Intestinal Bacteria Destroy Intestinal Barrier And Aggravate Immune Liver Injury In Patients With Autoimmune Hepatitis

Objective The pathogenesis of autoimmune hepatitis(AIH)is not clear.However,the causal relationship between the imbalance of intestinal flora and the pathogenesis of AIH is not clear.The paper aim to discuss the effect of intestinal flora imbalance caused by vancomycin on concanavalin A(Con A)-induced acute immune liver injury and its possible mechanism,and then compare the intestinal flora differences between AIH patients and healthy people.The fecal supernatant was given to mice by fecal microbiota transplantation(FMT).Finally,the effect and potential mechanism of intestinal flora from different sources on mouse liver were analyzed.Method First part,the effect of vancomycin induced imbalance of intestinal flora on immune liver injury in mice Female C57BL/6 mice(8 weeks old)under SPF conditions were randomly divided into three groups: normal control(CTRL),vancomycin(VANC),Con A and Con A+VANC.Vancomycin hydrochloride was given free drinking water of 0.5mg/ml for 2 weeks in the VANC treated group and the sterile water was given the non VANC treated group.All Con A intervention groups were given Con A(13mg/kg)tail vein injection 12 hours before the mice were killed,and the other groups were given phosphate buffer solution(PBS)as the control.Four hours before the mice were killed,FITC-dextran was given by gavage.After the mice were killed,serum ALT and AST levels were measured to evaluate the liver function and hematoxylin and eosin(HE)was used to evaluate the degree of inflammation and necrosis of liver tissue,the concentration of FITC-dextran in plasma was measured by spectrophotometer to evaluate the intestinal permeability.Gas chromatography was used to detect the level of short chain fatty acids in cecal contents.The expression levels of IL-6,IL-10,IFN-γ and TNF-α were detected by PCR,and the expression levels of autophagy related proteins such as SQSTM1/p62,LC3B-I and LC3B-II were detected by Western blot.Second part,the intestinal microorganisms in AIH patients increase the mice intestinal permeability and cause bacterial translocation 5 AIH patients were collected from the outpatient department of digestive department of Tianjin Medical University General Hospital from July 2019 to October 2019.5 healthy volunteers matched in age and gender were collected at the same time.The feces of each participant were collected in sterile containers,and the feces of 5 AIH and 5 healthy controls were mixed in relatively sterile environment to extract the fecal supernatant.Before the experiment,female C57BL/6 mice(6 weeks old)under SPF conditions were pretreated with four kinds of antibiotics.They were randomly divided into the following groups and corresponding treatments:(1)AIH group(AIH,n=8): fecal supernatant of AIH patients was given to mice by gavage;(2)healthy control group(Healthy,n=8): fecal supernatant of healthy volunteers was given to mice by gavage;(3)blank control group(Control,n=6): sterile water was given by gavage;(4)AIH+DSS group(n=8): DSS drinking water was given intermittently;(5)Healthy+DSS group(n=8);(6)Control+DSS group(n=6);(7)AIH+DSS+Con A group(n=8);(8)Healthy+DSS+Con A group(n=8);(9)Control+DSS+Con A group(n=6).16 times gavage treatment were given in two months respectively,and 16 S r RNA sequencing was used to detect the composition of the bacteria in the fecal supernatant of the two groups.Four hours before the execution,FITC-dextran was given by gavage.Mice with Con A intervention were injected with Con A in tail vein at a dose of 13mg/kg 12 hours before execution.The whole blood and tissues of the mice were collected for subsequent detection.After the mice were killed,the serum ALT and AST levels were measured to evaluate the liver function;FITC-dextran concentration in the plasma was measured by spectrophotometer to evaluate the intestinal permeability;the infiltration of macrophages in the liver of mice was analyzed by flow cytometry;the aerobic and anaerobic bacteria in mice’s liver,spleen and lymph nodes were culture to observed the translocation of intestinal flora.Result First part,vancomycin induced imbalance of intestinal flora aggravates immune liver injury in mice Compared with the Con A group,the levels of serum ALT and AST in Con A+VANC group were significantly higher(P<0.01),the liver pathological results showed that infiltration of inflammatory cells in the portal area and necrosis of hepatocytes were found in Con A+VANC group,and the infiltration of inflammatory cells in the portal area and edema of hepatocytes were significantly increased in Con A+VANC group compared with Con A group.The serum FITC concentration of Con A+VANC group was higher than that of Con A group and normal control group(P<0.05).Among the six kinds of short chain fatty acids(acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid and isovaleric acid)detected in cecal contents,the detection level of butyric acid in Con A+VANC group and VANC group was significantly lower than that in Con A group and normal control group(P<0.05),and the other five kinds of short chain fatty acids also had a downward trend.The expression level of IL-6 in liver of Con A+VANC group was significantly higher than that of Con A group(P<0.05),the IFN-γ and IL-10 were increased too,but there was no statistical difference.The expression of LC3B-II and SQSTM1/p62 in liver cells of Con A+VANC group increased,and the expression level of SQSTM1/p62 was significantly higher than that of Con A group(P<0.01).Second part,the intestinal microorganisms in AIH patients increase the mice intestinal permeability and cause bacterial translocation 16 S r RNA gene sequencing of fecal supernatant of AIH patients and healthy controls showed that the abundance of Proteobacteria and Firmicutes in intestinal microflora of AIH patients was significantly higher than that of healthy people.At the same time,at the department level,the intestinal microbial diversity of AIH patients decreased significantly.The serum ALT and AST levels of AIH group were significantly higher than those of Control group and Healthy group,but there was no statistical difference.There was no significant difference in liver related transaminase levels between the groups of mice gavage with stool supernatant or DSS water alone.The level of FITC-dextran in AIH group was significantly higher than that in the other two groups(P<0.05).The results of flow cytometry showed that CD11 b positive cells in AIH+DSS+Con A group were significantly more than those in Healthy+DSS+Con A group,suggesting that macrophages infiltrated from the liver tissue of AIH+Con A group increased.The results of culture and identification of bacteria in liver,spleen and lymph nodes of mice showed that compared with Control group and Healthy group,more aerobic and anaerobic bacteria could be cultured in the liver,spleen and lymph nodes of mice in AIH group,among which,the specific aerobic bacteria in AIH group were Lactococcus Formosa,and the anaerobic bacteria were Lactobacillus sp.ara2.In the Control group,except for Lactobacillus rhamnoides/Lactobacillus animalis in the liver of a mouse,no bacteria were cultured in the spleen and lymph nodes.In addition,Lactobacillus rhamnoides/Lactobacillus animalis could be found in the liver or lymph nodes of each group of mice.Conclusion(1)The imbalance of intestinal flora induced by VANC can aggravate the acute liver injury induced by Con A.VANC induced imbalance of intestinal flora led to the decrease of the level of short chain fatty acids,which in turn affected the intestinal barrier function,increased intestinal permeability and increased the amount of intestinal antigens entering the liver.The mechanism of aggravating liver injury is related to the increase of IL-6 and IFN-γ levels.At the same time,compared with other groups,the autophagy level of liver cells in Con A+VANC group was up-regulated.The role and influence of Con A+VANC in liver injury still need to be further explored.(2)Compared with healthy people,the abundance of Proteobacteria and Firmicutes in AIH patients increased.The acute liver injury induced by Con A could be aggravated by gavage of fecal supernatant of AIH patients.The intestinal permeability in AIH group was significantly higher than that in Healthy group and Control group.Compared with the other two groups,the number of macrophages in AIH group increased significantly.More strains can be identified in the liver,spleen and lymph nodes of AIH group mice,among which Lactococcus Formosa,Lactobacillus sp.ara2 are the special strains of AIH group.