A Newly Developed Synbiotic Alleviates Autoimmune Hepatitis In Mice And Its Mechanism

Objective:Autoimmune hepatitis(AIH)is a chronic inflammatory liver disease characterised by the destruction of liver parenchyma due to the failure of hepatic immune tolerance.Currently,the pathogenesis of AIH is unclear.In addition to genetic factors,various environmental factors are also associated with the development of AIH.In recent years,the gut microbiota has been identified as a major environmental risk factor for AIH.Therefore,in this study,a synbiotic preparation made from Bifidobacterium,Lactobacillus acidophilus and konjac dextran was used to intervene in mice with a chronic AIH model,with the aim of exploring the effect of this novel synbiotic on alleviating liver injury in AIH mice and the possible mechanisms involved,with a view to providing a new approach for the prevention and treatment of AIH.Methods:Forty-three male specific-pathogen-free(SPF)C57BL/6 mice(5 weeks of age;20g)were randomly divided into four groups:11 for control group(Ctrl group),11 for AIH model group(AIH group),11 for synbiotic group(Syn group)and 10 for prednisone group(Pred group).For the establishment of the AIH model,except for the Ctrl group,all mice were injected with a dose of 1 mL antigen(0.5 mL of syngeneic S-100 antigen was emulsified in an equal volume 0.5 mL of complete Freund’s adjuvant[CFA]on day 7 and day 14).From the first day of the experiment,the Ctrl group and AIH group mice were gavaged with 0.2 mL of sterile PBS daily,the Syn group mice were gavaged with 0.2 mL of synbiotics(1.5×10~9/CFU L.acidophilus,1.5×10~9/CFU B.infantis,and 2 g/kg of body weight konjac dextran)daily,and the Pred group mice was gavaged with prednisone(1.5mg/kg).On day 42 of the experiment,mice were anaesthetised and executed after aseptic collection of faeces,and blood,spleen,ileum,colon and liver tissues were collected for subsequent experiments.1.Centrifugation of the collected blood,leaving the serum to be tested for alanine aminotransferase(ALT)and aspartate aminotransferase(AST);2.Recording of liver weight;pathological sectioning of the liver with HE staining;immunofluorescence for F4/80 expression in the liver;immunohistochemistry for IFN-γ,IL-6,IL-17A,IL-1βand NLRP3 and caspase-1 expression in the liver;3.The proportion of Tregs cells in the spleen was detect by flow cytometry;4.LPS levels in the serum and liver homogenate were measured using an ELISA kit,Western-blot to detect TLR4,NF-κB,IκB,P-IκB expression levels in liver;5.16S rRNA microbiome sequencing to detect the composition of intestinal microbiota and the abundance of each species in mouse faeces and correlation analysis.6.Pathological sections of mouse ileum were subjected to H&E staining;immunofluorescence and Real-time PCR assay for mouse ileal tight junction proteins ZO-1and occludin.Results:1.A comparison of the appearance of the livers of the mice in each group revealed that the livers of the Ctrl group were bright red in colour,soft in texture and smooth on the surface,while the livers of the AIH group showed abnormally lobulated and paler in color,and the livers of the Syn and Pred groups improved in appearance with an overall red colour and slightly covered with white material.Meanwhile,HE results showed that the livers of mice in the AIH group were infiltrated with inflammatory cells,the livers of mice in the Pred group showed significant edema,and no abnormalities were found in the livers of the Ctrl and Syn groups.The serum ALT and AST results showed that the mice in the AIH group had significantly elevated serum ALT and AST,and Syn and Pred group effectively reduced this change.Flow cytometry results showed that the proportion of Treg cells in the spleen of mice in the AIH group was elevated compared to the Ctrl group,while Syn and Pred suppressed this change.2.Immunohistochemical results showed that the levels of inflammatory factors IFN-γ,IL-6,IL-17A and IL-1β in liver tissues were significantly higher in the AIH group compared to the Ctrl group,while the levels of these inflammatory factors were reduced in the Syn and Pred groups.Immunofluorescence results showed that the fluorescence intensity of liver tissue macrophages in the AIH group was significantly higher than that in the Ctrl group,while the fluorescence intensity of macrophages in the Syn and Pred groups was significantly lower than that in the AIH group.3.Western-blot results showed that TLR4/NF-κB signaling pathway was activated in AIH mice,and TLR4,NF-κB and P-IκB levels were increased and IκB levels were decreased in the liver,while Syn and Pred inhibited this change.At the same time,immunohistochemical results showed that the levels of NLRP3 and caspase-1 in the livers of AIH mice were also increased,while the levels of NLRP3 and caspase-1 in the livers of Syn mice returned to normal.4.The results of 16S r RNA microbiome sequencing in mouse faeces showed that the alpha diversity of intestinal flora was significantly lower in the AIH group compared to the Ctrl group.And after Syn intervention,the alpha diversity was significantly increased.Meanwhile,based on the OTU abundance information,the results of Venn diagram analysis showed that the number of OTUs in the AIH group decreased compared with the Ctrl group,and supplementation with Syn significantly increased the number of OTUs.Principal component analysis(PCA)and principal coordinate analysis(PCo A)were used to further analyse the degree of differences between groups of mouse intestinal flora,and the results showed that the intestinal microbiota was significantly separated between the four groups,with differences in the structural composition of the microbiota.At the phylum level,Bacteroidota and Firmicutes accounted for 52.19%and 45.12%of the faeces in the Ctrl group,while Bacteroidota,Firmicutes,Actinobacteriota and Proteobacteria dominated the faeces of mice in the AIH group,accounting for 53.82%,32.68%,6.30%and 6.06%respectively.Bacteroidota,Firmicutes and Actinobacteriota dominated the faeces of the Syn mice,accounting for 57.18%,33.03%and 8.08%respectively.At the species level,Escherichis＿coli＿g＿Escherichia-Shigella was elevated in the AIH group of mice compared to the Ctrl group among the 15 dominant species,and decreased significantly after the Syn intervention.Differences in faecal flora abundance between the four groups were identified using LEf Se(Line Discriminant Analysis(LDA)Effect Size)and the analysis showed that in the Ctrl group,the significantly different species were Clostridia,Lachnospirales,Lachnospiraceae In the AIH group,the significantly different species were Lactobacillales,Lactobacillus,Lactobacillaceae,etc;in the Syn group,the significantly different species were Actinobacteriota,Bifidobacteriaceae,Actinobacteria,etc;in the Pred group,the significantly different species were Lactobacillales,Lactobacillus,Lactobacillaceae,etc.In the Pred group,the significantly different species was Muribaculaceae.5.The HE results of mouse ileum showed that the ileal villus were intact and tightly arranged in the Ctrl group,while the ileal villus were broken and sparse in the AIH group,and the ratio of villus length to crypt depth decreased significantly compared with the Ctrl group,both of which were improved after Syn and Pred interventions.Meanwhile,the results of immunofluorescence and Real-time PCR for the tight junction proteins ZO-1 and occludin showed that the levels of ZO-1 and occludin in the ileal tissues of AIH mice were lower than those of the Ctrl group,while the levels of ZO-1 and occludin in the Syn and Pred groups were higher than those of AIH mice.Conclusion:We found that this novel synbiotic could reduce liver injury and improve liver function in AIH mice,while reducing liver inflammation and scorching;it could regulate intestinal microbial composition and intestinal barrier function by inhibiting hepatic TLR4/NF-k B/NLRP3 scorching signalling pathway,which not only improved intestinal ecological dysregulation,but also enhanced intestinal barrier integrity.The novel synbiotic proposed in this study is comparable to prednisone in the treatment of AIH and does not cause hepatic oedema.Therefore,this novel synbiotic provides a new direction for the clinical treatment of AIH.