The Protective Effect And Mechanism Of Akkermansia Muciniphila On Chronic Pancreatitis

Chronic pancreatitis(CP)is a progressive fibrous inflammatory disease of the exocrine glands in which the parenchymal portion of the pancreas is destroyed and replaced by fibrous tissue,eventually leading to damage to the gland.If the disease spreads,it leads to the simultaneous failure of both the exocrine and endocrine glands.Many studies have demonstrated that the intestinal microbiota of CP patients differs from that of healthy patients and complex probiotic intervention could alleviate CP.Akkermansia muciniphila(A.muciniphila),a highly promising next-generation probiotic,has been shown to have beneficial effects on obesity and type 2 diabetes.Interestingly,pasteurized A.muciniphila still has its beneficial effect.Our previous study has shown that exogenous administration of short-chain fatty acids(SCFAs)to mice could alleviate the pathogenesis of CP.However the effect of A.muciniphila,a SCFAs-producing bacteria,on CP is not known.Therefore,in this paper,we used caerulein to establish a CP mice model and chose A.muciniphila standard strain ATCC BAA-835 to investigate the effect and potential mechanism of A.muciniphila on caerulein-induced chronic pancreatitis in mice.Male C57BL/6J at the age of 8 weeks were randomly divided into 4 groups(n=7-10):control group,model group,live AKK group and pasteurized AKK group.CP was established by using repeated injections of caeruelin(50μg/kg)with six consecutive injections per day,three days per week,for four weeks.During the modeling period,mice were treated with 200μL 5×10~9 CFU/m L of A.muciniphila bacterial solution by gavage daily in the live AKK group.In the pasteurized AKK group,mice were given A.muciniphila bacterial solution by pasteurization at 70°C for 30 min.The pancreatic atrophy,the weight-to-body weight ratio of pancreas,the degree of pancreatic fibrosis and the gene expression levels of cytokines and macrophages were examined to determine whether the live A.muciniphila preparation and the pasteurized bacterial preparation had a protective effect on the pancreatic injury in CP or not.The intestinal permeability was characterized by detecting the level of Lipopolysaccharide(LPS)and FITC-Detrain in serum.Staining and labeling of type II mucin(Muc2)in mouse colon was performed using immunofluorescence.Protein expression of tight junction proteins ZO-2,Occludin and Claudin-1 in colon was detected using Western-blot to identify whether live A.muciniphila as well as pasteurized A.muciniphila have a protective effect on the intestinal barrier in mice.The composition of mouse intestinal flora was analyzed by 16S DNA amplicon sequencing,and the contents of SCFAs in pancreas,serum and colon,as well as histone deacetylases(HDACs)and G-protein-coupled receptors(GPCRs)GPR41,GPR43and GPR109A in pancreatic and colon tissues were measured.Additionally,MAPK/NF-κB inflammatory signaling pathway was examined to live A.muciniphila preparation and the pasteurized bacterial potential mechanisms of action.The results showed that both live and pasteurized A.muciniphila preparations could effectively alleviate chronic pancreatitis in mice by reducing the degree of pancreatic atrophy decreasing the level of pancreatic inflammatory factors,inhibiting macrophage infiltration and reducing pancreatic fibrosis.Meanwhile,A.muciniphila treatment significantly reduced intestinal permeability and increased the secretion of Muc2 and live A.muciniphila increased the expression of tight junction proteins ZO-2,Occludin and Claudin-1 in colon.In addition,live A.muciniphila treatment altered intestinal microbiota homeostasis and increased the content of SCFAs in the colon,serum and pancreas.It inhibited HDAC1 and activated GPR41and GPR43 in pancreas and colon and suppressed the activation of MAPK(p38,JNK)/NF-κB signaling pathway in pancreatic tissues.In conclusion,live A.muciniphila treatment significantly increased the concentration of intestinal metabolites SCFAs in the colon and migrated to the pancreas via peripheral blood.The pancreatic SCFAs inhibited HDAC1 and activated GPR41 and GPR43,thus alleviating the condition of CP in mice.Pasteurized bacterial interventions also have some effect,and the mechanisms by which pasteurized bacteria work will be explored subsequently.The study is the first time to confirm that A.muciniphila treatment has a protective effect on CP in mice and to reveal the role of probiotics and prebiotics in pancreatic-associated diseases and a new therapy for clinical CP.