Nonalcoholic fatty liver disease (NAFLD, now referred to as MAFLD) refers to a clinicopathological syndrome characterized primarily by excessive fat deposition in hepatocytes, occurring in the absence of alcohol consumption and other identified causes of liver injury. In its early stages, NAFLD typically exhibits no overt clinical symptoms, but it can subsequently progress to nonalcoholic steatohepatitis (NASH, now referred to as MASH), a liver disease marked by inflammation and fibrosis. Further progression of MASH can lead to cirrhosis, and ultimately, to hepatocellular carcinoma (HCC) or liver failure in patients.

The pathogenesis of MAFLD remains without a definitive explanation, and the mainstream hypotheses include the traditional "two-hit" hypothesis and the recently proposed "multiple-hit" hypothesis. In the traditional "two-hit" hypothesis, the first hit involves the accumulation of intrahepatic fat, primarily triglycerides (TG), resulting from a sedentary lifestyle, poor nutritional habits, and insulin resistance (IR). The second hit is the excessive production of lipid-induced reactive oxidative metabolites, which is exacerbated by the interplay of multiple factors such as cytokine-mediated inflammation, free fatty acid oxidation, apoptosis, necroinflammation, and fibrosis. However, it is widely acknowledged that the "two-hit" hypothesis is overly simplistic and fails to capture the complexity of the MAFLD pathogenesis. Studies have demonstrated that oxidative stress does not necessarily accompany lipid accumulation and can induce steatosis on its own. Based on this reasoning, it is more accurate to say that these events occur simultaneously, contributing to the onset and progression of the disease, which is the essence of the "multiple-hit" hypothesis.

The "multiple-hit" hypothesis describes the "integrated response" of the genetically susceptible host to a high-calorie diet, overeating, and a sedentary lifestyle, which may lead to metabolic syndrome and obesity. Research has shown that an imbalance in the gut microbiome after excessive eating leads to an increase in bacterial products in the portal circulation, activating the innate immune system. These events, coupled with IR in muscle tissues in response to elevated levels of circulating free fatty acids, are among the critical processes in the pathogenesis of MAFLD-MASH. All factors, including IR, oxidative stress, inflammation, obesity, metabolic syndrome, T2DM, hormones secreted by adipose tissue, gut microbiota, and epigenetics, are considered to play pivotal roles in the onset and progression of MAFLD.

1. Lipid Metabolism Disorder

Hepatic steatosis is one of the common characteristics of MASH. Insulin resistance promotes the breakdown of adipose tissue, leading to the deposition of free fatty acids (FFA) in tissues in the form of triglycerides (TG), causing damage. The accumulation of TG is the first step in the pathological development of MASH. On one hand, several highly expressed fatty acid transporters, fatty acid-binding proteins, and caveolins in the liver facilitate the uptake of FFA by hepatocytes, contributing to lipid accumulation. One such transporter, CD36, also promotes FFA uptake and intracellular transport, serving as a common target for liver X receptor (LXR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor gamma (PPARγ) in the liver. On the other hand, hepatocytes primarily rely on fatty acid β-oxidation for energy production, but excessive FFA uptake impairs mitochondrial oxidative phosphorylation, resulting in incomplete oxidation of FFA and promoting the synthesis and accumulation of toxic lipid intermediates such as ceramides and diacylglycerols. Furthermore, hepatic steatosis is also associated with the synthesis and secretion of very low-density lipoprotein (VLDL).

2.Inflammatory Response

The liver initiates a series of inflammatory responses to eliminate hepatocellular damage caused by lipotoxicity. This process is triggered by the death of hepatocytes, releasing signals that activate the innate immune system and recruit bone marrow-derived cells. The specific process involves the recruitment of immune cells and the activation of pro-inflammatory signaling pathways, such as tumor necrosis factor-α (TNF-α), nuclear transcription factor-κB (NF-κB), activator protein-1 (AP-1), Toll-like receptor 4 (TLR4), and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). In the pathogenesis of MASH, a high-fat diet leads to hyperplasia and hypertrophy of adipocytes, releasing a large amount of FFA, which can bind to TLR4 to activate the innate immune pathway, subsequently triggering endoplasmic reticulum stress and inflammatory responses. Additionally, TLR4 is also involved in the process of liver fibrosis induced by various factors.

Moreover, immune cells like natural killer T cells (NKT) and macrophages contribute to the development of MASH. In individuals with mild hepatic steatosis, the number of NKT cells is relatively low, but in those with MASH-induced cirrhosis, a large number of NKT cells accumulate. Furthermore, macrophages activate hepatic stellate cells by releasing transforming growth factor-β (TGF-β) and interleukin-6 (IL-6), leading to persistent inflammation and fibrosis in the liver.

3.Oxidative Stress

Long-term metabolic disorders and impaired mitochondrial function in the liver stimulate hepatocytes to produce reactive oxygen species (ROS), triggering an oxidative stress response. Due to metabolic abnormalities, excessive FFA enters mitochondria, leading to increased permeability of the mitochondrial inner membrane, decreased mitochondrial membrane potential, and loss of adenosine triphosphate (ATP) synthesis capacity, ultimately damaging mitochondrial function and increasing ROS production. This process, in turn, exacerbates hepatocellular metabolic disorders and generates a massive amount of ROS, creating a vicious cycle that leads to high levels of oxidative stress, which can then trigger inflammatory responses or apoptosis. Additionally, impaired intracellular antioxidant mechanisms contribute to increased metabolism related to fatty acid metabolism, resulting in the accumulation of non-metabolic fatty acids and disruption of hepatic fatty acid homeostasis, ultimately triggering hepatic steatosis and metabolic stress.

Furthermore, the interaction of ROS with unsaturated fatty acids generates highly reactive aldehydes, such as malondialdehyde and 4-hydroxynonenal, which can mutate mitochondrial DNA, promoting hepatocyte apoptosis. TLR4 activates X-box binding protein 1 (XBP-1) through ROS, leading to NF-κB activation and the production of pro-inflammatory cytokines, thereby mediating the transition from benign steatosis to MASH.

4.Fibrosis

Fibrosis is one of the pathological characteristics of MASH. Studies have revealed that hepatic stellate cells (HSCs) are the key mediators of the fibrotic response, undergoing genotypic and phenotypic changes when hepatocytes are damaged. The activation process of HSCs is complex, involving paracrine stimulation from neighboring cells such as Kupffer cells, hepatocytes, cholangiocytes, platelets, and sinusoidal endothelial cells, mediated by several cytokines. Subsequently, autocrine stimulation leads to increased proliferation of activated HSCs, significantly increasing the number of profibrotic cells in the liver. Insulin also contributes to fibrosis by promoting the formation of connective tissue growth factor, activating HSCs, and accelerating fibrosis.

Moreover, leptin and adiponectin are also implicated in the development of hepatic fibrosis. Leptin, a profibrotic molecule, upregulates the expression of TGF-β and participates in HSC activation through paracrine mechanisms. It also stimulates the production of tissue inhibitor of metalloproteinase-1 (TIMP-1), inhibiting the expression of matrix metalloproteinase-1 (MMP-1), activating the Hedgehog pathway, and subsequently activating HSCs, thereby promoting fibrosis. In contrast, adiponectin, upon binding to its cognate receptors 1 and 2, exerts antifibrotic effects on hepatic fibrosis through multiple mechanisms, including direct antifibrotic actions on HSCs and indirect antifibrotic effects related to its anti-inflammatory activity.

5.Bile Acid Metabolism

Bile acids (BAs) not only aid in the digestion and absorption of intestinal fat but also function as signaling molecules regulating glucose and lipid metabolism, as well as energy homeostasis. During their reabsorption in the ileum, BAs stimulate ileal cells to secrete fibroblast growth factor 19 (FGF19), which, upon entering the portal circulation, binds to fibroblast growth factor receptor 4 (FGFR4), activating the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. This leads to decreased expression of cholesterol 7α-hydroxylase (CYP7A1), inhibiting BA synthesis. Studies have found that the farnesoid X receptor (FXR) serves as both a nuclear receptor for BAs and an inhibitor of BA de novo synthesis. The absence of FXR increases triglyceride (TG) content, hepatic steatosis, inflammatory infiltration, and fibrosis, promoting the development of MASH.

Another membrane-bound BA receptor, G-protein-coupled receptor 5 (TGR5), highly expressed in Kupffer cells, exerts anti-inflammatory effects by inhibiting NF-κB and cytokine release upon activation. Furthermore, the vitamin D receptor, which is a common receptor for both vitamin D and lithocholic acid, a BA metabolite, plays a crucial role. Vitamin D deficiency promotes MASH development and upregulates the gene expression of TLRs2, TLRs4, and TLRs9, exacerbating hepatic steatosis and inflammatory responses.

6.Genes and Genetics

Studies have shown that genetic diversity can also influence the occurrence and progression of MASH. A genome-wide association study identified a common variant in the patatin-like phospholipase domain containing 3 (PNPLA3) gene that is closely associated with increased hepatic fat content and elevated serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Patients with the PNPLA3 allele have been found to exhibit significant inflammatory responses and fibrosis upon pathological examination. Furthermore, data from human HSC cell lines suggest that PNPLA3 is an enzyme necessary for HSC activation.

Another gene variant associated with hepatic steatosis, elevated transaminases, and advanced liver fibrosis is transmembrane 6 superfamily member 2 (TM6SF2). Additionally, the expression of microRNAs (miRNAs) such as miR-122, miR-335, miR-29c, miR-34a, miR-155, and miR-200b in the liver has been implicated in the pathogenesis of MASH, potentially serving as biomarkers. These miRNAs offer insights into the molecular mechanisms underlying MASH and could aid in its diagnosis and management.

7.Intestinal Microbial Dysbiosis

A growing body of research indicates that intestinal microbial dysbiosis contributes to the progression of MASH. It has been reported that the gut microbiota can modulate BA-mediated FXR signaling, leading to the production of endogenous ethanol. Furthermore, dysbiosis can increase lipoprotein lipolytic activity and TG accumulation by reducing choline levels or elevating trimethylamine levels, thereby promoting MASH development.

In addition, alterations in intestinal permeability play a potential role in the development of MASH. Microbial dysbiosis and damage to the intestinal barrier allow bacteria to translocate to the liver via the portal vein, where they are recognized by specific receptors, activating the immune system. This activation triggers pathways such as the mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), interferon regulatory factor 3 (IRF3), and NF-κB, leading to inflammation, hepatic steatosis, and fibrosis. Ultimately, these processes exacerbate liver damage and accelerate the progression of MASH.

References

[1] Guo Jifen, Hu Lei, Xu Sai, et al. Research Progress on the Mechanisms and Therapeutic Drugs for Non-alcoholic Steatohepatitis [J]. Pharmaceutical Biotechnology, 2020, 27(05): 479-485.

[2] Jin Rui, Wang Xiaoxiao, Liu Feng, et al. Advances in Pharmacological Treatment of Non-alcoholic Fatty Liver Disease [J]. Journal of Clinical Hepatology, 2022, 38(07): 1634-1640.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.