Intestinal Flora Metabolite Milnacipran Reduces Intestinal Ischemia/Reperfusion Injury By Activating Aryl Hydrocarbon Receptor To Promote The Release Of IL-22 From Type 3 Innate Lymphoid Cells

Background:Intestinal barrier damage caused by intestinal ischemia/reperfusion(I/R)is one of the causes of perioperative death in patients,and intestinal flora and metabolites play an important role in maintaining intestinal barrier homeostasis.Early metabolomics results suggest that milnacipran is a potential metabolite of intestinal flora,but its role in intestinal I/R injury is currently unclear.The tryptophan metabolites metabolized by the intestinal flora can induce the activation of aryl hydrocarbon receptor(AHR)and promote the release of IL-22 from innate lymphoid cells(ILC3).IL-22 has been proven to regulate immune response and maintain the homeostasis of the intestinal mucosal epithelial barrier.However,the role of AHR/ILC3/IL-22 signaling in intestinal I/R is currently unclear.In this study,we hypothesized that milnacipran can activate AHR and promote the release of IL-22 from ILC3 to improve intestinal I/R damage.Methods:Use 16S rRNA gene sequencing and metabonomic sequencing technology to detect changes in intestinal flora and metabolites.In vivo and in vitro experiments,wild-type C57BL/6J mice(WT),intestinal epithelial AHR gene conditional knockout mice(AHRfl/fl/villin-Cre),IL-22-/-mice were used to establish mouse intestinal I/R model,mouse small intestine organoids and ILC3 co-culture system hypoxia/reoxygenation(H/R)model in vitro.To detect the 7-day survival rate of mice and HE staining of small intestine tissues after I/R,detect the viability of organoids and the level of lactate dehydrogenase(LDH)in the culture medium after H/R;to detect the expression of intestinal mucosal mechanical barrier markers Occludin and ZO-1,the expression of chemical barrier marker Muc2,the expression of immune barrier markers antibacterial peptides Reg3b and Reg3g,intestinal stem cell proliferation activity markers Lgr5 and Ki67 expression,Paneth cell marker Lyzl expression,enteroendocrine cell marker Chga expression,AHR activation marker Cyp1a1 expression and IL-22 levels,in mouse small intestine tissues and organoids.Results:The results of non-targeted metabolomics sequencing showed that compared with the sham group,the content of milnacipran in the cecum content of mice after intestinal I/R was significantly reduced.These results suggest that milnacipran is a potential metabolite of intestinal flora and may be related to intestinal I/R damage;then we used targeted metabolomics experiments to find that compared with the control group,the content of milnacipran in the cecum of mice in the antibiotic treatment group was significantly reduced;intestinal I/R caused a significant decrease in the milnacipran content in the cecum of mice,while the milnacipran content in the cecum was significantly increased in milnacipran-treated mice after intestinal I/R.The results of in vivo and in vitro experiments showed that milnacipran treatment significantly inhibited the decrease in survival rate of wild-type mice and organoid activity,the increase of HE pathological damage,the intestinal mucosal barrier disorder,the decrease in intestinal stem cell activity and proliferation,caused by intestinal I/R.But these protective effects of milnacipran on intestinal I/R injury were not found in AHRfl/fl/villin-Cre mice and IL-22-/-mice.In addition,we found that recombinant mouse-derived IL-22 factor reduced intestinal I/R damage by promoting STAT3 phosphorylation.Conclusion:Intestinal flora metabolite milnacipran reduces intestinal ischemia-reperfusion injury by activating aryl hydrocarbon receptor to promote the release of IL-22 from ILC3s.