Literature Review on the Regulatory Effects of Hydrogen Sulfide in Non-Alcoholic Fatty Liver Disease (NAFLD)

Introduction

Research Background and Significance

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver and has become one of the most common chronic liver diseases globally, with a prevalence rate as high as 32.4%.¹ It is characterized by abnormal lipid accumulation in the liver and is closely associated with metabolic syndrome, obesity, and insulin resistance. With the increasing incidence of obesity and metabolic syndrome, NAFLD not only affects liver health but also significantly increases the risk of cirrhosis, liver cancer, and cardiovascular events, imposing a heavy burden on both patients’ health and the economy. However, current treatment strategies for NAFLD are limited in effectiveness. Therefore, in-depth research into its pathogenesis and exploration of new therapeutic targets are of great significance.

Hydrogen sulfide (H₂S), as the third gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO), plays an important role in physiological and pathological processes.² Studies have shown that H₂S is involved in regulating intracellular redox status, inflammatory responses, energy metabolism, and lipid metabolism, and has protective effects on various diseases. In recent years, the role of H₂S in the occurrence and development of NAFLD has gradually attracted attention, and exploring its regulatory mechanisms in NAFLD may provide possibilities for developing new therapeutic strategies.

Overview of Non-Alcoholic Fatty Liver Disease Research

NAFLD is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. Its pathological features include excessive fat deposition in hepatocytes, with a wide disease spectrum ranging from simple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH), NASH-related liver fibrosis, cirrhosis, and hepatocellular carcinoma. The pathophysiological mechanisms of NAFLD involve multiple cellular pathways and molecular factors, mainly including insulin resistance, lipid metabolism disorders, chronic inflammatory responses, and mitochondrial dysfunction.³

Insulin Resistance and Lipid Metabolism Disorders

Insulin resistance is one of the core pathophysiological mechanisms of NAFLD. Insulin resistance leads to decreased sensitivity of the liver and peripheral tissues to insulin, thereby causing lipid metabolism disorders. Excessive deposition of fatty acids in the liver is a major characteristic of NAFLD, which is closely related to insulin resistance.⁴ In addition, overconsumption of nutrients and gastrointestinal dysbiosis can also exacerbate lipid metabolism abnormalities.⁵

Chronic Inflammatory Responses

NAFLD is considered a chronic low-grade inflammatory state. The increase in pro-inflammatory factors and abnormalities in cytokine signaling play important roles in the occurrence and development of the disease. For example, the activation of Kupffer cells and apoptosis of hepatocytes can trigger inflammatory responses, further leading to the development of non-alcoholic steatohepatitis (NASH).^6,7

Mitochondrial Dysfunction

Mitochondrial dysfunction in the liver plays an important role in the pathogenesis of NAFLD. Abnormal mitochondrial function can lead to decreased fatty acid oxidation capacity, thereby exacerbating fat deposition in hepatocytes. In addition, mitochondrial dysfunction can also cause oxidative stress, further aggravating hepatocyte injury.⁸ In the pathogenesis of NAFLD, insulin resistance leads to increased hepatic lipid synthesis and deposition, which is an important link in the development of the disease. Meanwhile, mitochondrial dysfunction induces oxidative stress and lipid peroxidation, further worsening hepatic steatosis and inflammation. Moreover, the expression and release of inflammatory factors promote hepatic inflammatory responses, accelerating the progression of NAFLD to NASH and liver fibrosis.

Biological Characteristics and Physiological Functions of Hydrogen Sulfide

Hydrogen sulfide (H₂S) is a colorless, flammable, and toxic gas. However, in biological organisms, it functions as an important gaseous signaling molecule with a wide range of biological functions. H₂S is mainly synthesized in mammals from L-cysteine through the catalysis of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). Its biosynthesis and metabolic mechanisms are complex, involving multiple enzymes and regulatory pathways.

Biosynthesis of H₂S

Hydrogen sulfide is primarily synthesized through the following four enzymatic pathways in the body (as shown in Figure 1).

Figure 1 Endogenous Hydrogen Sulfide Production Pathways. Notes: The figure illustrates four enzymatic pathways of H2S synthesis in mammals: (1) CBS catalyzes cysteine and homocysteine to generate H2S; (2) CSE decomposes cystathionine to produce H2S; (3) 3-MST converts 3-mercaptopyruvate to H2S in the nervous system; (4) DAO oxidizes D-cysteine to 3-mercaptopyruvate, which is metabolized to H2S by mitochondrial 3-MST (predominantly in the brain and kidneys).

Cystathionine β-Synthase (CBS)

CBS is one of the main enzymes for H₂S synthesis, utilizing cysteine and homocysteine as substrates to generate cystathionine and H₂S.

Cystathionine γ-Lyase (CSE)

CSE generates cysteine, α-ketobutyrate, and H₂S by breaking down cystathionine, representing another important pathway for H₂S synthesis.

3-Mercaptopyruvate Sulfurtransferase (3-MST)

3-MST catalyzes the generation of H₂S from 3-mercaptopyruvate, mainly functioning in the nervous system.⁹

DAO/3-MST Pathway

Kimura¹⁰ first identified a fourth pathway for H₂S generation in the body. In the presence of DAO, D-cysteine is oxidized to generate 3-mercaptopyruvate (3-MP), ammonia (NH₃), and hydrogen peroxide (H₂O₂). Subsequently, 3-MP enters the mitochondria and is metabolized to H₂S by 3-MST. Since DAO is mainly distributed in the brain and kidneys, this H₂S generation pathway is believed to exist only in these two organs.

Metabolism of H₂S

The metabolism of hydrogen sulfide mainly occurs through the following pathways.

Oxidative Metabolism

H₂S can be oxidized in the body to form thiosulfate, sulfite, and sulfate, which are eventually excreted in urine.

Methylation Metabolism

H₂S can be methylated to form methanethiol and dimethyl sulfide, which are further metabolized and excreted.

Binding with Metal Ions

H₂S can bind with metal ions such as iron and copper to form insoluble sulfides, thereby reducing its toxicity. H₂S can freely permeate biological membranes and exert effects on a series of biological targets, participating in the regulation of intracellular redox status, inflammatory responses, energy metabolism, and lipid metabolism.⁹

Physiological Functions of H₂S

Anti-Inflammatory and Anti-Apoptotic Effects

Hydrogen sulfide protects cells from damage by inhibiting inflammatory responses and apoptosis. For example, SPRC (a garlic-derived compound) can slowly release hydrogen sulfide, enhancing anti-inflammatory and anti-apoptotic effects.¹¹

Metabolic Regulation

Hydrogen sulfide plays an important role in regulating energy and lipid metabolism. It can regulate insulin secretion and oxidative stress, thereby influencing the energy balance and metabolic state of cells. Additionally, hydrogen sulfide can delay cell aging and maintain cell function by regulating the generation of persulfides. It also promotes fatty acid β-oxidation and inhibits lipid synthesis, reducing lipid accumulation in the liver. Studies have shown that H₂S can shift the metabolism of free fatty acids (FFAs) from lipid accumulation to β-oxidation, thereby improving lipid metabolism disorders.^12,13

Signal Transduction in the Central Nervous System

In the central nervous system, hydrogen sulfide regulates homeostasis and cell signaling, playing an important physiological role. It is considered an emerging gaseous neurotransmitter that can regulate neuronal transmission and cell function.^14,15

Antioxidant Effects

Hydrogen sulfide delays cell aging and maintains cell function by scavenging reactive oxygen species (ROS) and regulating the generation of persulfides. Studies have shown that persulfidation is an evolutionarily conserved modification that prevents cysteine oxidation, preserving protein function. Hydrogen sulfide can activate ATP-sensitive potassium channels in endothelial cells, dilate blood vessels, inhibit adhesion between endothelial cells and leukocytes, reduce oxidative stress, and maintain vascular homeostasis.¹⁶

In summary, H₂S, as an important gaseous signaling molecule, participates in cell signaling, regulating physiological and pathophysiological processes of cells through various mechanisms. Its roles in energy metabolism, anti-inflammatory, anti-apoptotic, neuronal transmission, and vascular regulation make it an important target for the study of non-alcoholic fatty liver disease (NAFLD) and lipid metabolism. Future research can further explore the potential applications of hydrogen sulfide in disease treatment.

Current Treatment Strategies for NAFLD

At present, the treatment of NAFLD mainly includes lifestyle interventions and pharmacological treatments. Lifestyle interventions such as dietary adjustments, increased physical activity, and weight loss are the foundation of NAFLD management. In terms of pharmacological treatment, insulin sensitizers, antioxidants, lipid-lowering drugs, etc., are used to improve metabolic disorders and alleviate liver injury.¹⁷

Pharmacological Therapies

Biguanides

Biguanides are the first-line drugs for the treatment of type II diabetes, especially for overweight and obese patients. Metformin is the most commonly used biguanide, which improves insulin resistance by reducing hepatic glucose production and increasing peripheral tissue sensitivity to insulin.^18,19 Additionally, metformin has cardiovascular protective effects and can reduce the risk of diabetic complications.

Thiazolidinediones (TZDs)

TZDs improve insulin sensitivity by activating peroxisome proliferator-activated receptor γ (PPAR-γ). These drugs increase glucose uptake in adipose tissue and muscle, thereby reducing blood glucose levels.²⁰ Common drugs include rosiglitazone and pioglitazone.

GLP-1 Receptor Agonists

GLP-1 receptor agonists increase insulin secretion, inhibit glucagon secretion, and delay gastric emptying by mimicking the action of the gut hormone GLP-1. These drugs not only improve glycemic control but also help with weight loss.²¹ Common drugs include exenatide and liraglutide.

Vitamin E

Vitamin E, as an antioxidant, is thought to improve insulin sensitivity and reduce oxidative stress in some studies, although its role in diabetes treatment requires further research.²² Vitamin E is usually used as an adjunctive therapy to improve the overall health of patients with metabolic syndrome.²³

Gut Microbiota and Its Metabolites

Regulating the structure and function of the gut microbiota is closely related to the prevention and treatment of NAFLD.²⁴ Taking a combination of Bifidobacterium can increase the number of beneficial bacteria in the gut. Through the mutual restraint between different bacterial species, the structure of the gut microbiota is optimized. This helps reduce the functional damage caused by endotoxins produced by harmful bacteria such as Escherichia coli and Enterococcus to various organs and tissues, and aids in restoring the gut’s immune barrier function, thereby alleviating liver inflammation and enhancing the overall therapeutic effect.²⁵ Additionally, gut microbiota regulation as an adjunctive therapy can effectively improve liver function, serum adiponectin, fasting insulin, blood lipids, BMI levels, and Fibroscan controlled attenuation parameters in patients with non-alcoholic fatty liver disease.²⁶

Other Treatment Strategies

Other relevant literature has explored various treatment strategies for non-alcoholic fatty liver disease (NAFLD), including lifestyle changes, the application of bioactive substances, and pharmacological treatments.²⁷ In terms of lifestyle adjustments, particular emphasis is placed on the Mediterranean diet and regular physical exercise. The Mediterranean diet promotes weight loss and alleviates NAFLD symptoms by restricting the intake of high-calorie, high-sugar, and high-fat foods; while physical exercise helps significantly reduce liver fat content and the risk of progression from NAFLD to non-alcoholic steatohepatitis (NASH). In the exploration of bioactive substances, peptides, alkaloids, polyphenols, silymarin, and other components are mentioned for their potential value in NAFLD treatment. These substances exert multiple effects through antioxidant, anti-inflammatory, and lipid metabolism regulation.²⁸ In terms of pharmacological treatments, the application status and research progress of statins, peroxisome proliferator-activated receptor (PPAR) agonists, cenicriviroc, and farnesoid X receptor (FXR) agonists are detailed.²⁹

Challenges and Prospects for H₂S-Based Therapies

Multiple reports have highlighted the diverse regulatory effects of hydrogen sulfide (H₂S) in NAFLD. There is ongoing research and development of H₂S-related drugs. However, given the transient release and oxidative susceptibility of H₂S, its practical application faces many challenges. These challenges have spurred researchers to develop donors that can achieve controlled, sustained, and targeted release of H₂S. Currently,³⁰ some compounds that simulate endogenous H₂S release have attracted widespread attention. These include the pioneering H₂S donor compound GYY4137 in the field of H₂S donors; the naproxen H₂S donor derivative ATB-34 with anti-inflammatory effects, which can be used as a sedative for colonoscopy; the H₂S donor of mebeverine maleate for colonoscopy sedation; the biological GIC-1001; and the cardioprotective SG1002.³¹

Association Between NAFLD and H₂S Levels

In non-alcoholic fatty liver disease (NAFLD), the accumulation of triglycerides in hepatocytes is the main pathological change, leading to hepatic steatosis. The main causes can be summarized into four aspects: excessive uptake of free fatty acids from the blood by the liver; enhanced de novo synthesis of fatty acids; weakened fatty acid oxidation; and impaired output of triglycerides from the liver in the form of lipoproteins. When the liver’s capacity to take up triglycerides exceeds its clearance capacity, triglycerides accumulate in hepatocytes, leading to NAFLD. Hydrogen sulfide has dual effects on adipose tissue and the liver. On one hand, hydrogen sulfide accelerates the storage of fatty acids in adipose tissue and inhibits lipid synthesis in the liver. On the other hand, hydrogen sulfide increases fatty acid breakdown and oxidation in the liver, alleviating lipid accumulation in the liver. The activity of CSE directly affects the generation of H₂S, and its expression levels are usually reduced in patients with NAFLD. Supplementation with H₂S or its precursors can restore its function.^9,32

Non-alcoholic steatohepatitis (NASH) is a disease characterized by hepatic steatosis, fibrosis, and inflammation in the absence of other liver-damaging factors such as excessive alcohol consumption or drugs. The protective effects of hydrogen sulfide on NASH may be realized through antioxidant stress, anti-inflammatory, and anti-fibrotic mechanisms. In an MCD-induced NASH rat model, the hepatoprotective effects of endogenous hydrogen sulfide were observed. Specifically, the expression of CBS and CSE was significantly reduced, leading to decreased liver hydrogen sulfide levels, resulting in increased liver fat accumulation, oxidative stress, and expression of inflammatory factors in mice. Administration of exogenous hydrogen sulfide donors significantly improved liver function, reduced liver lipid deposition, and decreased hepatocyte apoptosis.³¹

In liver fibrosis, it has been reported in CCl4-induced liver fibrosis models that the protein expression of CSE and CBS is inhibited, leading to decreased H₂S content. Exogenous H₂S donors (such as NaHS) can improve liver function and reduce liver fibrosis. Moreover, H₂S alleviates oxidative stress and inflammatory responses through various mechanisms, inhibits the activation of hepatic stellate cells, reduces collagen deposition, and thereby alleviates liver fibrosis.³³

Research Review on the Mechanisms of Action Between Non-Alcoholic Fatty Liver Disease and Hydrogen Sulfide

In recent years, numerous studies have made significant progress in exploring the mechanisms of hydrogen sulfide in NAFLD through in vitro and in vivo experiments. The following is a detailed analysis of its specific effects and regulatory mechanisms.

Role in Hepatic Inflammation

Hydrogen sulfide alleviates liver inflammation through various mechanisms, including antioxidant and anti-inflammatory effects. H₂S can scavenge reactive oxygen species (ROS) and nitrogen radicals in the body, reducing oxidative stress-induced hepatocyte damage. Studies have shown that exogenous H₂S donors can significantly reduce oxidative stress levels in NAFLD models, protecting hepatocytes from lipid peroxidation damage. Additionally, H₂S inhibits the expression and release of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby alleviating liver inflammation and reducing the severity of NAFLD.³⁴ H₂S exerts its anti-inflammatory and anti-fibrotic effects by activating signaling pathways such as AMPK and Nrf2.^12,35

Role in Hepatic Fibrosis

Liver fibrosis is a key step in the progression of NAFLD to cirrhosis, and H₂S exhibits significant anti-fibrotic effects in this process.

Inhibition of Fibrosis-Related Gene Expression

H₂S downregulates the expression of pro-fibrotic factors such as TGF-β, reducing collagen deposition and thereby inhibiting fibrosis.^36,37

Promotion of Hepatocyte Regeneration

H₂S enhances the regenerative capacity of hepatocytes, repairing damaged tissues and thereby slowing the progression of fibrosis.^9,38

Epigenetic Regulation

H₂S may also regulate the expression of genes related to inflammation and fibrosis through mechanisms such as DNA methylation and histone modification.^9,38 H₂S can enhance the binding ability of NFYB transcription factor to the promoter regions of Tet1 and Tet2 genes by directly mediating the sulfhydryl modification of its thiol group, thereby up-regulating the expression levels of TET1/2. Subsequently, TET enzymes catalyze the oxidation of 5mC to 5hmC, promoting the active demethylation process at key gene loci such as Foxp3.³⁹

Metabolic Pathway Regulation

H₂S improves liver metabolic functions by influencing lipid and carbohydrate metabolism, indirectly reducing the risk of fibrosis.^9,40

Regulation of Lipid Metabolism

Hydrogen sulfide plays an important role in regulating lipid metabolism. Studies have shown that H₂S promotes fatty acid β-oxidation, thereby reducing lipid accumulation in the liver. Additionally, H₂S regulates the expression of lipid metabolism-related genes, such as peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element-binding protein 1c (SREBP1c), improving the balance of lipid metabolism in the liver and thereby alleviating the steatosis of non-alcoholic fatty liver disease (NAFLD).⁴¹ In methionine choline-deficient (MCD) induced non-alcoholic steatohepatitis (NASH) models, H₂S donors (such as NaHS) significantly reduced liver triglyceride and cholesterol levels by increasing PPARα expression and decreasing SREBP-1c gene expression, thereby alleviating steatosis. Moreover, H₂S activates the AMPK-mTOR signaling pathway to promote fatty acid β-oxidation and reduce lipid accumulation in the liver. In high-fat diet (HFD) induced NAFLD models, H₂S donors significantly reduced hypertriglyceridemia and improved NAFLD by activating hepatic autophagy flux and the AMPK-mTOR signaling pathway.⁴² H₂S can also regulate other mechanisms in lipid metabolism. For example, by inhibiting the expression of 3-mercaptopyruvate sulfurtransferase (MPST) to reduce H₂S generation, SREBP-1c is upregulated, promoting fat synthesis. Conversely, inhibiting MPST can reduce free fatty acid (FFAs) levels and increase H₂S generation, thereby improving lipid metabolism.⁴³

Inhibition of Cell Apoptosis

Apoptosis of hepatocytes is an important mechanism in the progression of NAFLD. Studies have shown that H₂S can inhibit the apoptotic process of hepatocytes, protecting them from death signals. H₂S regulates the expression of apoptosis-related genes such as B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax), thereby inhibiting hepatocyte apoptosis and alleviating the condition of NAFLD.⁴⁴

Regulation of Autophagy

Autophagy is a self-degradation process within cells that is important for maintaining cell homeostasis and responding to stress stimuli. Studies have shown that H₂S can induce autophagy in hepatocytes, promoting the clearance and regeneration of damaged organelles, thereby alleviating the condition of NAFLD. H₂S regulates the expression of autophagy-related genes such as microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 5 (Atg5), thereby promoting autophagy in hepatocytes and improving liver function.⁴⁵

Improvement of Insulin Resistance

Insulin resistance is an important mechanism in the development of NAFLD. Studies have shown that H₂S can enhance insulin signaling pathways, improving insulin resistance and thereby alleviating the condition of NAFLD. H₂S regulates the expression of insulin signaling-related genes such as insulin receptor substrate 1 (IRS1) and protein kinase B (Akt), thereby enhancing insulin sensitivity, promoting glucose uptake and utilization, and improving metabolic abnormalities in NAFLD.^46,47

In summary, H₂S exerts protective effects in various tissues and organs, including the cardiovascular system, nervous system, digestive system, and liver. In the liver, H₂S alleviates hepatocyte damage through antioxidant and anti-inflammatory effects, improves hepatic steatosis by regulating lipid metabolism, protects hepatocyte survival by inhibiting apoptosis, and promotes the clearance and regeneration of damaged organelles by inducing autophagy. Additionally, H₂S enhances insulin signaling pathways, improving insulin resistance and thereby alleviating the condition of NAFLD.

Discussion

Research Evaluation and Prospects

Research Evaluation

Current research on the role of H₂S in NAFLD has made significant progress, yet several limitations persist. For instance, although H₂S is known to modulate multiple signaling pathways—such as AMPK, Nrf2, and TGF-β—the precise molecular mechanisms and cross-talk among these pathways remain incompletely understood.^12,35–37 Existing studies often focus on single pathways in isolation, and integrative models are lacking.

Moreover, individual differences in H₂S metabolism and response have been observed but are underexplored. For example, variations in the expression of H₂S-synthesizing enzymes (CBS, CSE, 3-MST) due to genetic or epigenetic factors may influence disease progression and treatment efficacy.^9,38 Clinical and preclinical studies also suggest that H₂S levels fluctuate across NAFLD stages, with lower endogenous H₂S in advanced fibrosis, yet how these changes affect therapeutic outcomes is not well characterized.^31,33

These gaps highlight the need for more stratified research—considering disease stage, genetic background, and metabolic phenotype—to clarify H₂S’s role in NAFLD and inform personalized treatment approaches.

Future Prospects

1. Stage-specific mechanism exploration: Investigate H₂S’s regulatory differences in NAFLD stages (eg, whether it inhibits lipogenesis in NAFL and fibrosis in NASH) using clinical samples and stratified animal models.

2. H₂S donor optimization: Develop biomaterial-based targeted delivery systems (eg, liver-specific nanoparticles) to achieve on-demand H₂S release at lesions, reducing systemic toxicity.

3. Combination therapy development: Combine H₂S donors with:

3.1 Lifestyle interventions: Mediterranean diet + H₂S donors to synergistically improve insulin resistance;

3.2 Existing drugs: Metformin + H₂S donors to enhance hepatic lipid metabolism;

3.3 Gut microbiota modulators: Bifidobacterium + H₂S donors to amplify anti-inflammatory effects.

Conclusion

Hydrogen sulfide exerts protective regulatory effects in NAFLD via antioxidation, anti-inflammation, lipid metabolism modulation, apoptosis inhibition, autophagy induction, and insulin resistance improvement, making it a promising therapeutic target. However, current research is limited by unclear multi-signaling pathway crosstalk, unelucidated stage-specific and individual differences, and unvalidated H₂S donor safety/efficacy in large-scale clinical trials. Future studies should focus on stage-specific mechanisms, targeted donor development, and evidence-based combination therapies to advance H₂S-based NAFLD treatments and address the global burden of this disease. However, current research has limitations: the specific regulatory mechanisms of H₂S in multiple signaling pathways remain unclear, and its differential roles across individuals and NAFLD stages need further exploration. Additionally, the safety, efficacy, and targeted delivery of H₂S donor drugs (to avoid gas toxicity and achieve controlled release at lesions) require validation through large-scale clinical trials.