**2. The development of a novel animal model of NASH and the mechanism underlying the progression of NASH in this model**

The mechanisms of the pathogenesis of NASH are not completely understood, partly due to a lack of ideal animal models with histological patterns that resemble human NASH. Matsuzawa et al. showed that an HFC diet (an atherogenic, high-fat diet containing 1.25% cholesterol and 60% fat) induced steatohepatitis, cellular ballooning, and fibrosis in the livers of male C57Bl/6J mice [21]. We previously established an HFC diet-induced NASH model using hypertensive SHRSP5/Dmcr rats [20].

intake (300 mg) was removed from the 2015–2020 Dietary Guidelines for Americans [3], it is still recommended that individuals minimize cholesterol consumption. Animal foods such as egg yolk, meats, dairy products, fish, and poultry are major sources of dietary cholesterol. Meanwhile, dietary cholesterol is not found in plant foods. Instead, many plants contain phytosterols, which are chemically similar to cholesterol, and can therefore compete with it and decrease its absorption in the intestinal tract [4]. The effect of dietary cholesterol on plasma cholesterol levels remains undetermined, since the body may suppress endogenous cholesterol synthesis in response to additional cholesterol ingestion [1]. Some studies have suggested that dietary cholesterol increases serum total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, as well as the ratio of LDL to high-density lipoprotein cholesterol [5–8],

Dietary cholesterol is also linked to the pathogenesis of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) [9, 10]. NAFLD is one of the most common chronic liver diseases worldwide and comprises a spectrum of liver damage, from simple steatosis (a benign non-progressive condition) to NASH, the advanced form that may progress to hepatic cirrhosis or hepatocellular carcinoma [11]. The pathological characteristics of NASH include steatosis, hepatocellular ballooning, lobular inflammation, and hepatic fibrosis. Cholesterol may contribute to NASH development by being catabolized in the liver into bile acids (BAs), which are hepatotoxic and cause liver damage [12]. Li et al. demonstrated that dietary cholesterol exacerbates liver damage and hepatic inflammation in mice fed a high-fat diet [13]. Subramanian et al. reported that an LDL receptor-deficient mouse fed a high-fat, high-carbohydrate diet was a good animal model of NAFLD/NASH, and showed that dietary cholesterol worsened hepatic steatosis and inflammation in this model [9].

In addition, NAFLD/NASH was described as a hepatic manifestation of metabolic syndrome, and its development was associated with hypertension, obesity, diabetes, and hyperlipidemia [14, 15]. Some studies have shown an increased prevalence of NAFLD/NASH among hypertensive patients [16–18]. Using spontaneously hypertensive (SHR) rats fed a choline-deficient diet as a hypertensive animal model of NASH, and its normotensive control, the Wistar Kyoto (WKY) rat [19], Ikuta et al. revealed that hypertension enhances the progression of NASH. We previously developed a novel animal model of hypertension-associated NASH by feeding stroke-prone spontaneously hypertensive5/Dmcr (SHRSP5/Dmcr) rats a high fat and cholesterol (HFC) diet [20]. Further studies from our group suggested that dietary cholesterol may have a potential effect on the development of hypertension-associated NASH (unpublished). In this chapter, we will discuss the crucial role of dietary cholesterol in the progression of

**2. The development of a novel animal model of NASH and the mechanism underlying the progression of NASH in this model**

The mechanisms of the pathogenesis of NASH are not completely understood, partly due to a lack of ideal animal models with histological patterns that resemble human NASH. Matsuzawa et al. showed that an HFC diet (an atherogenic, high-fat diet containing 1.25%

which are considered to be associated with risk of vascular diseases.

hypertension-associated NASH.

110 Cholesterol - Good, Bad and the Heart

SHRSP5/Dmcr rats are the fifth substrain of the stroke-prone spontaneously hypertensive (SHRSP) rat [20, 22], which is derived from the SHR strain [23]. To establish this strain, SHRSP rats were fed an HFC diet for 1 week, then those with high serum cholesterol levels (600–900 mg/dL in females and 300–600 mg/dL in males) were selected for brother–sister inbreeding. Selective inbreeding was repeated and offspring with increased hypercholesterolemic responses were obtained. Although the SHRSP5/Dmcr rats, formally known as arteriolipidosis-prone rats, were developed as an animal model of arteriosclerosis, marked enlargement and an abnormal whitish color of the liver were noted in the 47th generation. These findings prompted our studies on HFC diet-induced liver damage in this strain.

In order to determine whether the HFC diet-fed SHRSP5/Dmcr strain was a suitable model of NASH, we investigated hepatic histopathological changes following HFC feeding [20]. Male SHRSP5/Dmcr rats at 10 weeks of age were fed either an HFC (35.3% crude lipid and 5% cholesterol) or control diet (4.8% crude lipid and no additional cholesterol) for 2, 8, and 14 weeks. We found that the HFC diet induced microvesicular steatosis and lymphocyte infiltration at 2 weeks. Macrovesicular steatosis, ballooned hepatocytes with eosinophilic Mallory-Denk bodies, and multilobular necrosis were observed in the livers of rats fed an HFC diet at 8 weeks. The severity of steatosis and hepatocyte ballooning was further increased at 14 weeks. Meanwhile, a progressive deterioration of hepatic fibrosis occurred during HFC feeding. Slight pericellular and perivenular fibrotic changes, bridging fibrosis, and end-stage honeycomb fibrosis were observed at 2, 8, and 14 weeks, respectively. In addition, the HFC diet induced a progressive increase in indicators of liver damage, including serum levels of alanine transaminase (AST), aspartate transaminase (ALT), and γ-glutamyltranspeptidase (γ-GTP). Matteoni et al. classified human NAFLD into four types according to histological analysis of liver biopsy specimens: type 1, fatty liver alone; type 2, fat accumulation and lobular inflammation; type 3, fat accumulation and ballooning degeneration; and type 4, fat accumulation, ballooning degeneration, and hepatic fibrosis [24]. The histological characteristics observed in the liver of the SHRSP5/Dmcr strain at 2, 8, and 14 weeks of HFC feedings were very similar to those in type 2, type 3 or 4, and type 4 human NAFLD, respectively. Therefore, all pathological stages of NAFLD can be observed in the SHRSP5/Dmcr strain during HFC feeding. In addition, obesity, insulin resistance, and diabetes were not observed in this model. Therefore, it represents an excellent model of NAFLD/NASH without obesity and diabetes, and is useful for studying the pathogenesis and therapeutics of this disease.

We further investigated the molecular mechanisms underlying the progression of HFCinduced NASH in the SHRSP5/Dmcr strain [25]. Rats were fed either an HFC or control diet for 2, 8, and 16 weeks, and expression of genes involved in inflammation and hepatic fibrosis was evaluated. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine, was reported to be upregulated in the livers of NASH patients [26]. We showed that the HFC diet increased the hepatic expression of TNF-α in SHRSP5/Dmcr rats at all time points. Nuclear factor κB (NF-κB; p50/p65) and inhibitor of κBα, the proteins involved in NF-κB signaling, which is regulated by TNF-α and plays an important role in inflammatory response, were also upregulated by the HFC diet. Hepatocyte injury and inflammation led to hepatic fibrosis via hepatic stellate cell (HSC) activation, which results in the production and deposition of extracellular matrix (ECM) [27]. The HFC diet induced the upregulation of transforming growth factor-β1 (TGFβ1), a profibrotic cytokine that promotes HSC activation, prior to the appearance of obvious hepatic fibrosis (at 2 weeks). Its upregulation was also observed at subsequent stages (at 8 and 16 weeks). Expression of alpha smooth muscle actin (α-SMA) and platelet-derived growth factor-B, involved in hepatic fibrosis, were elevated at 8 weeks of HFC feeding, indicating extensive activation of HSC at this time point. Alpha-1 type I collagen, the major component of ECM, was produced by activated HSC (myofibroblast) and was markedly elevated at 8 and 16 weeks, corresponding to the appearance of extensive liver fibrosis observed at the same time points.

strain. The severity of the hepatic fibrosis observed in the SHRSP5/Dmcr strain was even higher compared with that of the SHR strain. The HFC diet induced elevation of serum inflammatory cytokines, TNF-α and TGF-β1, in the hypertensive strains, whereas an increase in TGF-β1 was not observed in the normotensive rats. The combination of TNF-α and TGF-β1 may trigger a more severe inflammatory response in the hypertensive rats by regulating the activation of downstream inflammatory signaling such as NF-κB and mitogen-activated protein kinase (MAPK) pathways. Increased activation of NF-κB and MAPK (p38 and JNK) signaling occurred in the hypertensive strains, which may have contributed to the more severe lobular inflammation observed in these rats. In addition, oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and their elimination by antioxidant defenses, may lead to cellular injury and chronic inflammation [30]. An increase in oxidative stress in NASH patients was previously reported [31, 32]. We measured serum thiobarbituric acid reactive substances levels and found that oxidative stress was significantly elevated in hypertensive strains fed an HFC diet but not in normotensive rats (unpublished data). Meanwhile, in hypertensive rats, the HFC diet suppressed the nuclear factor erythroid 2-related factor 2 (Nrf2)/ Kelch-like ECH-associated protein 1 (Keap1) pathway, involved in antioxidative defenses [33]. We also found that hepatic levels of superoxide dismutase-1 (SOD-1) [25] and SOD-2 [28], that contribute to antioxidant defense by catalyzing the dismutation of superoxide anions [34], were decreased in hypertensive SHRSP5/Dmcr rats fed the HFC diet. The decrease in SOD-2 expression induced by HFC feeding was not observed in normotensive WKY and hypertensive SHR strains [28]. This could suggest that an increase in oxidative stress and a lower antioxidative capacity may trigger a more severe inflammatory response and liver damage in hypertensive

The Role of Cholesterol in the Pathogenesis of Hypertension-Associated Nonalcoholic…

http://dx.doi.org/10.5772/intechopen.76199

113

rat strains following HFC feeding, compared with normotensive strains.

**associated NASH**

**4. The role of cholesterol in the development of hypertension-**

As previously stated, dietary cholesterol intake is considered a risk factor for NAFLD/NASH.The liver is a crucial organ implicated in the regulation of cholesterol metabolism, including the synthesis and secretion of cholesterol, as well as the synthesis of BAs from cholesterol (a major pathway for hepatic cholesterol catabolism) and BA detoxification [35]. Disturbed cholesterol homeostasis in the liver is thought to be associated with the pathogenesis of NAFLD/NASH [35]. Our study showed that the HFC diet increased serum and hepatic levels of TC in the hypertensive SHR and SHRSP5/Dmcr strains, as well as the normotensive WKY strain [28]. It is worth noting that the increase in hepatic TC levels in the hypertensive rats was significantly lower than those in the normotensive WKY strain. Therefore, we postulated that more cholesterol was consumed for the synthesis of BAs in the livers of the hypertensive rats. In addition, serum TC levels in the hypertensive strains fed the control diet were markedly lower compared with those of the normotensive WKY strain, suggesting that the dysregulation of cholesterol metabolism may play an important role in the progression of hypertension-associated NASH.

In order to investigate the kinetics of cholesterol during the development of HFC-induced NASH in our hypertensive SHRSP5/Dmcr rat model, we evaluated the expression of proteins

In order to investigate the role of dietary cholesterol in the pathogenesis of HFC diet-induced NASH in SHRSP5/Dmcr rats, we compared hepatic histological changes induced by a high fat (HF) diet and those by an HFC diet (unpublished). As described above, the HFC diet induced severe steatosis, lymphocyte infiltration, ballooned hepatocytes, and fibrosis in the livers of the rats. In contrast, HF feeding only led to mild hepatic steatosis and lymphocyte infiltration, while liver fibrosis was not observed. It was suggested that dietary cholesterol may play a key role in the transition from simple steatosis to fibrotic steatohepatitis, the progressive stage, during the progression of NAFLD/NASH.
