Mechanisms Of Innate Lymphocyte Flora Changes And Tissue Repair In Antibiotic-Associated Diarrhea

Objective.By establishing a mouse model of antibiotic-associated diarrhea(AAD),the proportion of bacteria in the feces of mice,the content of short-chain fatty acids(SCFAs)metabolite of bacteria and the damage of intestinal mucosal barrier in mice were observed;To determine the correlation between AAD and the changes of three groups of innate lymphoid cells(ILC3s)in intestinal mucosal innate immunity;To explore the mechanism that IL-22 produced by ILC3 s mediates the repair of intestinal epithelial cells(IECs)after AAD injury.Methods.1.The model of antibiotic-related diarrhea was established and divided into Nacl group(normal saline control group),AAD group(antibiotic continuous administration group),AAD+IL-22 group(antibiotic+IL-22 continuous administration group).The weight change,diarrhea rate,fecal water content and mortality of mice in each group were monitored.2.Collect the fecal samples of mice before and after AAD,detect the abundance change of intestinal flora through high-throughput sequencing,and detect the content change of short-chain fatty acids by gas chromatography-mass spectrometry.3.The mice were killed and fresh colon tissue was taken to prepare single cell suspension,and ILC3 s were quantitatively detected by flow cytometry.The content of IL-22 in colon tissue was detected by enzyme-linked immunosorbent assay(ELISA).4.The colon tissue of the killed mice was taken for HE staining and immunohistochemical staining,and the morphology of the colon tissue,the positive expression level of AQP8,Claudin-1 and other intestinal injury markers were observed under the microscope.5.The colon tissue of the killed mice was taken for real-time fluorescent quantitative PCR(RT-q PCR)and Western-blot to detect the intestinal injury related factors AQP8 and Claudin-1,the short chain fatty acid downstream gene GPR41 m RNA,and HIF1 α MRNA and phosphorylation level of STAT3 signal pathway related to intestinal epithelial cell repair.Results.Changes after AAD in mice of continuous antibiotic administration group(Group AAD):1.Compared with the normal saline group(Na Cl group),the mice in the continuous antibiotic administration group(AAD group)were in poor overall state,significantly reduced body weight,increased diarrhea rate and mortality,increased fecal water content,decreased intestinal microflora abundance,and decreased microbial metabolite-short chain fatty acid content.The difference was statistically significant(P<0.05).2.Fresh mice colon samples were taken to prepare single cell suspension,and the changes of ILC3 s were detected by flow cytometry.The results showed that the percentage of ILC3 s in all lymphocytes in AAD group was lower than that in Nacl group,and the difference was statistically significant(P<0.05);The content of IL-22 in fresh colon tissue was detected by enzyme-linked immunosorbent assay(ELISA).It was found that the level of IL-22 in the intestine of AAD group was lower than that of Nacl group,and the difference was statistically significant(P<0.05).3.The colon tissue of the killed mice was taken.It was found that the intestinal tube of AAD group was darker than that of Nacl group,and the elasticity of the tube wall was poor.After HE staining,the intestinal mucosa of AAD group was damaged,and the intestinal structure of Nacl group was intact.4.The mice were killed and the colonic tissues were taken.The expression of intestinal injury related factors AQP8 and Claudin-1 m RNA and protein were detected by immunohistochemical staining,PCR and Western-blot methods.The expression of intestinal Claudin-1 and AQP8 in AAD group was lower than that in Nacl group;Short chain fatty acid downstream gene GPR41,HIF1 α MRNA expression in AAD group was lower than that in Nacl group.The above differences were statistically significant(P<0.05).Changes after AAD in mice of antibiotic+IL-22 continuous administration group(group AAD+IL-22):1.The mice in AAD+IL-22 group were generally in good condition,with slight weight loss,lower diarrhea rate and mortality rate than those in AAD group,and the above differences were statistically significant(P<0.05).2.The colon tissue of the killed mice was taken.It was found that the color of the colon in AAD+IL-22 group was darker,and the elasticity of the wall was still poor.After HE staining,the microscopic observation showed that the intestinal mucosa damage degree of AAD+IL-22 group was better than that of AAD group.3.The mice were killed and the colonic tissues were taken.The expression of m RNA and protein of intestinal injury related factors AQP8 and Claudin-1 were detected by immunohistochemistry,RT-q PCR and Western-blot methods.The expression of Claudin-1 and AQP8 m RNA and protein in the intestine of AAD+IL-22 group was higher than that of AAD group;Western-blot method was used to detect the phosphorylation of STAT3 signal pathway related to the repair of intestinal epithelial cells.It was found that the expression of intestinal phospho STAT3/STAT3(STAT3phosphorylation)protein in AAD+IL-22 group was higher than that in AAD group(P<0.05).The above differences were statistically significant(P<0.05).Conclusions:1.When AAD occurs,the abundance of gut microbiota and SCFA content in mice significantly decrease,intestinal mucosal damage occurs,and the expression of Claudin-1 and AQP8 in the intestine is impaired.2.HIF1 downstream pathway of SCFA metabolite in mouse intestinal microbiota during AAD occurrence、 The expression of GPR41 decreased.3.The occurrence of AAD affects the innate immunity of the mouse intestine,resulting in a decrease in the number of ILC3 s and a significant decrease in the content of IL-22 in its intestinal tissue.4.Exogenous administration of IL-22 resists intestinal mucosal damage caused by AAD,upregulating the expression of Claudin-1 and AQP8 in the intestine,which may be repaired through the STAT3 phosphorylation mechanism.