The Role Of Calcium Activated Chloride Channel ANO1 In Regulating Bacteria-bile Acid Metabolism In Mice Colitis

BackgroundInflammatory bowel disease（IBD）is a chronic immune-mediated intestinal inflammatory disease,which clinically contains Ulcerative colitis（UC）and Crohn’s disease（CD）.The main symptoms of the patient are diarrhea,abdominal pain,bloody stool and weight loss.Long-term inflammation can affect the whole body organs.Moderate to severe long-term ulcerative colitis is one of the high risk factors of colon cancer.The pathogenesis of IBD is complex,which is related to genetic and environmental factors.The imbalance of interaction between intestinal microbiota and intestinal immunity is one of the important pathogenesis of IBD.A large number of studies on clinical subjects have shown that there are differences in intestinal microbiota between IBD patients and healthy individuals.The consistent observation of changes in intestinal microbiota composition in IBD patients is that microbial diversity is reduced,Firmicutes are reduced and Proteobacteria are increased.Some studies also found that the levels of Bacteroides、Clostridium、Ruminococcus、Bifidobacterium、Lactobacillus and Fecalbacilli in patients decreased,while the levels of Escherichia coli and Fusobacterium increased.Calcium activated chloride channel ANO1（anostatin-1）,also known as TMEM16A or DOG1,is an ion channel composed of ten transmembrane domains,which is widely expressed in a variety of cells,including epithelial cells,airway and vascular smooth muscle cells,vascular endothelial cells,myocardial cells,Cajal interstitial cells,etc.,and plays a regulatory role in the occurrence and development of breast cancer,head and neck squamous cell carcinoma,esophageal cancer,gastric cancer,colorectal cancer and other cancers.As a chloride channel,the main physiological function of ANO1 is to assist in regulating the mucus secretion of airway or digestive tract epithelial cells.Intestinal epithelial cells secrete mucus and build mucus barrier,which is crucial for the colonization,migration and migration of intestinal microbiota and is an important barrier to prevent and control the invasion of pathogenic microbes.This study intends to explore the regulatory role of ANO1 on intestinal microbiota,as well as its role and corresponding mechanism in IBD.ObjectiveThis study intends to build a mice colitis model to simulate human UC:1.To explore the regulatory effect of ANO1 on intestinal microbiota in mice.2.To explore the role and mechanism of ANO1 in mice colitis.Methods1.Experimental animals and animal model preparation:Wild-type SPF grade C57BL/6J mice（male,6-8W,18-22g）were purchased from Liaoning Changsheng Biotechnology Co.,Ltd.（license number:SCXK（Liao）2020-0001）.Cre-loxp system was used to construct ANO1 intestinal epithelial cells-specific knockout mice(ANO1^IEC-KO or ANO1 knockout)by mouse hybridization.The mouse colitis model was prepared by drinking 2.5%dextran sodium sulfate（DSS）freely for seven days.Detect the disease activity index（DAI）,colon length,colon histology,and intestinal mucosal barrier to determine whether the model preparation is successful（ethics number:AEE20045）Pseudo-germ-free mice（PGF）were constructed by free drinking mixed antibiotics（ampicillin,neomycin sulfate,metronidazole,vancomycin）.2.Effect of ANO1 on intestinal microbiota in miceThe composition of intestinal microbiota was detected by 16s RNA gene sequencing.Based on the fluorescence in situ hybridization（FISH）protocol,the invasion of intestinal microbiota to the upper cortex was explored.3.Effect of ANO1 on bile acid metabolism in miceThe changes of bile acid metabolism in mice colon contents were detected by bile acid metabolomics.Content collection:After the mice were killed with CO₂,all intestinal segments from the anus to the cecum were cut,cut longitudinally,and the content particles were quickly removed,and frozen with liquid nitrogen.Results1.ANO1 knockout significantly increased the severity of colitis in mice:ANO1^IEC-KOmodel group showed more severe colitis symptoms,with DAI significantly increased（P<0.05）,colon length shortened（P<0.01）,and colon histological score significantly increased（P<0.05）.The results showed that ANO1 intestinal epithelial cells-specific knockout could significantly increase the susceptibility of mice to DSS-induced experimental colitis.2.ANO1 knockout affects the secretion and differentiation of goblet cells and damages the intestinal barrier:The intestinal mucus layer of ANO1^IEC-KO mice became thinner（P<0.001）,the expression of intestinal tight junction protein occludin was decreased,and the blood recovery rate was significantly increased after intragastric administration of fluorescent dye FD-4（P<0.001）.After modeling,the thickness of mucus layer in ANO1^IEC-KO model group decreased significantly（P<0.05）.After ANO1 knockout,the secretion of mucin MUC2 in the colon of mice decreased,and the levels of transcription factors Hes1（p<0.01）,Klf4（p<0.001）,Math1（p<0.001）,Spdef（p<0.001）of goblet cell differentiation and maturation decreased.The results suggest that the specific knockout of ANO1 not only destroys the secretory function of goblet cells,but also inhibits their differentiation and maturation.3.ANO1 knockout leads to intestinal bacterial disorder and bacterial invasion to epithelial cells.Compared with WT control group mice,the alpha diversity and beta diversity of intestinal microbiota decreased（P<0.01）.The ratio of Firmicutes to bacteroides decreased.At the genus level,the beneficial bacteria such as Enterorhabdus and Turiciactor（P<0.01）decreased significantly,and the invasion depth of bacteria to the upper cortex increased.After the intestinal microbiota of ANO1^IEC-KO mice was transplanted into PGF mice,the severity of colitis in mice increased significantly.4.ANO1 knockout changes bile acid metabolism of intestinal microbiota,leading to intestinal inflammation:The bile acid metabolomics test showed that the total bile acid profile of the feces of the ANO1 knockout mice increased,the taurine conjugated bile acids increased（P<0.05）,23 bile acids were significantly up-regulated,and 3 bile acids were significantly down-regulated.ConclusionThis study proved that ANO1 intestinal epithelial cells-specific knockout can increase the symptoms of colitis in mice,destroy the secretion function of goblet cells,reduce the differentiation of goblet cells,and lead to the damage of intestinal mucus barrier,and then lead to the disturbance of intestinal microbiota and the increase of bacterial invasion depth to the upper cortex.The disorder of intestinal microbiota caused by ANO1 can lead to the imbalance of intestinal microbiota-bile acid metabolism induce intestinal inflammation,and aggravate the severity of colitis in mice.This study provides scientific basis and theoretical basis for revealing the role of ANO1in the occurrence and development of IBD.