Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), reportedly affects the health status of approximately one in four people worldwide, according to a recent meta-analysis [1]. The prevalence of this disease is projected to increase in the future due to the growing numbers of people with obesity, dyslipidemia, hypertension, and type 2 diabetes (T2DM). NAFLD is one of the most common causes of liver failure necessitating transplantation in the U.S. [2]. Due to the global increase in metabolic syndrome and the advancements in therapies for viral hepatitis, NAFLD is expected to become the most common cause of liver cirrhosis and liver cancer in the near future, surpassing viral etiologies [3].

NAFLD can be categorized histologically into nonalcoholic fatty liver (NAFL) and NASH. While NAFL is defined as the presence of hepatic steatosis without necroinflammation, the key pathological features for the diagnosis of NASH are inflammation, necrosis, fibrosis in the liver, the ballooning degeneration of hepatocytes, and hepatic steatosis [4]. Without therapeutic interventions, 7–30% of NAFL cases may progress to the necroinflammatory form, NASH ^[1][3][1,3]. In a recent meta-analysis of more than 4000 patients with NAFLD, liver fibrosis was tightly associated with all-cause mortality as well as liver-related mortality ^[1][5][1,5]. Therefore, liver fibrosis is the most important factor contributing to the prognosis of patients with NAFLD and has been a major focus of the emergence of targeted therapies for this disease [6]. A prospective study yielded the same results and concluded that fibrosis stage is positively associated with risks of liver-related complications and death [7]. NASH and advanced fibrosis need to be identified in order to prevent the related complications.

Nevertheless, there are increased risks of all-cause mortality, liver-related mortality, and cardiovascular events in patients with NAFLD compared with the general population ^[1][5][8][1,5,8]. Apart from diet and weight loss, there is no established treatment for NAFLD, despite the various clinical trials that have been conducted. Of note, liver fibrosis can serve as a surrogate endpoint for the outcomes in clinical trials. Although potential pharmacotherapeutic agents have been tested in clinical trials, most of them have failed to achieve the expected outcomes of steatohepatitis resolution or reversal of fibrosis. Now there is a large unmet need to gain a deeper understanding of the underlying mechanisms in NAFLD and to identify and develop effective treatment options for the benefit of patients. Therefore, it is important to establish therapeutic agents and preventive interventions with the potential to reduce the risk of the progression of NAFLD, particularly approaches that combat liver cirrhosis and hepatocarcinogenesis.

2. Factors Associated with Nonalcoholic Fatty Liver Disease (NAFLD) Pathophysiology

NAFLD is associated with obesity, dyslipidemia, hypertension and T2DM, which are clinical features of metabolic syndrome and are associated with insulin resistance and adipose tissue dysfunction ^[1][9][1,9]. Obesity is considered the most important factor influencing the development of fatty liver [10]. The severity of hepatic steatosis is significantly correlated with body mass index, visceral fat thickness and insulin resistance. The incidence of NAFLD is significantly higher among patients with T2DM compared with the general population [11]. In addition, T2DM increases the risk of cirrhosis and HCC among patients with NAFLD [12]. The gut microbiota is associated with advanced fibrosis in NAFLD patients. During the progression of NAFLD to advanced fibrosis, the abundance of the Proteobacteria phylum significantly increases, while that of the Firmicutes phylum decreases [13]. In patients with NAFLD-related cirrhosis, the gut microbiota profile and systemic inflammation are significantly correlated and are involved in hepatocarcinogenesis [14]. Genetic predisposition also has an important impact on the development of NAFLD. In 2008, for the first time, genome-wide association studies reported the I148M single nucleotide polymorphism (SNP; rs738409, C > G) in the patatin-like phospholipase domain containing protein 3 (PNPLA3), which is a susceptibility gene involved in the development of NAFLD [15]. Adiponutrin encoded by PNPLA3 is expressed on adipose cell membranes and promotes lipase activity, predominantly during lipid metabolism [16]. PNPLA3 I148M increases the activity of lysophosphatidic acid acyltransferase, which regulates hepatic liposynthesis, a major determinant of lipogenesis in the liver. PNPLA3 I148M is widely involved in the pathogenesis of NAFLD and contributes to fibrosis progression and hepatocarcinogenesis ^[17][18][19][17,18,19]. Transmembrane 6 superfamily member 2 (TM6SF2) activity is required for normal VLDL secretion, and impaired TM6SF2 activity causally contributes to NAFLD and is associated with steatosis, inflammation and fibrosis ^[20][21][20,21]. A splice variant (rs72613567) of the 17-β-hydroxysteroid dehydrogenase 13 gene (HSD17B13) has been associated with increased steatosis, although it decreases inflammation and ballooning degeneration via its inherent hepatic retinol dehydrogenase activity. This rs72613567 variant attenuates the progression of fibrosis and may reduce the risk of NAFLD-related cirrhosis [22]. Other SNP variants, such as glucokinase gene regulator (GCKR), GATA zinc finger domain containing 2A (GATAD2A), membrane bound O-acyltransferase domain-containing 7 (MBOAT7), and phosphatidylethanolamine N-methyltransferase (PEMT), may also be implicated in the pathophysiology of NAFLD ^{[23][24][25][26]}[23,24,25,26]. Notably, the genetic alterations and their consequences can be potential targets for the treatment of NAFLD. Currently, a clinical study of an antisense medicine AZD2693, which was designed to inhibit the production of PNPLA3 protein, is ongoing to investigate its effects on patients with NASH, fibrosis stage 0 to 3, who are homozygous for the PNPLA3 148M risk allele (ClinicalTrials.gov identifier: NCT04483947).

3. Pathophysiology of NAFLD

Notably, the most important factor for the disease progression of NAFLD and for the development of its treatment is liver fibrosis. Various pathways have been implicated in fibrosis progression in patients with NAFLD. Clinical studies have shown that factors such as older age, insulin resistance, diabetes mellitus, circulating adipokines, and serum ferritin levels are predictive of fibrosis progression [27]. Furthermore, multiple factors are proposed to interact with each other to increase oxidative stress, induce inflammation, and contribute to fibrosis progression. A mechanism of hepatic fibrosis is excessive production of collagen via the activation of hepatic stellate cells (HSCs). Normally quiescent HSCs, which are essentially vitamin-A-storing cells, transdifferentiate into activated HSCs in injured liver tissues and differentiate into proliferative fibrogenic myofibroblast-like cells that, via constitutive and induced expression, generate cytokines and growth factors such as PDGF and TGF-β ^{[27][28][29][30]}[27,28,29,30]. Activation of HSCs via extracellular signals from resident and inflammatory cells, including macrophages, hepatocytes, endothelial cells, T cells, B cells, and other immune cells, is also an important factor [27]. Autophagy is a metabolic process that degrades and recycles intracellular organelles and their constituents, and it has many roles in human pathological conditions, including liver diseases. Recent studies have suggested a role of autophagy in the development of NAFLD [31] and have shown that autophagy can induce lipid droplet degradation in hepatocytes, a process called lipophagy [32]. Increased autophagic flux was observed in activated HSCs, and accordingly, treatment with bafilomycin A1—an autophagy inhibitor—was shown to suppress the expression of profibrotic markers and inhibit cellular proliferation in HSCs [33]. Understanding the mechanism of autophagy in the activation process of HSCs may lead to new therapeutic strategies for liver fibrosis. As activated HSCs play a pivotal role in progressive hepatic fibrogenesis [34], they represent a potential target for antifibrotic therapeutics. Despite the numerous advances made towards understanding the pathogenesis of liver fibrosis, to date there are no specific antifibrotic drugs that have received U.S. Food and Drug Administration (FDA) approval, and effective treatments are lacking. The major challenge to overcome in the treatment of liver fibrosis pertains to its slow rate of progression to cirrhosis. Chronic mechanisms give rise to modifications of liver vascularization, the composition of the extracellular matrix, and drug metabolism. The currently available therapies to treat these conditions remain inadequate [27], and there is an urgent unmet need for new clinically effective and safe therapies.