Correlation Between Intestinal Mucosal Barrier Injury And Intestinal Mucosal Microbiota Dysbiosis In NASH Mice

Objective1.The non-alcoholic steatohepatitis(NASH)mouse model is established by high-fat diet(HFD)and methionine-choline-deficient diet(MCD).The changes of intestinal mucosa microbiota in the two model mice are detected by 16s rRNA gene sequencing.2.To detect and evaluate the injury of intestinal mucosal barrier in the two models and the correlation with intestinal mucosal microbiota dysbiosis is analyzed.Methods1.Establishment of NASH mice model:NASH mice model was established by HFD and MCD,respectively.After the mice were sacrificed,the relevant specimens were taken.2.Hematoxylin and eosin(HE)staining and Oil red O staining were used to observe the pathology of liver tissue.3.To detect serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),diamine oxidase(Diamine oxidase,DAO),D-lactic acid and bacterial endotoxin.4.HE staining was used to observe the pathology of colonic mucosa.Immunohistochemical(IHC)staining was used to observe the expression and distribution of tight junction protein occludin.5.Flow cytometry was used to detect the quantity of colonic macrophage(M(?))and myeloid-derived suppressor cells(MDSCs).6.16s rRNA gene sequencing was used to detect the intestinal mucosal microbiota in mice of each group.Results1.Successfully established two NASH mice models:Compared with ND group,body weight,liver weight,liver index,ALT and AST of HFD group all increased(P<0.05).Compared with CON group,ALT and AST of MCD group also increased significantly(P<0.05).Liver HE staining and oil red O staining show the liver tissues of ND group and CON group were evenly colored,the liver cells were structurally intact,and no lipid droplets and inflammatory cells were found.However,hepatic steatosis and inflammatory reaction were observed in the liver of HFD group and MCD group.The above results show that two NASH mice models were successfully established.2.Function of intestinal mucosal barrier impaired in NASH mice:Compared with control groups,the villi of colon tissue in HFD group and MCD group were loosely arranged,and some of the villi appeared atrophy,defect and fracture.Even the epithelial layer was separated from the lamina propria.The model groups were lower in expression of occludin protein than the control group,and the distribution was abnormal.Furthermore,the results of flow cytometry showed that the quantity of M(?)and MDSCs in the intestinal mucosa of the model group were significantly higher than that of the control group(P<0.001).At the same time,DAO,D-lactic acid and bacterial endotoxin in HFD group and MCD group were also significantly higher than the control groups(P<0.05).3.Intestinal mucosal microbiota dysbiosis:At the level of phylum,the abundance of Proteobacteria in HFD group was higher than that in ND group(P=0.08),and the abundance of Actinobacteria and Verrucomicrobia were decreased(P<0.05).Compared with CON group,the abundance of Deferribacteres was increased(P<0.05),the abundance of Bacteroidetes and Verrucomicrobia were decreased in the MCD group(P<0.05).Verrucomicrobia was decreased in both HFD group and MCD group.According to the microbiota that was increased or decreased in both models,we found that the Akkermansia genus was significantly decreased in both models(P<0.05),and the bacterium belonged to Verrucomicrobia.4.Correlation analysis between intestinal mucosal barrier index and abundance of Akkermansia:DAO(r=-0.879,P<0.05),D-lactic acid(r=-0.898,P<0.05)and bacterial endotoxin(r=-0.936,P<0.05)in HFD group were negatively correlated with the abundance of Akkermansia respectively.Similarly,DAO(r=-0.957,P<0.05),D-lactic acid(r=-0.891,P<0.05)and bacterial endotoxin(r=-0.880,P<0.05)in MCD group were also negatively correlated with the abundance of Akkermansia respectively.Conclusion1.Both models can be seen with impaired mechanical barrier,intestinal mucosal injury,intestinal villus shortening and sparse,expression of tight junction protein occludin decreased,permeability increased,endotoxin translocated.Intestinal mucosal immune barrier function was activated.The quantity of M(?)and MDSCs has increased.Intestinal mucosal barrier injury may play an important role in the development of NASH.2.Intestinal mucosal microbiota dysbiosis occurred in both models.Akkermansia abundance decreased in both models,and correlation analysis with intestinal mucosal barrier index showed that the relative abundance of Akkermansia was negatively correlated with DAO,D-lactic acid and bacterial endotoxin respectively,indicating that the decrease of the bacteria may aggravate the injury of the intestinal mucosal barrier.