| 【Background】The global mortality rate of gastric cancer(GC)ranks fourth among all types of cancers,ranking second in both new cases and deaths among malignant tumors in China.In the Correa cascade,carcinogenesis progresses from chronic gastritis to atrophy,intestinal metaplasia(IM),and dysplasia,and finally resulting in GC.In this process,IM and dysplasia are considered precancerous lesions of GC,and IM is associated with a tenfold increased risk of GC in the general population.However,there is a lack of drugs that can safely and effectively treat IM.A meta-analysis revealed a significant association between metformin treatment and a reduced incidence of GC in patients with type 2 diabetes(HR=0.763,95%CI:0.642-0.905).Notably,the risk reduction was particularly pronounced in Taiwan(HR=0.514,95%CI: 0.384-0.688).Furthermore,certain investigations have demonstrated that the utilization of insulin and sulfonylureas does not impact the prognosis of individuals with GC,suggesting that the cancer protective effect of metformin was independent of glycemic control.Additionally,metformin has been reported to alter the gut microbiota community and regulate bile acid levels within the gastrointestinal tract.We previously identified a positive association between serum total bile acid level and GC risk,and significant variations in serum bile acid profiles were observed among patients with different types of gastric mucosal lesions.The above evidence suggests that metformin may be a potential candidate drug for the chemoprevention of GC in diabetic patients.However,most of the current evidence on metformin’s potential benefits for cancer comes from observational studies in patients with type 2 diabetes,lacking evaluation of its efficacy in reversing IM in general population.Therefore,clinical trials are needed in non-diabetic patients to verify the chemopreventive effect of metformin against GC and further elucidate the underlying mechanism by which metformin counteracts bile acid-induced gastric IM.【Objective】To evaluate the safety and efficacy of metformin in treating gastric IM,and further to elucidate its mechanisms in reversing bile acid-induced IM.【Methods】1.Risk factors associated with gastric IM in northwest ChinaPatients who attended the gastroenterology outpatient department of Xijing Hospital from April 1,2022 to April 30,2023 were included in this study.Based on endoscopy and/or pathology results,the participants were categorized into two groups: chronic gastritis group and IM group.Questionnaires were administered to collect data from the patients,including baseline information as well as endoscopy and pathological findings.The relationship between patients’ demographic data,endoscopic/pathological examination results,and IM was analyzed.By employing a binomial logistic regression model,the associated risk factors of IM patients in northwest China were identified.2.Efficacy and safety of metformin in the reversal of gastric IMAn open-label,prospective,randomized controlled trial was performed over a 6-month period.Patients diagnosed with IM were recruited from the outpatients of the First Affiliated Hospital of Air Force Medical University between April 2022 and May 2023.All study subjects were randomly assigned in a 1:1 ratio by computer to receive either metformin at a dose of 500 mg once-daily or folate at a dose of 5 mg three times-daily for 6 months.Participants were required to undergo gastroscopy at baseline and after completing 6 months of treatment.The primary outcome was defined as the regression rate and the progression rate of IM based on OLGIM stage in different groups before and after 6 months of treatment.Safety outcomes included percentages of adverse events.3.Mechanism of metformin in the reversal of bile acid-induced gastric IMThe present experiment employed bile acid as a stimulant for human gastric epithelial cells to establish an IM model.Subsequently,the impact of metformin on the proliferative capacity of gastric IM cells was investigated at varying concentrations and time intervals.The metabolites exhibiting significant differences before and after the administration of metformin were identified through non-targeted metabolomics analysis,while the key pathway underlying metformin’s impact on IM was determined via multi-omics association analysis.The Metabo Analyst 5.0 and MBROLE 2.0 software was utilized for conducting pathway analysis and enrichment analysis of metabolome data,as well as performing cytoscape network analysis to identify the interactions among differential metabolites.The identification of key pathways was achieved by integrating the results obtained from network analysis and pathway enrichment analyses of diverse metabolites.4.Effect of metformin on serum bile acid profiles in patients with IMSerum samples were collected at baseline and 6 months post-treatment from patients with IM in both the metformin and folate groups.Simultaneously,patients with chronic nonatrophic gastritis(CNAG)who met the same exclusion criteria as those in the second part were selected from the Xijing Hospital of Digestive Disease during the same period,and their serum samples were also collected.Using UPLC-MS/MS,serum samples from the CNAG group and IM group were analyzed using targeted metabolomics at baseline and 6months post-treatment.By comparing the changes in serum bile acid profiles between patients with CNAG and those with IM,as well as before and after administration of metformin/ folate in patients with IM,we conducted a comprehensive analysis to investigate the impact of metformin on the serum bile acid profiles in patients with IM,aiming to elucidate its potential mechanism in reversing bile acid-induced IM.【Results】1.Risk factors associated with gastric IM in northwest ChinaThe present study enrolled a total of 588 patients from April 2022 to April 2023,meeting the clinical assessment criteria and providing voluntary informed consent.Among them,there was a total of 430 individuals who received a diagnosis of chronic gastritis,while IM was observed in 158 patients.The mean age of patients with IM(55.70 ± 8.19years)was significantly higher compared to the chronic gastritis group(49.42 ± 11.82 years),with 89.24% of them being over the age of 45,and a majority being male(62.66%).In addition,over 90% of patients with IM have been infected with Helicobacter pylori(Hp),which is significantly higher than the average rate of Hp infection in China.When comparing the results of gastroscopy between the two groups,there was a higher proportion of bile reflux observed in the IM group compared to the chronic gastritis group.A logistic regression model using binary variables was employed to investigate the risk factors contributing to the risk of IM.The analysis revealed that age over 45(OR=6.71,95%CI3.22-14.74),consumption of dairy products(OR=1.95,95%CI 1.14-3.38),a history of Hp infection(OR=10.50,95%CI 5.38-21.99),and bile reflux(OR=3.70,95%CI 1.47-9.35)were independent risk factors for IM.2.Efficacy and safety of metformin in the reversal of gastric IMA total of 140 patients were enrolled in the study and were randomly allocated to either the metformin group or the control group at a 1:1 ratio.After excluding 6 patients from the metformin group and 5 patients from the control group,a total of 129 patients were included for statistical analysis.There were no significant differences observed between the two groups at baseline regarding basic demographic characteristics such as gender,age,BMI,smoking and alcohol consumption,family history of gastric cancer,and dietary habits.After a 6-month treatment with metformin and folate,respectively,the OLGIM stage decreased by at least one stage(RR=1.57,95% CI 1.04-2.37)in 33 out of 64 patients in the metformin group(53.12%)and in 22 out of 65 patients in the control group(33.85%).The statistical analysis showed a significant difference between the two groups(P=0.027),suggesting that metformin has potential for reversing gastric IM.After 6 months of treatment with metformin and folate,respectively,the metformin group showed a lower incidence of progression in gastric mucosa atrophy compared to the control group(20.31%vs 38.46%;RR=0.53,95% CI 0.30-0.94;P=0.024),indicating that metformin significantly reduces the risk of disease progression.In terms of safety,no adverse reactions were observed in the control group,while the metformin group exhibited a lower incidence of mild adverse reactions that could be self-alleviated.3.Mechanism of metformin in the reversal of bile acid-induced gastric IMGES-1 gastric epithelial cells were stimulated with DCA to establish an IM cell model.Treatment with 200μM DCA significantly upregulated the m RNA and protein expression levels of CDX2,KLF4,MUC2,and VILLIN1.Based on these findings,metformin was employed for the treatment of IM cells,which resulted in a significant increase in AMPKα2m RNA levels.Moreover,metformin significantly reduced DCA-induced CDX2 m RNA and protein expression,an effect that could be preempted by the AMPK inhibitor Compound C.These results suggest that the inhibition of DCA-induced CDX2 upregulation by metformin may be dependent on AMPK activity.Additionally,metformin was found to inhibit the induction of DCA on HNF4α expression,and the inhibition effect of metformin on HNF4αprotein expression was reduced by AMPK inhibitor Compound C.This suggests that AMPK is involved in metformin’s ability to inhibit DCA-induced HNF4α expression.These findings indicate that metformin suppresses the expression of HNF4α/CDX2 by activating the AMPK pathway and effectively reverses gastric IM.The non-targeted metabolomic analysis revealed differential changes in metabolites of IM cells under the influence of metformin.The Venn diagram showed the number of corporate or particular metabolites in DMSO vs DCA group and DCA vs DCA+ metformin group,and 25 corporate metabolites were identified,including L-Palmitoylcarnitine,Stearoylcarnitine,L-Acetylcarnitine,9-cis-Retinoic acid,D-sphingosine and other metabolites.MBROLE 2.0 was utilized for the analysis of the KEGG pathway associated with differential metabolites.We observed that 8 corporate pathways were involved in DCA vs DMSO group and DCA+ metformin vs DCA group,including PPAR signaling pathway,phenylalanine,beta-Alanine metabolism,retinol metabolism,tyrosine and tryptophan biosynthesis,intestinal immune network for Ig A production,pantothenate and Co A biosynthesis,adipocytokine signaling pathway and secondary bile acid biosynthesis.These findings suggest that aberrant metabolic dysregulation may play a pivotal role in the initiation and perpetuation of IM.4.Effect of metformin on serum bile acid profiles in patients with IMCompared to the CNAG group,the IM group exhibited an increased relative abundance of secondary bile acids,while a decreased relative abundance of primary bile acids was observed;however,these differences did not reach statistical significance.Additionally,there were significant differences observed in the serum bile acid profiles between patients diagnosed with severe IM(OLGIM IV)and those diagnosed with CNAG.After 6 months of metformin treatment,a comparison of serum samples from patients with IM revealed significant differences in the distribution of serum bile acid profiles between the baseline and post-treatment groups.Specifically,there was a decrease in the relative abundance of secondary bile acids and an increase in primary bile acids compared to baseline(P < 0.001).The patients in the IM group who responded well to metformin treatment(based on OLGIM staging,where the pathological diagnosis of gastric mucosa showed at least a 1-stage improvement compared to baseline after treatment)underwent further screening.Serum samples were collected at baseline and 6 months post-treatment,ensuring that paired serum samples from the same patient were used for analysis.After 6 months of metformin treatment,the relative abundance of secondary bile acids decreased compared to the baseline,while there was a significant increase in the relative abundance of primary bile acids(P <0.05).Finally,10 metabolites exhibiting significant differences after metformin treatment were identified,namely CA,CDCA,GCDCA-3S,b MCA,HCA,Nor CA,muro CA,7-Keto LCA,7-DHCA and 12-DHCA;the levels of these metabolites demonstrated a substantial increase.【Conclusions】1.The study revealed that age ≥ 45,consumption of dairy products,a history of Hp infection,and bile reflux were identified as independent risk factors for patients with IM in Northwest China.2.Metformin(500 mg/day)can safely and effectively reverse IM in non-diabetic patients without Hp infection,particularly in patients older than 45 years,those with a history of Hp infection,or those with OLGIM stage III/IV.Furthermore,metformin significantly reduces the risk of progression of gastric mucosa atrophy.3.Metformin suppresses the expression of HNF4α/CDX2 by activating the AMPK pathway and effectively reverses gastric IM.Additionally,treatment with metformin induced significant alterations in the differential metabolite profile of IM cells,particularly enriching the PPAR metabolic pathway.These findings suggest a crucial role for aberrant fatty acid metabolism in both the initiation and maintenance of IM.4.Significant disparities were observed in the serum bile acid profiles between patients diagnosed with IM and those suffering from CNAG.Metformin has the potential to modulate the serum bile acid profiles of IM patients,specifically by increasing levels of various secondary bile acids that exhibit inhibitory effects on FXR signaling.This mechanism could potentially contribute to the therapeutic efficacy of metformin in reversing IM. |
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