**7.3 With or without 'iron deposition in hepatocyte and/or Kupffer cell'**

Although iron is indispensable for normal physiology and biochemical reactions, excess iron is toxic and harmful because it can accelerate the Fenton reaction that generates noxious reactive oxygen species (ROS) and severely damages cells and tissues in the human body. Thus, maintenance of body iron homeostasis is pivotal, particularly because there is no physiological pathway for removal of excessive iron from the body [109]. Systemic iron regulation is mediated via the liver-secreted iron regulating hormone hepcidin under normal physiological conditions [110]. Several studies have reported the fibrosis-enhancing effects of iron. For instance, induced collagen deposition in gerbil [111], iron elevated collagen gene expression in HSCs and increased TGF-β expression in rats [112], and promoted cirrhosis in mice [113]. Ramm et al. [114] demonstrated the correlation between LIC and HSC-activation in humans, resulting in increased expression of α-SMA and collagen deposition in patients with hemochromatosis for the first time. Similar results were observed in rat HSCs, wherein iron increased HSC-cell proliferation and selectively increased collagen synthesis without affecting non-collagen proteins [115].

In the pathogenesis of NAFLD, iron has been widely implicated, therefore represents a potential target for treatment. Correlations between the serum ferritin concentration and NAFLD are noted in most studies, although serum ferritin is an indistinct measure of iron loading. A large number of mechanisms underlying the pathogenic role of hepatic iron in NAFLD have been demonstrated in animal and cell culture models. However, the human data linking hepatic iron to liver injury in NAFLD is not so clear, with seemingly conflicting evidence, supporting either an

effect of iron in hepatocytes or within reticuloendothelial cells. Adipose tissue has emerged as a key site where iron may have a pathogenic role in NAFLD [116].

An investigation of the serum ferritin level and histological findings including iron deposition in 628 patients with NAFLD was performed by Kowdley et al. [105]. This large cross-sectional study revealed that elevated serum ferritin (>1.5 × UNL) was associated with advanced hepatic fibrosis (odds ratio [OR], 1.66; 95% confidence interval [CI], 1.05–2.62; P = 0.028) and a higher NAS (OR, 1.99; 95% CI, 1.06–3.75; P = 0.033). Elevated serum ferritin levels (seen in approximately 20% of the subjects) were associated with greater iron accumulation in the body (i.e., a high serum iron and transferrin-iron saturation) and greater hepatic iron deposition in both the reticuloendothelial system and hepatocytes.

It was also elucidated that the patients with increased serum ferritin levels also had higher serum transaminases and gamma-glutamyl transferase and a lower platelet count. Interestingly, even in patients without a hepatic iron overload on histology, higher serum ferritin was correlated with advanced stage of the disease.

## **7.4 With or without 'inflammation and elevation of cytokines'**

One of modifying factors in NAFLD/NASH is hepatic iron content (HIC) [117]. Iron accumulation exacerbates hepatic oxidative stress and can, therefore, affect susceptibility to oxidant stress induced by fatty acid oxidation [118]. HIC is susceptive to factors that differ among individuals (polymorphisms in genes such as HFE) and factors that might change during the lifetime of individuals, including sex-related factors (menstruation or pregnancy) and diet (consumption of greasy meal, roughage or red meat) [119]. The lipotoxic outcomes of identical fatty acid exposures can, therefore, differ based on complicated factors that modulate hepatic iron content. Inflammation is required to clear damage-related debris and stimulate local accumulation of other wound-healing cells, such as liver progenitors and myofibroblasts. However, excessive inflammation can compromise the viability of residual hepatocytes and promote over-growth of progenitors and myofibroblasts, laying the groundwork for progressive fibrosis to liver cirrhosis and carcinogenesis. Therefore, the liver is variably repopulated with relatively immature or dysfunctional hepatocytes as long as wound-healing responses are active and continuing. This potentiates metabolic stress and increases the risk of liver cancer [120].

Recent studies have suggested several possibilities in progressing liver fibrosis, involving inflammation caused by OS associated with lipid peroxidation, endogenous toxins of fructose metabolites, cytokine activation and NO [9]. Mitochondrial dysfunction not only facilitated the production of ROS but also contributed to the progression of NAFLD by inducing hepatic inflammatory cytokines. The network of obesity, IR and adipokine/cytokine has been hypothesised to induce both liver fat accumulation and NASH development [121]. ROS along with products of lipid peroxidation leads to increased release of several cytokines (tumour necrosis factoralpha (TNF-α), Fas ligand), which play a key role in cell death, inflammation and fibrosis [107]. Lipid peroxidation, release of inflammatory cytokines and cell death are the consequences of ROS-mediated mechanisms. Biologically active lipid peroxidation products and cytokines take action together by inducing hepatic inflammation, leading to the development of diverse hepatic lesions associated with NASH. The inflammatory response is induced because of the upregulation of pro-inflammatory cytokines including TNF-α, interleukin (IL) 1 and IL-6 [122], which play an essential role in directing polymorpho-and mono-nuclear leukocytes into flamed tissue. The
