Role Of Aquaporin 4 On DSS-induced Experimental Colitis In Mice

Ulcerative colitis（UC）is a type of chronic,recurrent and idiopathic inflammatory bowel disease.It mostly involves the sigmoid colon and rectum,and may extend to proximal colon in a continuous manner,or even the entire colon.UC usually afflicts 30 to 40-year-old adults and causes work disability.Over time,many patients with UC experience serious complications such as fibrosis,stenosis,fistulas,and even colorectal cancer.At present,the incidence and prevalence rates of UC have been on the rise in different areas throughout the world,indicating that it has become a global health problem.Exact etiology of UC remains largely elusive,involving with environmental factors,genetic and psychological predisposition,as well as microbial dysbiosis and impaired mucosal immune system.Changes in fluid flow across epithelial cells in UC patients also contribute to a―leaky‖intestinal epithelium,increasing susceptibility to immune-induced injury.Aquaporins（AQPs）represent a family of ubiquitous membrane proteins responsible for efficient and selective transport of water and other small molecule solutes across the hydrophobic cell membrane through osmotic gradients.Extensive data from transgenic mice have revealed AQPs,acting as water channel proteins,also involve in several biological events including inflammatory response,cell migration,adipocyte metabolism,and tumor-related edema.AQP4,as an isoform of aquaporins,plays an important role in regulating brain edema,neuroexcitation and human astrocytoma in the central nervous system.Accumulating evidence supports the abundant expression of AQP4 in human and animal intestine,and localization in the basolateral membrane of the colon epithelial cells.Several studies have shown that AQP4 mRNA and AQP4 protein expression levels are rapidly down-regulated under inflammatory conditions and slowly return to normal levels following cessation of inflammatory stimulation.These indicate that AQP4 is involved in the pathophysiological process of intestinal inflammation.However,the down-regulation of AQP4 expression may be the consequence of inflammation,or the defense mechanism against inflammatory reaction is not yet clear.Recently,the role of intestinal microbiota in IBD has attracted widespread attention.The interaction between gut microbiome and mucosal immune cells plays a crucial role in initiating and regulating the immune system.Studies have shown that abnormal microbial colonization of the intestine is an essential factor in driving inflammation and mucosal damage in UC,and certain microorganisms may aggravate inflammation,while some others may mitigate inflammation.Dysbiosis and decreased complexity in the intestinal microbial ecosystem are common characteristics of IBD patients.Data demonstrate that expression of AQP4 in ileum and colon is decreased in germ-free mice,but the level of AQP4 returns to normal upon bacterial colonization,which indicating that intestinal microbiota affect the expression of AQP4.However,it remains unclear whether AQP4 is involved in intestinal microflora in mice with colitis.In the present study,therefore,experimental colitis was induced in wild-type and AQP4 knockout CD1 mice to decipher the biological functions and related mechanisms of AQP4 in the inflammatory state.The potential role of the intestinal microbiota using 16S rRNA high-throughput sequencing in the AQP4-mediated colitis was also discussed.The findings preliminarily clarify the role and mechanism of AQP4 in colitis;deepen the understanding of the pathogenesis of UC,and provid a theoretical basis and experimental evidence for using AQP4 as a target for UC prevention and treatment.Part Ⅰ Effect and related mechanism of AQP4 on experimental colitis in CD1 miceObjectives:To decipher the biological functions and related mechanisms of AQP4in mice with the colitis.Methods:AQP4 deficiency(AQP4^-/-)mice are viable,fertile,and without any phenotypic defects in appearance.Eight-to 10-week-old male wild-type and AQP4^-/-mice（25³5g）were used for the experiments.To induce colitis,wild-type and AQP4^-/-mice were received 2.5%DSS dissolved in drinking water for 7 days followed by 5 days of regular drinking water.Mice in the control groups(wild-type and AQP4^-/-mice)received regular drinking water for 12 days.Mice were monitored daily for their body weight loss,stool consistency,and rectal bleeding throughout the entire experiment.On the 13th day of the experiment,the mice were sacrificed and blood samples,colon and feces were collected.Serum levels of TNF-α,IL-6 and IL-12 were also measured using the enzyme-linked immunosorbent assay.Colon length,and histological score were assessed.Expression of CD 3,CD177,F4/80 and p65 were determined by immunohistochemistry assay.Total protein,cytoplasmic protein and nucleoprotein of the distal colon specimen were extracted and the expression of p65 and p-p65（Ser536）were western blotted.Results:AQP4^-/-mice exhibited higher tolerance to experimental colitis compared to wild-type mice following administration of 2.5%DSS,as assessed by disease activity index（DAI）,colon length,tissue damage.Additionally,deletion of AQP4significantly reduced the serum levels of serum IL-6 and TNFα,as well as infiltration of CD3⁺T cells,CD177⁺neutrophils and F4/80⁺macrophages in colon-inflamed mucosa.A significantly increased abundance of the NF-κB p65 in WT mice caused by DSS treatment was observed.AQP4 deficiency markedly inhibited the expression of p65 in colonic inflamed epithelial cells and down-regulated the activation of NF-κB,which marked by translocation of subunit p65 from cytosol to nucleus.Meanwhile,WT mice treated with DSS displayed a sharp increased expression of phosphorylated p65（p-p65）in nucleus,as assessed by western blot.In contrast,AQP4 deficiency markedly curtailed the nuclear translocation of p65 in colonic tissues in response to DSS treatment.Conlusion:AQP4 plays a novel part in driving experimental colitis via NF-κB signaling pathwayPart Ⅱ Role of AQP4 for regulating intestinal microbiota in mice with colitisObjectives:To explore the potential role of the intestinal microbiota in the AQP4-mediated colitis.Metods:The contents in the colon of mice in each group were collected and total bacterial DNA was extracted.16S rRNA high-throughput sequencing upon Illumina MiSeq platform was used to analyse differences in intestinal microbiota.Results:There was no significant difference in relative abundance and diversity of intestinal microbiota between the AQP4^-/-and AQP4^WT groups treated without DSS.Compared with the AQP4^WT+DSS group,the Chao index,ACE index of the AQP4^-/-+DSS group increased（P<0.05）,the Shannon-wiener index increased（P<0.05）,and the Simpson index decreased（P<0.05）,indicating that alpha diversity in the AQP4^-/-+DSS group was significantly higher than that in the AQP4^WT+DSS group.RDA analysis indicated that environmental factors IL-6,TNF-a were related to gut flora in AQP4^WT+DSS group,while IL-12 was associated with AQP4^-/-+DSS.Additionally,the relative abundances of Lachnospiraceae,Lactobacillaceae,Ruminococcaceae,and Erysipelotrichaceae are significantly different among four groups,and Erysipelotrichaceae with greatest difference（P=0.01653）.AQP4^-/-significantly promoted the abundance of Erysipelotrichaceae and the Firmicutes/Bacteroidetes ratio in experimental colitis mice.Conlusion:AQP4 reduces the diversity of intestinal microbiota,the abundance of Erysipelotrichaceae and Firmicutes/Bacteroidetes ratio in mice with experimental colitic,aggravating colon inflammation.