BACKGROUND:Autoimmune hepatitis (AIH) is characterised by death of hepatocytes and chronic inflammation. Patients with deleterious variants in A20 are identified with AIH presentations and immune activation. However, it remains unclear how A20 dysregulation contributes to AIH pathogenesis. OBJECTIVE:This study elucidates the role and mechanism of A20 in AIH progression and its therapeutic potential. DESIGN:A20 expression was analysed in patient sample and AIH mouse model. A20 function was explored in mice with hepatocyte-specific A20 knockout and overactivation and wild type controls. Liver samples were assessed by histology, immunoblot and electron microscopy. Proteomics, mass spectrometry, co-immunoprecipitation and site-specific mutation experiments were used to elucidate underlying mechanisms. AdipoRon was used to identify the effects of pharmacological induction of A20 on AIH. RESULTS:A20 expression is reduced in livers of individuals with AIH. In experimental AIH, hepatocyte-specific A20 depletion aggravates hepatic inflammation and fibrosis, enhances ferroptosis of hepatocytes and increases sensitivity to ferroptosis inhibitors, while A20 overexpression ameliorates AIH pathology and reduces hepatocyte ferroptosis. Mechanistically, A20 is found to interact with KEAP1 and mediate its K48-linked polyubiquitination degradation, promoting NRF2 accumulation and nuclear translocation to enhance antioxidant and antiferroptosis effects. Pharmacological induction of A20 confers protective effects in AIH. The clinical correlation among A20, KEAP1, NRF2 and ferroptosis is validated in human AIH samples. CONCLUSIONS:Our findings elucidate a previously unrecognised A20-KEAP1-NRF2 axis that orchestrates hepatocyte ferroptosis in AIH. Targeting A20 may provide a promising therapeutic strategy for AIH.

BACKGROUND & AIMS:Autoimmune hepatitis (AIH) is a chronic progressive inflammatory liver disease, with its incidence increasing continuously worldwide. The mechanisms underlying the regulation of immune tolerance in AIH remain largely unknown. This study investigated a novel regulatory pathway of regulatory T cell/T helper 17 (Treg/Th17) balance involving the ubiquitin-conjugating enzyme E2O (UBE2O) and underlying mechanisms. METHODS:UBE2O expression was analyzed in patients and mice with AIH. In vivo UBE2O overexpression in a chronic AIH model and naïve CD4+ T differentiation induction assays were used to investigate the exact role of UBE2O in AIH. Liver samples were assessed by histology, immunochemistry, immunoblot, flow cytometry, and enzyme-linked immunosorbant assays. Mass spectrometry, co-immunoprecipitation, cytokine microarray, and site-specific mutation experiments were utilized to elucidate underlying molecular mechanisms. RESULTS:We detected a reduction of UBE2O expression in livers of patients and mice with AIH. Low expression of UBE2O indicated severe liver inflammatory injury. Hepatic UBE2O overexpression experiments demonstrated that UBE2O alleviated hepatic injury, inflammation, and fibrosis, and restored Treg/Th17 balance in experimental AIH. Mechanistically, UBE2O was found to interact with and promote ubiquitination degradation of YBX1 at lysine 135 (K135), leading to the reduction of interleukin-6 transcription and secretion in hepatocytes, thus rewiring naïve CD4+ T cells differentiation into Tregs. Furthermore, YBX1 partially reversed the protective effects of UBE2O overexpression in AIH. CONCLUSIONS:Our study revealed a previously unrecognized hepatocellular UBE2O/YBX1/interleukin-6 axis in AIH that primes hepatocytes to restore immune tolerance. Targeting UBE2O might provide a promising therapeutic target for AIH by linking posttranslational modification and hepatic immune tolerance.

Autoimmune hepatitis (AIH) is a chronic progressive inflammatory liver disease with a rising global incidence. The treatment of AIH remains challenging because first-line drugs show limited efficacy and systemic side effects. Gut microbiota plays a crucial role in the pathogenesis of AIH, leading to growing interest in developing probiotic-based therapies. In this study, we used multi-omics analysis to investigate the therapeutic effects of a quadruple probiotic mixture (Probiotic-quad) consisting of Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus in a well-established chronic AIH murine model. Our results showed that Probiotic-quad treatment significantly alleviated AIH progression, as evidenced by lower serum liver enzyme levels, ameliorated hepatic inflammatory infiltration and histopathological damage. Metagenomic sequencing results showed that gut dysbiosis in AIH mice was partially reversed after Probiotic-quad administration. Additionally, the integrity of the intestinal epithelial barrier was restored, accompanied by a reduction in serum lipopolysaccharide levels. Untargeted metabolomic and transcriptomic analysis revealed that Probiotic-quad treatment was linked to alterations in hepatic metabolism, including the citrate cycle and tryptophan metabolism, and was associated with reduced activation of the NF-κB and NOD-like receptor signaling pathways. These findings suggest that Probiotic-quad treatment ameliorates AIH severity and is potentially associated with changes in hepatic immune responses, metabolism, gut microbiota, and intestinal barrier function, highlighting its potential as an adjuvant therapy for AIH.

The conversion of primary bile acids to secondary bile acids by the gut microbiota has been implicated in colonic inflammation. This study investigated the role of gut microbiota related bile acid metabolism in colonic inflammation in both patients with inflammatory bowel disease (IBD) and a murine model of dextran sulfate sodium (DSS)-induced colitis. Bile acids in fecal samples from patients with IBD and DSS-induced colitis mice, with and without antibiotic treatment, were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). The composition of the microbiota in fecal samples from IBD patients and DSS-colitis mice was characterized via Illumina MiSeq sequencing of the bacterial 16S rRNA gene V3-V4 region. Metagenomic profiling further identified metabolism-related gene signatures in stool samples from DSS-colitis mice. Histological analysis, quantitative PCR (qPCR) and Western Blotting were conducted on colonic samples from DSS-induced colitis mice to assess colonic inflammation, mucosal barrier integrity, and associated signaling pathways. The multivariate analysis of bile acids was conducted using Soft Independent Modelling of Class Analogy (SIMCA, Umetrics, Sweden). The relation between the relative abundance of specific phyla/genera and bile acid concentration was assess through Spearman's correlation analyses. Finally, lithocholic acid (LCA), the key bile acid, was administered via gavage to evaluate its effect on colonic inflammation and mucosal barrier integrity. In patients with IBD, the composition of colonic bile acids and gut microbiota was altered. Moreover, changes in the gut microbiota further modulate the composition of bile acids in the intestine. As the gut microbiota continues to shift, the bile acid profile undergoes additional alterations. The aforementioned alterations were also observed in mice with DSS-induced colitis. The study revealed a correlation between dysbiosis of the gut microbiota and modifications in the profile of colonic bile acids, notably LCA observed in both patients with IBD and mice with DSS-induced colitis. Through multivariate analysis, LCA was identified as the key bile acid that significantly affects colonic inflammation and the integrity of mucosal barrier. Subsequent experiments confirmed that LCA supplementation effectively mitigated the inhibitory effects of gut microbiota on colitis progression in mice, primarily through the activation of the sphingosine-1-phosphate receptor 2 (S1PR2)/NF-κB p65 signaling pathway. Analysis of the microbiome and metagenomic data revealed changes in the gut microbiota, notably an increased abundance of an unclassified genus within the family Prevotellaceae in DSS-induced colitis mice. Furthermore, a positive correlation was observed between the relative abundance of Prevotellaceae and bile acid biosynthesis pathways, as well as colonic LCA level. These findings suggest that LCA and its positively correlated gut bacteria, Prevotellaceae, are closely associated with intestinal inflammation. Targeting colonic inflammation may involve inhibiting LCA and members of the Prevotellaceae family as potential therapeutic strategies.

BACKGROUND AND AIMS:Treatment of autoimmune hepatitis (AIH) remains challenging and primarily relies on corticosteroid therapy. Regulatory T cells (Tregs), essential for maintaining immune homeostasis and preventing various autoimmune diseases, present a potential therapeutic target for AIH. However, the role of Tregs in AIH pathogenesis remains unclear. APPROACH AND RESULTS:In a well-established AIH model induced by hydrodynamic transfection of cytochrome P450 2D6 (CYP2D6) plasmid into mouse liver, Tregs were found to increase in number as the disease progressed. Despite the increased hepatic Treg presence, the proportions of effector T cells also rose, contributing to disease progression. Systemic Treg depletion using FoxP3-DTR/eGFP mice exacerbated AIH progression, while adoptive Treg transfer failed to alleviate liver inflammation and fibrosis, highlighting intrahepatic Treg dysfunction. Single-cell RNA sequencing of hepatic Tregs using the 10× Genomics platform identified impaired suppressive function and a shift toward proinflammatory phenotypes, contrasting with enhanced suppressive capacity observed in peripheral Tregs. This intrahepatic Treg dysfunction was associated with elevated hepatic serum amyloid A1 (SAA1) levels, which impaired Tregs through toll-like receptor 2. Liver-specific AAV8-mediated shSAA1 therapy restored Treg function and ameliorated AIH symptoms, while the adoptive transfer of Tregs lacking toll-like receptor 2 significantly improved disease outcomes. CONCLUSIONS:Intrahepatic Tregs exhibit a diminished suppressive phenotype and an enhanced effector phenotype, failing to control the escalating inflammation in AIH. AIH treatment should not only focus on increasing Treg numbers but also on restoring their functional capacity.

ABSTRACT Background and Aim Acute‐on‐chronic liver failure (ACLF) is characterized by fast progression and high mortality, with systemic inflammation and immune paralysis as its key events. While natural killer (NK) cells are key innate immune cells, their unique function and subpopulation heterogeneity in ACLF have not been fully elucidated. This study aimed to investigate the characteristics of NK cell subsets in the peripheral blood of patients with ACLF and determine their roles in the inflammatory responses. Methods Circulating NK cells (14 751 cells) from patients with ACLF and healthy controls (HCs) were subjected to single‐cell RNA sequencing (scRNA‐seq). Clustering and annotation were used to identify the features of NK cell subsets and the characteristics of disease progression in ACLF. Results Four NK cell subsets were obtained, including adaptive NK cells, mature NK cells, inflamed NK cells, and CD56 bright NK cells. Compared with the HCs, the patients with ACLF had a significantly lower proportion of Mature NK cells and a higher proportion of Inflamed NK cells. Quasi‐temporal analysis showed that Inflamed NK cells were highly enriched in the late quasi‐temporal sequence, and genes related to pro‐inflammatory were significantly up‐regulated in Inflamed NK cells. In addition, scRNA‐seq and flow cytometry confirmed that the expression level of cell migration inducing hyaluronidase 2 ( CEMIP2 ) in NK cells progressively increased from the HC group to the ACLF survival group and then to the ACLF death group. Conclusions scRNA‐seq reveals that Inflamed NK cell subsets are associated with ACLF progression and poor prognosis. CEMIP2 may be a molecular marker for ACLF progression.

Antitumor drugs (such as chemotherapy, targeted therapy, immunotherapy, etc.) and local treatments like surgery and radiotherapy are widely used in cancer treatment, but they often carry the risk of liver injury, which seriously affects the prognosis and quality of life of patients. Therefore, liver protection is of crucial importance in cancer treatment. Relevant experts organized by the Oncology Hepatology Committee of China Anti-Cancer Association Association formulated this guideline based on the latest research achievements and clinical practice experience at home and abroad, aiming to provide comprehensive and systematic guidance for clinicians on liver protection. This guideline elaborates on the importance of liver protection during cancer treatment, comprehensively introduces the pathophysiological mechanisms, diagnosis, treatment, and preventive measures of antitumor treatment-related liver injury. This guideline is helpful for clinicians to formulate individualized treatment plans, improve the treatment outcome and quality of life of cancer patients, and provides an important reference for the clinical practice of liver protection in cancer treatment.

Background:Aspirin is widely used to prevent and treat cardiovascular diseases. The most common side effect is gastrointestinal damage. In recent years, aspirin-associated enteropathy has received increasing attention. This study aimed to establish a chronic model of aspirin-associated enteropathy, investigate the effect of enteric-coated aspirin on the intestinal flora, and explore the specific molecular mechanism of small intestinal damage. Methods:C57BL/6J mice were given aspirin for 45 days to induce chronic small intestinal injury. The intestinal mucosal injury was observed macroscopically and microscopically. Intestinal mucus levels were assessed by PAS staining. The intestinal permeability was measured by FD4. The oxidative stress levels of the small intestine were detected by immunofluorescence and immunohistochemistry. The mRNA and protein levels of inflammatory factors, tight junctions, and antioxidant defense-related genes were measured by qRT-PCR and Western Blot. The MPO activity, SOD activity and MDA content in serum were measured. The mitochondrial morphology and paracellular space were observed under transmission electron microscopy. The fecal samples were analyzed by high-throughput sequencing of 16S rRNA V3-V4 amplicons. Results:Aspirin induced weight loss, reduced food intake and increased faecal occult blood in mice. Aspirin led to a shortened small intestine, macroscopic and microscopic damage to the intestinal mucosa, and local inflammation. Aspirin disrupted the intestinal barriers and increased the permeability of the small intestine. Aspirin destroyed mitochondrial structure and damaged antioxidant capacity, and aspirin may induce oxidative stress through Nrf2/Gpx4 signaling pathway. Intestinal flora analysis showed that aspirin could induce changes in the abundance of Akkermansia and Lactobacillus. Conclusion:Long-term administration of enteric-coated aspirin successfully established a chronic small intestinal injury model in mice. It increased oxidative stress in the small intestine by disrupting mitochondrial structure and impairing antioxidant capacity. This damaged the intestinal mucosal barrier, increased intestinal permeability, and triggered gut microbial dysbiosis and inflammation.

Background Myeloid differentiation factor 88 (MyD88) is the core adaptor for Toll-like receptors defending against microbial invasion and initiating a downstream immune response during microbiota-host interaction. However, the role of MyD88 in the pathogenesis of inflammatory bowel disease is controversial. This study aims to investigate the impact of MyD88 on intestinal inflammation and the underlying mechanism.Methods MyD88 knockout (MyD88-/-) mice and the MyD88 inhibitor (TJ-M2010-5) were used to investigate the impact of MyD88 on acute dextran sodium sulfate (DSS)-induced colitis. Disease activity index, colon length, histological score, and inflammatory cytokines were examined to evaluate the severity of colitis. RNA transcriptome analysis and 16S rDNA sequencing were used to detect the potential mechanism.Results In an acute DSS-colitis model, the severity of colitis was not alleviated in MyD88-/- mice and TJ-M2010-5-treated mice, despite significantly lower levels of NF-kappa B activation being exhibited compared to control mice. Meanwhile, 16S rDNA sequencing and RNA transcriptome analysis revealed a higher abundance of intestinal Proteobacteria and an up-regulation of the nucleotide oligomerization domain-like receptors (NLRs) signaling pathway in colitis mice following MyD88 suppression. Further blockade of the NLRs signaling pathway or elimination of gut microbiota with broad-spectrum antibiotics in DSS-induced colitis mice treated with TJ-M2010-5 ameliorated the disease severity, which was not improved solely by MyD88 inhibition. After treatment with broad-spectrum antibiotics, downregulation of the NLR signaling pathway was observed.Conclusion Our study suggests that the suppression of MyD88 might be associated with unfavorable changes in the composition of gut microbiota, leading to NLR-mediated immune activation and intestinal inflammation.

The gut microbiota is closely associated with human health, and alterations in gut microbiota can influence various physiological and pathological activities in the human body. Therefore, microbiota regulation has become an important strategy in current disease treatment, albeit facing numerous challenges. Nanomaterials, owing to their excellent protective properties, drug release capabilities, targeting abilities, and good biocompatibility, have been widely developed and utilized in pharmaceuticals and dietary fields. In recent years, significant progress has been made in research on utilizing nanomaterials to assist in regulating gut microbiota for disease intervention. This review explores the latest advancements in the application of nanomaterials for microbiota regulation and offers insights into the future development of nanomaterials in modulating gut microbiota.

AbstractAimsThe endoscopic treatment of esophagogastric varices is challenging, and the nationwide application of endoscopic therapies for various types of esophagogastric varices and different clinical scenarios remains unclear. This study investigated the use of endoscopic therapy for portal hypertension in China.MethodsThis study used a questionnaire survey initiated by the Liver Health Consortium in China to investigate the use of endoscopic therapies for portal hypertension. Questionnaires were released online from January 30, 2023 to February 28, 2023 and filled out by chief physicians or senior instructors responsible for endoscopic therapies in participating hospitals across 31 provinces (autonomous regions and municipalities) in China. Comparisons of guideline adherence between primary and referral medical centers were performed using the chi‐square test or Fisher's exact test.ResultsIn total, 836 hospitals participated in the survey. For primary and secondary prophylaxis of esophagogastric variceal bleeding (EGVB), adherence to the national guidelines was 72.5% (606/836) and 39.2% (328/836), respectively. Significant differences were observed in the rate of adherence between the primary and referral centers for primary (79.9% [111/139] vs. 71.0% [495/697], p = 0.033) and secondary prophylaxis (27.3% [38/139] vs. 41.6% [290/697], p = 0.002). Of the hospitals, 78.2% (654/836) preferred endoscopic therapies for acute EGVB, and the timing of endoscopy was usually within 12 h (48.5%, 317/654) and 12–24 h (36.9%, 241/654) after bleeding. Endoscopic therapy was more likely to be the first choice of treatment for acute EGVB in referral centers than in primary centers (82.6% [576/697] vs. 56.1% [78/139], p < 0.001). Regarding the management of gastroesophageal varices type 2 and isolated gastric varices type 1, the most prevalent procedures were cyanoacrylate injection combined with sclerotherapy (48.2% [403/836] and 29.9% [250/836], respectively); however, substantial hospitals preferred clip‐assisted therapies (12.4% [104/836] and 26.4% [221/836], respectively). Nonselective beta‐blockers were routinely used in 73.4% (614/836) of hospitals during the perioperative period of EGVB management, and propranolol (88.8%, 545/614) and carvedilol (37.5%, 230/614) were the most widely used nonselective beta‐blockers.ConclusionsThis survey clarified that various endoscopic procedures have been implemented nationwide in China. Participating hospitals have actively performed emergent endoscopy for acute EGVB; however, these hospitals do not adequately follow recommendations regarding primary and secondary prophylaxis of EGVB. In the future, standardizing the selection of endoscopic procedures and improving compliance with guidelines is crucial.

Helicobacter pylori eradication is crucial in the treatment of peptic ulcers caused by H. pylori infection, a disease highly prevalent in Asia. We present a pooled analysis of two randomized, double-blind, double-dummy, phase 3 studies evaluating the efficacy and safety of vonoprazan-based bismuth-containing quadruple therapy for H. pylori eradication. Patients aged >= 18 years with endoscopically confirmed duodenal or gastric ulcers were randomized 1 : 1 to receive vonoprazan 20 mg or lansoprazole 30 mg once daily for up to 6 (duodenal ulcers) or 8 weeks (gastric ulcers). H. pylori- positive patients received vonoprazan- or lansoprazole-based bismuth-containing quadruple therapy for the first 2 weeks. H. pylori eradication was determined using the carbon-13 urea breath test at a follow-up visit 4 weeks post-treatment. The H. pylori eradication rate was 90.6% with vonoprazan vs. 85.2% with lansoprazole (difference: 5.4%; 95% confidence interval (CI):- 0.1, 10.8). H. pylori eradication rates were 7.1% (95% CI: 1.4, 12.8) and 12.6% (95% CI: 3.9, 22.0) higher in patients aged < 65 years and current smokers, respectively, with vonoprazan vs. lansoprazole. In the Chinese subpopulation, the H. pylori eradication rate was 92.0% with vonoprazan vs. 86.0% with lansoprazole (difference: 6.1%; 95% CI: 0.5, 11.7). Treatment-emergent adverse events occurred in 72.7 vs. 62.6% of H. pylori- positive patients at baseline in the vonoprazan vs. lansoprazole arm. H. pylori eradication with vonoprazan-based quadruple therapy was noninferior to lansoprazole-based quadruple therapy and exceeded 90%, a clinically relevant threshold for determining the efficacy of H. pylori eradication regimens (ClinicalTrials.gov identifier: NCT03050359; NCT03050307).

The incidence of autoimmune hepatitis (AIH) has increased significantly worldwide. The present study aims to explore the protective effect of L-lysine supplementation against AIH and to investigate its potential underlying mechanisms. A chronic experimental AIH mouse model was established by repeated tail vein injection of human cytochrome P450 2D6 (CYP2D6) plasmid. Starting from day 14 of the modeling, mice in the CYP2D6-AIH +L-lysine group were given 200 µl of purified water containing 10 mg/kg L-lysine by gavage until day27, once a day, and mice in the healthy control group and model group were given an equal volume of purified water by gavage. Our results showed that L-lysine supplementation partially reversed the liver injury mediated by CYP2D6 overexpression. These effects were consistent with the restraining impacts of L-lysine supplementation on decreasing pro-inflammatory cytokines expression level and CD4+ and CD8+ T lymphocytes infiltration, as well as curbing hepatic oxidative stress. Furthermore, L-lysine supplement relieved liver fibrosis in the context of AIH. In conclusion, L-lysine supplementation attenuates CYP2D6-induced immune liver injury in mice, which may serve as a novel nutrition support approach for AIH.

Gut microbiota-derived extracellular vesicles (mEVs) are reported to regulate inflammatory response by delivering bacterial products into host cells. The complement receptor of the immunoglobulin superfamily macrophages (CRIg+ Mφ) could clear invading bacteria and their derivatives. Here, we investigate the role of CRIg+ Mφ and the mechanism by which mEVs regulate intestinal inflammation. We found that it is exacerbated in IBD patients and colitis mice by mEVs’ leakage from disturbed gut microbiota, enriching microbial DNA in the intestinal mucosa. CRIg+ Mφ significantly decrease in IBD patients, allowing the spread of mEVs into the mucosa. The microbial DNA within mEVs is the key trigger for inflammation and barrier function damage. The cGAS/STING pathway is crucial in mEVs-mediated inflammatory injury. Blocking cGAS/STING signaling effectively alleviates inflammation caused by mEVs leakage and CRIg+ Mφ deficiency. Microbial DNA-containing mEVs, along with CRIg+ Mφ deficiency, stimulate inflammation in IBD, with the cGAS/STING pathway playing a crucial role.

BackgroundCircadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear.MethodsWe used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining.ResultsWe found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect.ConclusionThis study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease.

Objective: To investigate the clinical potential and safety of Moluodan to reverse gastric precancerous lesions. Methods: Patients aged 18-70 years diagnosed with moderate-to-severe atrophy and/or moderate-to-severe intestinal metaplasia, with or without low-grade dysplasia, and negative for Helicobacter pylori were recruited in this randomized, double-blind, parallel-controlled trial. The primary outcome was the improvement of global histological diagnosis at 1-year follow-up endoscopy using the operative link for gastritis assessment, the operative link for gastric intestinal metaplasia assessment, and the disappearance rate of dysplasia. Results: Between November 3, 2017 and January 27, 2021, 166 subjects were randomly assigned to the Moluodan group, 168 to the folic acid group, 84 to the combination group, and 84 to the high-dose Moluodan group. The improvement in global histological diagnosis was achieved in 60 (39.5%) subjects receiving Moluodan, 59 (37.8%) receiving folic acid, 26 (32.1%) receiving the combined drugs, and 36 (47.4%) receiving high-dose Moluodan. Moluodan was non-inferior to folic acid (95% confidence interval: -9.2 to 12.5; P = 0.02). High-dose Moluodan had a trend for better protective efficacy, though there was no statistical significance. The disappearance rate of dysplasia was 82.8% in the Moluodan group, which was superior to folic acid (53.9%; P = 0.006). No drug-related serious adverse events were observed. Conclusions: One pack of Moluodan three times daily for 1 year was safe and effective in reversing gastric precancerous lesions, especially dysplasia. Doubling its dose showed a better efficacy trend.