SCFAs(Butyric Acid)Regulate Autophagy In Intestinal Epithelial Cells And Relieve Colitis By HIF-1α

BackgroundInflammatory bowel disease is a class of chronic nonspecific diseases involving the intestines of unknown etiology,including Crohn’s disease and ulcerative colitis.In recent years,with the wide application of genome-wide association study,several autophagy-related genes and their single nucleotide polymorphisms have been proved to be associated with IBD.Autophagy is a highly conservative evolutionary process in eukaryotes,which maintains intracellular dynamic balance by degrading and absorbing damaged organelles,useless proteins and aging nucleic acids.The pathological progress of IBD is closely related to autophagy.The lack of autophagy of intestinal epithelial cells in patients with IBD destroys the normal physiological balance of intestinal epithelial cells.There is a"physiological hypoxia"area between the oxygen-rich intestinal lamina propria and the anoxic intestinal lumen,and the idiopathic anaerobes located in this area maintain the"physiological hypoxia"environment of the intestine by increasing oxygen consumption through metabolites.Microcirculatory hypoxia is an important feature of IBD.Microvascular occlusion and thrombosis aggravate the hypoxia of intestinal epithelial cells under the condition of IBD.Hypoxia inducible factor-1αis a transcription factor that regulates a variety of genes under intracellular hypoxia.It can induce neovascularization,promote the expression of various related genes,and make cells adapt to hypoxia.More interestingly,intestinal"physiological hypoxia"can induce intestinal autophagy,thus preventing the invasion of pathogens.Therefore,we speculate that HIF-1αis closely related to autophagy.Some studies have found that the lack of HIF-1αin the intestinal epithelium of IBD patients and mice can lead to intestinal microecological disorders,mainly reflected in the decrease in the number of bacteria producing short-chain fatty acids and the decrease of SCFA content in the intestinal cavity.SCFA is a product of dietary fiber decomposed and fermented by intestinal bacteria,including acetic acid,propionic acid and butyric acid.SCFA,especially butyric acid,is the main energy source of intestinal epithelial cells.More and more studies have shown that butyric acid not only has anti-cancer and anti-inflammatory effects,but also can regulate intestinal water and electrolyte balance,and plays an important role in maintaining the dynamic balance of intestinal mucosal barrier.Previous scholars mainly believed that SCFA relieves symptoms by regulating intestinal immune function.However,recently,some scholars have proposed that SCFA can stabilize HIF-1αand make HIF-1αplay a role by increasing the oxygen consumption of intestinal epithelial cells and causing moderate hypoxia of intestinal epithelial cells.Therefore,this study assumes that the intestinal flora is disordered under the condition of IBD,which leads to the decrease of SCFA production and the relative deficiency of HIF-1αactivation in intestinal epithelial cells.By supplementing butyric acid in the intestine,the activation of HIF-1αin intestinal epithelial cells can be increased,thus the level of intestinal autophagy can be up-regulated,the disturbance of flora can be corrected,and intestinal injury can be recovered finally.In this study,we established a mouse colitis model induced by sodium dextran sulfate,and used immunohistochemistry,qPCR,Western Blot and transmission electron microscopy to explore the role of intestinal epithelial-derived HIF-1αin mouse colitis and the role of intestinal epithelial autophagy in the maintenance of intestinal microecology,to clarify the molecular mechanism of butyric acid in relieving colitis in mice,and to provide a new theoretical basis for the repair of intestinal mucosal barrier injury.Methods:1.The intestinal epithelial cell specific knockout HIF-1αmice(HIF-1α^?IEC mice)and their litter negative control mice(HIF-1α^loxP/loxP mice)were established to establish the colitis model induced by DSS and the colitis model induced by butyric acid fed with DSS.The weight of mice was measured,the fecal properties were observed,and the disease activity index,HE staining was used to observe the severity of colonic inflammation and to evaluate the protective effect of butyric acid on the intestines of mice.2.The expression of autophagy-related proteins ATG16L1,p62 and LC3 in intestinal tract of mice was observed by immunohistochemistry,WesternBlot,immunofluorescence and transmission electron microscope.3.The changes of intestinal flora in the feces of mice in each group were detected by 16S rDNA high-throughput sequencing,and the content of short-chain fatty acids in the feces of mice in each group were detected by high performance liquid chromatography（HPLC）.4.siRNA technique was used to interfere the expression of HIF-1αgene in human colon adenocarcinoma cell line HT29 cell and to observe the effect of butyric acid on autophagy of HT-29 cell under different conditions.Results:1.HIF-1α^?IEC mice were more sensitive to chemical damage caused by DSS,which was mainly characterized by more severe weight loss,shorter colon length,higher inflammatory score and higher disease activity index in HIF-1α^?IEC mice.2.Compared with HIF-1α^loxP/loxP mice,the expression of ATG16L1 and LC3 protein in intestinal epithelium and autophagosome in intestinal epithelium of HIF-1α^?IEC mice decreased significantly,while the expression of p62 protein increased.3.Compared with HIF-1α^loxP/loxP mice,the intestinal flora of HIF-1α^?IEC mice changed from anaerobes to facultative anaerobes,and the content of SCFA in feces decreased,which was consistent with the change trend of intestinal flora and its metabolites in mice after chemical injury induced by DSS.4.Under normal conditions,butyric acid of SCFA can promote the expression of ATG16L1 and LC3 proteins in HT-29 cells.However,after interfering with HIF-1αgene of HT29 cells by siRNA technique,the promoting effect of butyric acid on ATG16L1 and LC3proteins was significantly decreased.5.After DSS-induced colitis model mice were fed with butyric acid,the weight loss of HIF-1α^loxP/loxP mice became slower,and the inflammatory index and disease activity fraction decreased due to the shortening of colon length.However,compared with HIF-1α^loxP/loxPoxP/loxP mice,butyric acid significantly reduced the relieving effect of butyric acid on intestinal injury in HIF-1α^?IEC mice induced by DSS.Conclusion:1.HIF-1αplays an important role in the maintenance of intestinal homeostasis in mice.The lack of HIF-1αin intestinal epithelium will affect the intestinal microecological balance and increase the sensitivity of intestinal tract to injury;2.Under normal conditions,butyric acid of SCFA can promote autophagy of HT-29cells,and the ability of autophagy induced by butyric acid is decreased due to the lack of HIF-1α;3.Butyric acid can regulate autophagy in intestinal epithelium and relieve colitis by HIF-1α.