Protective Effects Of Irisin Against Sepsis-induced Intestinal Injury And Its Influence On Gut Microbiota In Mice

Objective:To investigate whether irisin has protective effects on sepsis-induced intestinal injury in Mice,and to explore possible mechanisms from the effects of irisin on intestinal barrier function and gut microbiota.Methods:Male C57BL/6 mice aged 8-12 weeks were randomly divided into three groups: control(Ctrl)group,sepsis(LPS)group,and sepsis + irisin(L.Iri)group.The Mice in the LPS group and L.Iri group were injected with lipopolysaccharide(LPS)10mg/kg intraperitoneally to establish a sepsis model,and the control group was intraperitoneally injected with an equal amount of saline.In the L.Iri group,1ug/kg of exogenous recombinant irisin(Pepro Tech100-65)was subsequently injected into the septic mice through the tail vein.The LPS group and the Ctrl group were injected with the same amount of saline through the tail vein.Ten mice in each of the three groups were used to observe the activity status during the 24 hours after being modeled and the 7-day survival of the mice.The surviving mice were sacrificed 24 hours after LPS injection,and their blood,colon tissues,and intestinal contents were harvested for subsequent analysis.The methods of Western blot and immunofluorescence staining were used to detect intestinal irisin/FNDC5 expression.The levels of inflammatory factors including IL-22,TNF-a,and IL-6 in serum,were measured by enzyme-linked immunosorbent assay(ELISA);the flora from peripheral blood was incubated on blood plates to observe bacterial translocation to assess the level of the translocation of intestinal microbiota.The intestinal histomorphology was observed by hematoxylin-eosin(H&E)staining.The expression of tight junction protein Occludin was measured by Western blot to evaluate intestinal barrier function.The intestinal Pro Caspase-3,Cleaved Caspase-3,Bax,Bcl-2 protein and apoptotic cells were respectively detected by Western blot and TUNEL method.Five samples of intestinal contents from each group were randomly selected,then fecal DNAs were extracted,and intestinal flora were analyzed by 16 S r DNA Amplicon Sequencing.The composition and abundance of intestinal microbiota of the in mice the three group were compared.The data were expressed as mean±standard deviation,and were analyzed by Oneway ANOVA.The difference in relative abundance between groups of intestinal flora was performed by Kruskal-Wallis method for non-parametric tests.The survival analysis used log-rank test Kaplan-Meier survival curve.P<0.05 was considered statistically significant.Results:The irisin/FNDC5 protein was highly expressed in the intestinal tissue of mice in the Ctrl group,and decreased in the LPS group,the difference was statistically significant(P<0.05),while the expression in the L.Iri group was significantly higher than that in the LPS group(P<0.05).There was no significant difference between the Ctrl group and the L.Iri group(P>0.05).The immunofluorescence of irisin/FNDC5 in the LPS group was weaker than that in the Ctrl group;while the fluorescence in the L.Iri group was stronger than that in the LPS group.All mice in the Ctrl group survived,two mice in the LPS group survived,and five mice in the L.Iri group survived.Compared with the Ctrl group,the 7-day survival rate of mice in the LPS group and the L.Iri group was significantly reduced(P<0.01 or P<0.05).The survival rate of mice in LPS group is 20%,and the survival rate of mice in L.Iri group(50%)was significantly higher than that in LPS group(P<0.05).Compared with the Ctrl group,the serum IL-22 decreased and the TNF-? and IL-6 increased in the LPS group,which had statistically significant differences(P < 0.01 or P < 0.05).Compared with the LPS group,the serum IL-22 increased and TNF-? decreased in the L.Iri group,and the difference was statistically significant(P<0.05 or P<0.01),but the difference of the IL-6 was not statistically significant(P>0.05).Compared with the Ctrl group,there was no statistically significant difference in serum IL-22,TNF-? and IL-6(P>0.05)in the L.Iri group.The results of peripheral blood incubation showed that the CFU of bacteremia in the LPS group was significantly increased compared with the Ctrl group(P<0.01),and the level in the L.Iri group was significantly lower than that in the LPS group(P<0.01)and not statistical different from the Ctrl group(P>0.05).H&E staining showed no obvious erosion loss in the intestinal mucosa of the three groups of mice: the colon mucosa epithelium of mice in the Ctrl group was intact,the goblet cells and glands were arranged regularly,and there was no infiltration of inflammatory cells;Mice in the LPS group showed reduced colonic epithelium goblet cells,disordered and deformed glands,damaged tight junction structures between intestinal epithelial cells,and extensive infiltration of inflammatory cells;The colonic epithelium of mice in the L.Iri group is basically intact,and the arrangement of goblet cells and glands is basically normal.The degree of inflammatory cell infiltration is also less than that in the LPS group.The expression of Occludin protein in the colon tissue of mice in the LPS group was lower than that in the Ctrl group and the L.Iri group(P<0.05 or P<0.01).Compared with the Ctrl group and L.Iri group,the expressions of apoptosis-related proteins Cleaved Caspase-3 and Bax in LPS group were increased(P<0.05 or P<0.01);Pro Caspase-3 and Bcl-2 were lower in the LPS group than those in Ctrl group and L.Iri group(P<0.05 or P<0.01);there was no significant difference in the expression of the above proteins between the Ctrl group and the L.Iri group(P>0.05).Compared with the Ctrl group and the L.Iri group,the positive rate of apoptotic cells in the LPS group was significantly increased(P<0.01);there was no significant difference in the positive rate of apoptotic cells between the Ctrl group and the L.Iri group(P>0.05).16 S r DNA Amplicon Sequencing of the intestinal flora revealed at the phylum level,and the relative abundance of Bacteroidetes and Firmicutes was no significant difference in the three groups(P>0.05).Proteobacteria,Verrucomicrobia and Deferribacteres in the LPS group increased significantly compared with the Ctrl group(P <0.05 or P<0.01).The levels in the L.Iri group were not statistically different from those in the Ctrl and LPS groups(P>0.05).There was no significant difference(P>0.05)in the relative abundance accounting for the top two of the family level in all the three groups,namely Muribaculaceae and Erysipelotrichaceae.In the LPS group,Lactobacillaceae were significantly reduced compared to the Ctrl group(P<0.05);Enterobacteriaceae and Akkermansiaceaewere significantly increased compared to the Ctrl group(P<0.05);There was no statistical difference between the Ctrl group and the L.Iri group(P>0.05),as well as between the LPS group and the L.Iri group(P>0.05).Conclusion:1.Irisin/FNDC5 in colon tissues is mainly expressed in intestinal epithelial cells.Sepsis can lead to decreased its expression,so intestinal irisin/FNDC5 may be a target for clinical treatment of sepsis.2.Irisin can inhibit the inflammatory response of sepsis mice,stabilize the intestinal barrier integrity and intestinal barrier function,reduce bacteremia caused by intestinal bacteria translocation,attenuate intestinal damage and reduce sepsis mortality.3.Irisin can normalize the gut microbiota structure of sepsis mice,and regulate the relative abundance between probiotics and harmful bacteria,thus decreasing the occurrence and development of sepsis.