Non-Alcoholic Fatty Liver Disease and Its Potential Therapeutic Strategies

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.

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 non-diabetic 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.

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.

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 reticulum-induced apoptosis pathway will be triggered, resulting in apoptosis. ER stress can also inhibit insulin signaling by activating UPR-corresponding kinases, such as IRE1α, 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 hypoxia-inducible 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 proliferator-activated 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 antigen-antibody 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 HBsAg-antibodies 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), 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 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 controlled-release 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, 63, 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 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 beconsidered 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.

Acknowledgments

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.