Use Of Gastric Acid Suppressants And Subsequent Risk Of Cholelithiasis:A Cohort And The Mechanism Study

Background and aims:Gastric acid suppressants,such as proton-pump-inhibitors（PPI）,are among the most commonly used medicines worldwide.The long-term safety of acid suppressants has raised considerable concerns recently.PPI have a major impact on the gut microbiome which in turn,may affect metabolic-related diseases,such as cholelithiasis.Cholelithiasis is a common health problem with a prevalence of 11%in China.Previous studies suggested that PPI can reduce the antibacterial ability of gastric acid and alter the normal flora structure of gastrointestinal tract.Besides,intestinal flora may promote the development of cholesterol stone.Therefore,gastric acid suppressants may increase the risk of cholelithiasis but the epidemiological evidence remain lacking.The aims of this study were to invesitgate the relationship between gastric acid suppressants and the cholelithiasis risk,and explore whether gut microbiota were involved in the acid suppressant-induced bile acid metabolism disorders and cholelithiasis.Methods:This study included 4 parts:1.The prospective cohort study included 477,293 participants who reported PPI or H2RA use and were free of cholelithiasis or cancer from the UK Biobank（aged37–73 years）.We evaluated hazard ratios（HRs）of regular use of PPI or H₂RA and risk of cholelithiasis adjusting for demographic factors,lifestyle habits,the presence of comorbidities,use of other medications,and clinical indications.2.One hundred twenty C57BL/6J female mice were divided into 6 groups.The control group and PPI group were fed with common fodder,the gallstone model group was fed with Tepperman gallstone modeling fodder,and the other three groups were fed with gallstone modeling fodder and PPI（10 mg/kg,30 mg/kg,and 40mg/kg）.We recorded the time of occurrence of gallstones and the number of mice with gallstones in each group.In addition,we explore the underlying mechanism.We using immunohistochemistry combined with q PCR and Western Blot techniques to detect key factors of bile acid metabolism pathways.3.We carried out a case-control study evaluating bile acids by PPI use.Bile acid profiles were detected in serum from 23 gallstone patients and 21 healthy controls by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（LC-MS/MS）.We tested the differences of bile acids between the two groups to find the characteristic bile acids associated with gallstones with the Wilcoxon rank sum test.4.Based on an ongoing cohort study,we used 16s-r RNA gene sequencing method to detect the intestinal microbial structure of 208 patients taking PPI.We also use LC-MS/MS to test bile acid spectrum of 97 blood samples.We analyzed the effect of gastric acid suppressants on intestinal flora and bile acid profile,and clarify the mechanism of"PPI use-intestinal flora-bile acid spectrum change".Results 1.We identified 12,870 cases of cholelithiasis over a median follow-up of 8.1 years.Regular use of PPI（HR 1.22,95%CI:1.16 to 1.29）or H₂RA（HR 1.16,95%CI:1.05 to 1.28）was associated with increased risk of choletithiasis after confounding adjustment.No major differences were shown for individuals used PPI or H2RA.The absolute risk of PPI-associated choletithiasis was increased with the baseline predicted risk evaluated by known environmental and genetic risk factors（Risk Differences in the lowest vs.the highest quartile:1.37 vs.4.29 per 1000person-years）.2.From the 6th week after Tepperman feed and/or PPI intervention,the mice were dissected to observe the formation of gallstones（3 mice/group each week）in each group.The development of gallstones were as follows:Week 6:30 mg/kg PPI（1/3）;Week 7:10 mg/kg PPI（1/3）,40 mg/kg PPI（1/3）;Week 8:30 mg/kg PPI（2/3）;Week 9:10 mg/kg PPI（1/3）;Week 10:30 mg/kg PPI（1/3）,40 mg/kg PPI（2/3）;Week11:30 mg/kg PPI（1/3）,40 mg/kg PPI（2/3）,control group（1/3）.There were no stones in the control group and only PPI feeding group.The expression levels of FXR and FGF15 in this group were also up-regulated,while the m RNA and protein levels of CYP7A1 were significantly down-regulated.3.23 patients with gallstones,and 21 healthy controls were included.We demonstrated CA,DCA,GCA,GDCA,and TDCA in the gallstone group significantly decreased when compared with the control group,while GCDCA and TCDCA significantly increased compared with the control group.（P<0.05）4.Use of PPI for more than 2 weeks resulted in changes of the gut micr obiome compared with those who did not use PPI.There were significant diffe rences in the microbiota between the two groups,such as p＿＿Bacteroidetes、c＿＿Bacteroidia、o＿＿Bacteroidales、g＿＿Bacteroides、f＿＿Bacteroidaceae、o＿＿Betapro teobacteriales、f＿＿Burkholderiaceae、g＿＿Lachnospira、f＿＿Acidaminococcaceae、g＿＿Phascolarctobacterium、g＿＿Prevotella＿2、g＿＿Lachnoclostridium、s＿＿butyrate-p roducing＿bacterium＿GM2/1.We also found PPI can increase the abundances of BSH bacteria and reduce the diversity of bacteria could code bai B in the intestinal tract.PPI could also lead to the change of bile acid metabolism and significantly increase GCDCA,GCA,UDCA,GUDCA,and TCDCA.Conclusions:This study demonstrated that gastric acid suppressants increase the risk of cholelithiasis and the association is causal.It may associate to the potential pathway of‘acid suppressants-gut microbiota-cholelithiasis related bile acid change-cholelithiasis’.The results of this study have a major impact on understanding the mechanism of gastric acid suppressant-induced gallstones,prevention of cholelithiasis and promoting appropriate use of acid-suppressing drugs.