| ObjectiveInflammatory bowel disease(IBD)is a chronic inflammatory bowel disease with a prevalence of nearly 0.3%in the general population and an increasing trend by year.IBD is mainly classified into ulcerative colitis(UC)and Crohn’s disease(CD)according to the characteristics and sites of onset.UC mainly presents as diffuse inflammation of the intestinal mucosa with varying degrees of proximal spread in the rectum.The etiology and pathogenesis of UC are still unclear,the imbalance of intestinal homeostasis due to intestinal epithelial barrier dysfunction was thought to be one of the potential pathogenesis of UC.As the main cellular entity of the intestinal barrier,intestinal epithelial cells(IECs)are essential for maintaining intestinal homeostasis.Identifying the role of IECs in the development of UC may help to improve the understanding of the inflammatory process of UC and explore potential therapeutic targets.Irisin,a novel myokine released into the bloodstream,is produced by cleavage of the type Ⅲ fibronectin domain-containing 5(FNDC5)protein and has 100%homology between humans and mice.FNDC5/Irisin has been shown to exert its pleiotropic effects in different diseases.A number of studies in recent years have suggested that FNDC5/Irisin may be a key factor in the pathophysiological process of inflammatory diseases,but the role of FNDC5/Irisin in UC,especially in the active phase of UC,the potential effect and mechanism remain unclear.In this study,we first clarified the expression of FNDC5/Irisin in peripheral blood and intestinal tissues of UC patients as well as colitis mouse models.Subsequently,we evaluated the phenotypic alterations in intestinal inflammation,intestinal epithelial barrier,and mitochondrial function in FNDC5/Irisin knockout colitis mouse model.In addition,we observed the effects of exogenous Irisin on colitis mouse model,colonic organoid and intestinal epithelial cell lines and explored the possible underlying mechanisms.Methods and Results(A)Differences in the expression of FNDC5/Irisin in UC patients and colitis mouse models1.The expression of FNDC5/Irisin was increased in the peripheral blood of UC patients,but decreased in the intestinal tissuesIn this study,35 patients diagnosed with UC and 33 healthy volunteers were recruited and their peripheral blood Irisin expression was examined.The results showed that the expression level of Irisin was significantly higher in the peripheral blood of patients with moderately and severely UC,while the expression level of Irisin in the peripheral blood of patients with severe activity was higher than that of patients with mild activity.The results of Western blot showed that the expression of FNDC5/Irisin was significantly decreased in the intestinal tissues of UC patients.2.FNDC5/Irisin expression was elevated in the peripheral blood but decreased in the intestinal tissues of colitis mouse modelsIn vivo DSS-induced colitis model was constructed in mouse by DSS induction.The ELISA results showed that the expression of Irisin was significantly higher in the peripheral blood and decreased in the intestinal tissues of the colitis mouse models compared with the normal mouse,both at the mRNA and protein levels.(B)FNDC5/Irisin knockout attenuated intestinal inflammation and intestinal epithelial barrier damage in DSS-induced colitis mouse model1.FNDC5/Irisin knockout alleviated intestinal inflammation in colitis mouse modelMouse in the FNDC5-/-+DSS group showed less weight loss,lower DAI scores,and longer colon length compared to mouse in the wild type DSS group.Assessment of colonic inflammation in mouse by RT-PCR,ELISA,and HE staining revealed that FNDC5/Irisin knockout attenuated the disruption of colonic tissue structure and the expression of inflammatory factors in the colitis mouse model compared with mouse in the wild type DSS group.2.FNDC5/Irisin knockout ameliorated intestinal epithelial barrier damage in the colitis mouse modelWe examined the expression levels of the tight junction protein Occludin and the adhesion junction protein E-Cadherin by RT-PCR,Western blot and immunofluorescence.The results showed that the expression of Occludin and E-Cadherin was elevated in the FNDC5-/-+DSS group compared with the DSS group.AB-PAS staining results of mouse colon tissue slides showed that mucin was increased and the degree of mucus barrier damage was reduced in the FNDC5-/-+DSS group compared with the wild type DSS group.The results of FITC-Dextran gavage experiment showed that intestinal permeability was decreased in the FNDC5-/-+DSS group of mouse compared with the wild type DSS group,as evidenced by a decrease in the level of FITC-Dextran in peripheral blood.3.FNDC5/Irisin knockout attenuated apoptosis,ROS content and mitochondrial damage in the intestinal cells of colitis mouse modelThe results of TUNEL staining and Western blot of mouse colon tissue slides showed that the number of apoptotic cells in the intestine of FNDC5-/-+DSS group was decreased compared with the wild type DSS group.Meanwhile,the expression of key apoptotic proteins cleaved-caspase3 and Bax were decreased,and the expression of Bcl-2 was increased compared with the wild type DSS group.The results of DHE staining and the assay of antioxidant enzymes revealed that the content of intestinal ROS and MDA were decreased in the FNDC5-/-+DSS group compared with the DSS group,while the expression of antioxidant enzymes SOD,GSH-PX and CAT were increased.Finally,we focused on the effect of FNDC5/Irisin on the mitochondrial function of intestinal cells.Measurements of ATP content in mouse intestinal tissues showed that ATP content was elevated in the FNDC5-/-+DSS group compared to the wild type DSS group.Transmission electron microscopy results showed that the overall mitochondrial damage in the FNDC5-/-+DSS group was significantly less than that in the DSS group.(C)Irisin aggravates intestinal inflammation and epithelial barrier dysfunction by inducing mitochondrial ROS production and promoting epithelial cell apoptosis1.Exogenous irisin aggravated intestinal inflammation in colitis mouse modelTo clarify the direct effect of irisin on UC,we administered recombinant Irisin intraperitoneally to mouse at the same time of DSS modeling.The results showed that the exogenous administration of irisin caused a further decrease in body weight,a significant increase in DAI score and a further shortening of colon length in the colitis mouse model.Meanwhile,the results of HE staining of intestinal tissues and the expression level of inflammatory factors showed that exogenous Irisin further damaged the intestinal tissue structure and the expression of inflammatory factors was promoted.2.The intestinal epithelial barrier damage was further aggravated by irisinThe results of RT-PCR,Western blot and immunofluorescence showed that the expression of intestinal tight junction protein Occludin as well as adhesion junction protein E-Cadherin continued to decrease in the DSS+Irisin group compared with the DSS group,and the tight junction structure of some villi was completely lost.The AB-PAS staining results showed that exogenous Irisin injection intraperitoneally further aggravated the damage to the colonic mucus barrier.The results of FITC-Dextran gavage experiment showed that intestinal permeability was increased in the DSS+Irisin group of mouse compared with the DSS group,as evidenced by a increase in the level of FITC-Dextran in peripheral blood.3.Irisin inhibited the proliferation of intestinal epithelial cells and aggravated the impairment of epithelial barrier in vitroTo further clarify the effects of Irisin on intestinal epithelial cells in vitro,we constructed in vitro models of intestinal epithelial inflammatory injury by stimulating Caco-2 cell lines and colonic organoids with TNF-α/IFN-γ.CCK-8 results showed that high concentrations of Irisin treatment further reduced the survival rate of Caco-2 cells.In colonic organoids,immunofluorescence and Western blot results showed that high concentrations of Irisin treatment further inhibited the proliferation of Caco-2 cells in the context of intestinal epithelial inflammatory injury.Western blot results showed that in the context of intestinal epithelial inflammatory injury,high concentrations of Irisin treatment further inhibited the proliferation and survival of colonic organoids and disrupted the integrity of the intestinal epithelial barrier.4.Irisin promotes apoptosis of intestinal epithelial cells through activation of p38 MAPK and NF-κBIn this part,we found that exogenous Irisin intraperitoneally promoted apoptosis in the intestinal cells of colitis mouse model by TUNEL staining and Western blot.Antioxidant enzyme assay and DHE staining showed that exogenous irisin further promoted ROS and MDA production in the intestinal epithelial cells of colitis mouse model.In colonic organoid and Caco2 cell line,we also obtained similar conclusions that high concentrations of irisin promoted apoptosis in intestinal epithelial cells in the presence of intestinal epithelial inflammatory damage by TUNEL istaining,key apoptotic proteins detection and flow cytometry.Subsequently,we evaluated mitochondrial activity and ROS content in intestinal epithelial cells by DCFH-DA,MitoSOX,and MitoTracker staining,the results showed that high concentrations of irisin treatment decreased mitochondrial activity and promoted mitochondrial ROS production in intestinal epithelial cells.(D)FNDC5/Irisin promotes apoptosis in intestinal epithelial cells by binding to integrin αV/β5 and activating the p38 MAPK-NF-κB pathway1.The FNDC5/Irisin receptor integrin αV/β5 was mainly expressed in intestinal epithelial cells,and the expression was increased in UC.We first examined the expression levels of integrin αV/β5 in the intestinal tissues of colitis mouse models by Western blot and immunofluorescence.The results showed that integrin αV/β5 were mainly expressed in intestinal epithelial cells and are elevated in the intestinal tissues of the colitis mouse model.Similarly,the protein expression levels of integrin αV/β5 were significantly higher in the intestinal tissues of UC patients compared to healthy volunteers.2.Irisin receptor integrin αV/β5 expression was elevated after TNF-α/IFN-γtreatmentWe examined the expression of Irisin receptor integrin αVβ5 by Western blot,and the results showed that the expression level of integrin αVβ5 was elevated after TNF-α/IFN-γ stimulation in Caco-2 cell lines as well as in the colonic organoid.This result suggests that irisin exerts intestinal epithelial regulatory effects through integrinαVβ5 mediation.3.RNA sequencing analysis of intestinal epithelial cells in colitis mouse modelWe isolated and extracted colonic epithelial cells from Control,wild type DSS,FNDC5-/-and FNDC5-/-+DSS groups of mouse and performed RNA sequencing.Functional enrichment analysis of the differential expressed genes showed that FNDC5/Irisin knockout in UC mainly affected the epithelial cell response to ROS and regulation of epithelial cell apoptotic process.KEGG and GSEA enrichment analysis suggested that MAPK and NF-κB signaling pathways might be potential key pathways for FNDC5/Irisin to regulate the function of UC intestinal epithelial cells.4.Irisin promotes the phosphorylation level of p38 MAPK and NF-κB to promote the apoptosis of intestinal epithelial cellsWe verified the phosphorylation levels of p38 MAPK and p65 NF-κB in mouse model as well as in organoid level.The results demonstrated that the phosphorylation levels of p38 MAPK and p65 NF-κB were decreased in the intestinal epithelial cells of FNDC5/Irisin knockout colitis mice.In colonic organoids,Irisin treatment further promoted the phosphorylation levels of the above molecules in the context of TNF-α/IFN-γ stimulation,while p38-MAPK inhibitor led to a decrease in the number of apoptotic cells in the organoids caused by combined TNF-α/IFN-γ and Irisin treatment.ConclusionThe expression of FNDC5/Irisin was elevated in the peripheral blood of UC patients and in colitis mouse models.Irisin exacerbates mitochondrial damage and induces ROS production,activates the p38 MAPK-NF-κB pathway and promotes apoptosis in intestinal epithelial cells through binding to the receptor integrin αVβ5 in the context of colonic inflammation,thereby disrupting intestinal epithelial barrier function. |
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