HCC, however, are yet to be demonstrated in human. Hepatocytes with abnormal fatty acid metabolism are susceptible to DNA damage caused by reactive oxygen species (ROS) resulting from mitochondrial dysfunction [76]. Hepatocytes are also affected by changes in the expression of particular metabolic enzymes, which reduces their ability to repair DNA damage [77]. Changes in inflammatory signaling are also a result of the metabolic failure; for example, elevated levels of IL-17 (a tumor-promoting cytokine) have been seen in human NASH [78]. A number of pathogenic lipids are produced as oncometabolites in NASH in addition to increased lipid production [79, 80]. When mTORC2 is continuously activated in mouse hepatocytes, a high level of glucosylceramide is produced, increasing ROS production, which can lead to HCC [79]. Alterations in cholesterol metabolism may also have a role in HCC pathogenesis [80], possibly by causing the generation of pro-tumorigenic nuclear receptor ligands. Although autophagy has antitumor properties, one study found that lipophagy (autophagic destruction of lipid droplets) plays a crucial role in HCC progression. Hepatocytes from NASH patients and a mouse model of HCC overexpress sequestosome 1 (also called p62), a lipophagy regulator [81]. Patients with NASH had a higher risk of HCC than those with NAFLD according to studies [6]. In one experiment, fatty acid-induced oxidative stress in hepatocytes increased the expression of STAT1 and STAT3, two pro-inflammatory transcription factors that generally operate in tandem [82]. Surprisingly, a high level of STAT1 promoted NASH progression in this mouse model, while a high level of STAT3 promoted HCC, both independently [82]. Accordingly, similar inflammatory signals may promote progression from NAFLD to NASH or HCC in different ways. This is because NAFLD is more common in the general population than NASH [6]; the data indicate the need to understand how NAFLD, regardless of NASH, can lead to HCC. When hepatocytes are overloaded with fatty acids, the increased ER stress, pathological lipophagy, ROS generation, and a lowered reducing power (low NADH or NADPH levels) may combine to generate oncogenic genetic changes and accelerate the development of malignant cells.

Based on transcriptomic-based phenotypic classes, hepatocellular carcinoma (HCC) can be divided into two primary molecular groupings [49, 64–67]. More aggressive tumors with weak histological differentiation, high vascular invasion, and higher levels of alpha-fetoprotein (AFP) belong to the proliferation class [50]. In S1 or iCluster 3 [64, 65], Wnt-TGF activation leads to an immune-exhausted phenotype [68], while in S2 or iCluster 1 [64, 65], stem cells markers (CK19, EPCAM) as well as IGF2 and EPCAM signaling pathways are expressed [50]. In hepatitis B virus (HBV)-associated tumors, cell proliferation pathways such as PI3K-AKT-mTOR, RAS-MAPK, MET, and IGF are usually activated. Furthermore, numerous TP53 mutations, high chromosomal instability, and widespread DNA hypomethylation are also

characteristics of this group. The nonproliferation class consists of tumors that are less aggressive, well-differentiated histologically, have low AFP levels, and have fewer vascular invasions [50]. These tumors can be caused by nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), or infection with hepatitis C virus (HCV) [49, 64–67]. This class is divided into two distinct subgroups: the WNT––catenin CTNNB1 subclass has frequent CTNNB1 mutations and activated WNT––catenin signaling, leading to an immune-excluded phenotype with low immune infiltration [49, 67, 68]; and the interferon subclass has a highly activated IL6-JAK-STAT signaling pathway, leading to a more inflamed microtumor with many TERT promoter mutations, and this class has chromosomal stability [63–68].

#### **17. Immune infiltration of fatty liver**

The histological characteristic of NASH is immune cell infiltration of the obese liver [72]. The establishment of animal models that accurately reproduce human HCC is critical for basic pathogenesis research as well as translational research [83–97]. Immune cells and cytokines have been found to have an essential role in the pathogenesis of HCC in several experimental types. In mouse models, for example, persistent NASH causes CD8+ T cell activation, which leads to hepatocyte destruction and HCC [98]. As a consequence of NAFLD, intrahepatic CD4+ T cells are selectively depleted, which are necessary to initiate an effective adaptive immune response against tumors [99]. Additionally, B cells, Treg cells, natural killer cells, and other myeloid cells have been associated with NASH-induced HCC [72, 73]. The activation and recruitment of platelets in the liver also contribute to HCC formation in mice, specifically via platelet glycoprotein Ib (GPIb) signaling, which is in line with clinical data [100], implying that this pathway has the therapeutic potential [101]. The causal function of NASH in HCC was also linked to a changed cytokine milieu [74]. NASH, for example, has been demonstrated to overexpress hepatic IL-6 and TNF-α, which are both causes of HCC in various etiologies [102].

On the background of fatty liver disease, all of the mechanisms described earlier could promote HCC at the same time. Their relative involvement to human HCC, however, is uncertain at this time. The comparison of mutational signatures in NASHassociated HCC versus HCC from other causes should aid in determining the relative contributions of different variables.

#### **18. Inflammation and HCC**

HCC is an archetypal inflammation-related malignancy, with chronic inflammation caused by viral hepatitis, excessive alcohol consumption, NAFLD, or NASH accounting for 90% of the HCC burden. In the development of HCC [103], the immunological microenvironment plays a critical role. Immune infiltrates are associated with a better prognosis in HCC, possibly due to more effective antitumor immunity [68, 104]. Immune signals such as IL-6, lymphotoxin-, and TNF-α have been shown to accelerate hepatocarcinogenesis and impact tumor aggressiveness in mouse models of HCC [47, 105], yet immune responses can also slow the course of liver cancer [103]. In addition, the liver has the greatest number of immune cells in the body and has a unique immunological state that allows it to survive the constant influx of inflammatory signals coming from the gut [103]. Understanding

#### *Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.105473*

this specific hepatic immune system is likely important given the intricate interplay between malignant hepatocytes and the liver immune system [103, 106]. A surprising finding in mice and humans is that VEGF released by malignant hepatocytes creates an immune-tolerant, pro-tumorigenic microenvironment [49, 107], suggesting that inhibiting the VEGF cascade might have a positive effect on liver immunity by modifying VEGF production. Interestingly, the combination of ICIs and certain targeted medicines such as VEGF inhibitors had greater survival advantages than the use of single agents [16, 108].

It has been shown that hepatocytes in chronically inflamed livers interact with numerous cell types including macrophages, endothelial cells, stellate cells, and various types of lymphocytes [103, 106]. Due to its importance in immuno-oncology therapy, researchers are paying more attention to the adaptive immune system's involvement. Mouse models have revealed that practically every immune cell type can play both pro-tumor and antitumor roles [103]. In addition to producing protumorigenic cytokines and growth factors that support tumor cell proliferation or inhibit apoptosis, immune cells also diminish nearby lymphocytes' antitumorigenic function. The NF-B and JAK-STAT pathways have been identified as major inflammatory signaling pathways implicated in the promotion of HCC in studies [109], and this assertion was confirmed in a transcriptomic analysis of human HCC [110]. Immune monitoring and the destruction of premalignant or completely changed malignant hepatocytes are the adaptive immune system's main antitumor functions [104].

#### **19. The role of adaptive immune system in HCC**

The main effectors of antitumor immunity are cytotoxic T (CD8+) cells. As a result, one study found that depleting these T cells in mice increased HCC burden [111], while another found that these T cells promote premalignant hepatocyte surveillance [112]. Several studies in mice have shown that the depletion of CD8+ T cells can also reduce tumor burden [98]. Analyses of human HCC samples suggest that some individuals have functional CD8+ T lymphocytes that produce antitumor effector molecules such as granzyme A, granzyme B, and perforin [113]. However, single-cell sequencing of human HCC T cells has revealed that the CD8+ T cells are often dysfunctional in HCC [114]. There is no clear understanding of the causes of CD8+ T cell dysfunction, which leads to diminished proliferation and the inability to generate cytotoxic effector molecules. Increasing numbers of Treg cells within the tumor are linked with poorer clinical outcomes in HCC, and Treg cells are thought to be a primary cause of T cell dysfunction [115]. Treg cells' immunosuppressive capabilities may be mediated by CD10 and TGF116 production, suggesting that blocking these cytokines could make HCC more susceptible to ICIs. HCC-infiltrating Treg cells are known to suppress immune responses through the hyaluronic acid receptor, layilin, which is interesting [116]. As a result of a layilin induction, CD8+ T cells exhibited dysfunction in human HCC, and layilin overexpression was associated with distinct mRNA expression signatures in lymphocytes [114].

Although B cells were once assumed to be innocent bystanders in cancer, new data suggest that they have an active role in the adaptive immune system's interaction with cancer [117]. B lymphocytes both stimulated and inhibited tumor growth in mice models of HCC [118]. Furthermore, one study found that IgA-expressing cells actively suppressed CD8+ T cell activity, which aided HCC growth [111]. Furthermore, studies in humans and mice have shown that tertiary lymphoid

structures, which are crucial for adaptive immune responses to cancer [119], have both pro-tumor and antitumor capacity in HCC [120, 121].

#### **20. The microenvironment of cirrhosis in HCC**

The risk of HCC is high enough to warrant surveillance once the patient has reached cirrhosis, even though some etiologies (for instance, HCV versus autoimmune hepatitis) are more likely to cause HCC than others [10, 11]. In response to chronic injury, hepatic stellate cells play an important role [122]. Upon activation, it undergoes phenotypic changes and synthesizes components of the extracellular matrix, mainly collagen, as well as growth factors, which promote neoangiogenesis, endothelial cell migration, and fibrosis [123]. Cirrhosis and portal hypertension have a histological substrate in which the hepatic architecture is distorted and the vasculature is disordered. Premalignant senescent hepatocytes respond to this condition by secreting chemokines that impair senescent surveillance and immune-mediated tumor suppression in vivo [112]. Experimental models have also demonstrated that CD4+ cells are relevant in promoting NAFLD-related HCC [99], and the interaction between the innate immune system and the intestinal microbiota plays a role in promoting the development of HCC [124, 125]. In HCC, the immune system, in addition to fibrosis, plays a significant role in the cancer field effect. The cancer field effect refers to the favorable microenvironment in cirrhosis that favors tumor formation. The primary molecular elements unregulated in this microenvironment have been identified through various genomic investigations. Several gene profiles obtained from cirrhotic tissue are associated with the probability of developing HCC and can be utilized to risk stratify patients [110, 126, 127]. The presence of these gene signatures is associated with cancer risk, the incidence of hepatic decompensation in patients, and overall survival [126, 127]. More research has been done on the genetic characteristics of the cirrhosis inflammatory milieu that contribute to HCC development [128]. In 50% of neighboring cirrhotic tissue from HCC patients, an immunemediated cancer field molecular subclass was observed. In addition to lymphocyte infiltration, this subclass can be further divided based on pro-inflammatory or immunosuppressive signal activation. In the immunosuppressive subclass, which accounted for 10% of patients and had a threefold higher risk of developing HCC, TGF signaling, T-cell exhaustion, and overexpression of immunological checkpoints (such as CTLA4, TIGIT, and LAG3) were shown to be more prevalent [128]. Modulating the tumor microenvironment's role in HCC's natural history would be a compelling reason for altering the dynamic crosstalk between hepatocytes and the hepatic immune system [103].

*Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.105473*

#### **Author details**

Rahmat Adetutu Adisa1 \* and Lateef Adegboyega Sulaimon1,2

1 Faculty of Basic Medical Sciences, Department of Biochemistry, Laboratory for Biomembranes, Toxicology and Cancer Research, College of Medicine, University of Lagos, Lagos, Nigeria

2 Department of Chemical Sciences, College of Natural and Applied Sciences, Crescent University, Abeokuta, Ogun State, Nigeria

\*Address all correspondence to: radisa@unilag.edu.ng

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## *Edited by Costin-Teodor Streba, Ion Rogoveanu and Cristin Constantin Vere*

This book discusses the various therapeutic agents and pathologies that can have a negative impact on liver function. The profound changes such agents may exert on the liver, an essential organ, can severely alter a patient's metabolism, negatively impacting the course of a disease and thus significantly shortening life expectancy and negatively affecting outcomes. This book provides a comprehensive overview of these selected issues with chapters on epidemiology, pathogenesis, clinical manifestations, diagnostic methods, and treatment options in the context of hepatotoxicity.

Published in London, UK © 2022 IntechOpen © Sinhyu / iStock

Hepatotoxicity

Hepatotoxicity

*Edited by Costin-Teodor Streba,* 

*Ion Rogoveanu and Cristin Constantin Vere*