The Role And Mechanism Of Leaky Gut In The Pathogenesis Of Autoimmune Hepatitis

Objective The incidence of autoimmune hepatitis(AIH)is increasing yearly,which has become an important cause of cirrhosis.However,the pathogenesis of AIH has not been fully clarified.In recent years,environmental factors,especially intestinal dysbiosis and barrier dysfunction,have been considered to be related with AIH,but the mechanism is still unclear.In this study,we evaluated the alterations of intestinal barrier in AIH patients at the early stage of disease,thus explored the role and mechanism of intestinal barrier dysfunction in the pathogenisis of AIH,and finally explored the therapeutic effect of intestinal barrier intervention in AIH.It provides a new perspective to elucidate the pathogenesis and prevention strategies of AIH.Methods1.To evaluate the alterations of intestinal barrier in patients with AIH,68 AIH patients including 39 patients without cirrhosis(AIH-n)and 29 patients with cirrhosis(AIH-c)were enrolled in this study.Fifteen controls(CTRL)were selected from the Health Management Center and matched the AIH patients in age and gender.Intestinal mucosal specimens,feces and blood were collected.The expressions of intestinal tight junction proteins(TJPs)and inflammatory factors were detected by RT-q PCR.The SCFA concentrations were determined by gas chromatography.The serum concentrations of LPS and zonulin were quantified with the ELISA kits.Furthermore,the expression of TJPs in Caco-2 cells stimulated by the facal supernatant from AIH-n patients was detected.2.To explore the role of intestinal barrier in the pathogenisis of AIH,we did animal experiments with 6-week-old C57BL/6 mice,which were randomly divided into four groups including CTRL group,DSS group,Con A group and DSS-Con A group.After7 days,mice were injected with Con A solution or the normal saline through tail vein.HE staining was used to evaluate the pathological changes of intestinal and liver tissues.The proportions of the liver macrophages were analyzed via flow cytometry.RT-q PCR,western blot and immunofluorescence were used to detect the activation of RIP3 signaling pathway.Another 12 mice were randomly divided into two groups including DSS-Con A group and GSK872 pretreated group to explore the effect of RIP3 signaling pathway on the activation and accumulation of liver macrophages.3.In order to explore the therapeutic effect of early intervention with probiotics in AIH,were randomly divided the mice into CTRL group,EAH group and B420 group.The EAH model was established by intraperitoneal injection of liver antigen S100.Mice in B420 group were gavaged with B420 since the first day of modeling.On the28 th day,the mice were sacrificed.The intestinal microbiota was detected by 16 S r RNA sequencing.RT-q PCR,western blot and immunofluorescence were used to detect the changes of intestinal TJPs and the activation of RIP3 signaling pathway.The proportion of Treg/Th17 cells in liver and spleen was measured by flow cytometry.4.To further clarify the role of intestinal microbiota in the pathogenesis of AIH,we divided mice into 3 groups including CTRL group,EAH group and Abx group.The mice in Abx group were given broad-spectrum antibiotic water for 2 weeks before modeling.After 4 weeks,mice were sacrificed and the liver pathological changes were evaluated by HE staining.The alterations of intestinal barrier and the activation of liver macrophages were detected by RT-q PCR and immunofluorescence.Results1.Compared with the CTRL group,the AIH-n group had focal loss of intestinal epithelial cells,less regular arrangement of the gland and more infiltration of inflammatory cells;the expression of Occludin decreased and the expression of TNF-α,IL-1β and IL-6 increased;the contents of fecal propionic acid,butyric acid,iso-valeric acid and valeric acid were significantly decreased;serum LPS and Zonulin levels were increased.Besides,the fecal supernatant from AIH-n patients significantly inhibited the expression of ZO-1 and Occludin in Caco-2 cells and destroyed the structure and morphology of tight junction.2.Compared with Con A group,the DSS-Con A group had increased infiltration of inflammatory cells in the portal area,higher serum transaminase levels and increased the expression of TNF-α,IL-1β,IL-6 and CCl2;the number of activated macrophages and the ratio of infiltrating macrophages to resident macrophages were significantly increased;the m RNA expression of RIP3 and MLKL and the protein expression of p-RIP3 and p-MLKL were further increased.Pretreated with GSK872 significantly inhibited the ratio of infiltrating macrophages to resident macrophages and decreased the expression of macrophage-related inflammatory cytokines and chemokines.3.Early intervention with B420 significantly improved liver injury in EAH mice,alleviated infiltration of inflammatory cells and the focal necrosis.B420 improved the diversity and composition of intestinal microbiota and increased the content of fecal butyric acid.B420 also increased the expression of TJPs in the intestinal tissues,thus decreased intestinal permeability and inhibited LPS translocation.The expression of RIP3 and MLKL in liver tissues was significantly decreased with lower expression of TNF-α,IL-6 and CCl2,the proportion of Th17 cells in liver and spleen was also decreased.In-vitro experiments demonstrated that the B420 supernatant significantly alleviated LPS-mediated down-regulation of ZO-1 and Occludin in Caco-2 cells,and decreased the expression of TNF-α and IL-6 in macrophages.However,the expression of RIP3 wasn’t inhibited.4.The application of broad-spectrum antibiotics could significantly reduce the richness and diversity of intestinal microbiota,improve the damage of intestinal barrier,inhibit the activation of liver macrophages and alleviate liver inflammation in EAH mice.ConclusionsIntestinal barrier destruction and increased intestinal permeability occurred in the early stage of AIH.Intestinal barrier dysfunction augmented activation and accumlation of liver macrophages in Con A-mediated liver injury,highlighting RIP3 as an important interface that mediated liver inflammation.Early intervention with probiotics could mitigate immune-mediated hepatitis through regulating intestinal barrier and liver immune microenvironment.Intestinal microbiota is necessary for destruction of intestinal barrier and imbalance of liver immune homeostasis in AIH.