Interleukin-17A Promotes Initiation And Development Of Colitis Fibrosis Through EMT

Objective:Crohn’s disease（Crohn’s disease,CD）is a cause of chronic nonspecific unclear inflammatory bowel disease,the disease was chronic,recurrent,and gradually formed the intestinal wall fibrosis,resulting in bowel function decline,even luminal stenosis,intestinal obstruction.Existing drugs can reduce inflammation and relieve symptoms,but can not relieve intestinal fibrosis.So explore the molecular mechanisms of pathogenesis of intestinal fibrosis strategies to delay or block the process of the development of fibrosis,has important practical significance.IL-17A is an inflammatory cytokine that is believed to be the expression of a variety of abnormal levels of acute and chronic diseases of the potential development of a diagnostic marker.Study confirmed that there are a lot of Th17 cell infiltration in the intestinal mucosa of the patients with CD,and IL-17A levels were significantly higher than normal mucosa.However,the development of IL-17A is involved in CD intestinal fibrosis currently rarely reported.This study investigated whether IL-17A participate CD-related intestinal fibrosis and its possible mechanism.Methods:1.Colonoscopy biopsies were collected from patients with a diagnosis of CD from August 2012 to March 2013 at the First Affiliated Hospital of China Medical University,a total of 14 cases.The 14 cases of patients with the clinical diagnosis of CD are chronic relapsing.Eight cases of control group was collected from patients with colonoscopy biopsies of normal mucosa which next to polyp.Detect the mRNA transcription and protein expression levels of IL-17A and EMT-related indicators of clinical tissue specimens.2 Cultured normal rat intestinal crypt epithelial cells IEC-6,use IL-17A and TGF-β1 to stimulate IEC-6,then observe the changes of EMT related indicators and fibrosis indicators.3.50 BALB/c mices were randomly divided into five groups,each 10,grouped as follows:（1）control group:normal diet,no special treatment;（2）TNBS model group:the 1^st and the 2ⁿdd week given to BALB/c mice TNBS 1.5mg/50%alcohol solution 100ul enema once a week;3^rdand 4^th week for BALB/c mice TNBS 2.0mg/50%alcohol solution 100ul enema once a week;5^thand 6^th week for BALB/c mice TNBS2.5mg/50%alcohol solution 100ul enema;（3）alcohol control group:gave the mice 50%alcohol solution enema 100ul once a week for 6 weeks.（4）anti-IL-17 neutralizing antibody treatment groups:administered to TNBS model group,and mice were given intravenous injection of prior to TNBS enema on the day of 22,29,36,and anti-IL-17A neutralizing antibodies 2μg/mouse;（5）IgG isotype control groups:administered to TNBS model group,and mice were given intravenous injection of IgG isotype antibody2μg/mouse on the day of 22,29,36 prior to TNBS enema.Saline control group and intestinal fibrosis model group mice were injected with an equal volume of saline.General observation of the mice during the experiment and calculating the change in DAI score in each group and evaluated the severity of colitis and fibrosis.After the end of the experiment,mice were sacrificed cervical amputation,abdominal anatomy,colon generally observed,measure the length.In the colon from the anus at 1cm 0.5cm long colon tissue clipping a few blocks part in 4%paraformaldehyde-fixed,paraffin-embedded sections,HE staining and Masson staining,I colon tissue frozen in liquid nitrogen,-80℃cryopreservation hydroxyproline content and Western blot analysis for detection.Results:1.IL-17A,vimentin and snail mRNA transcription levels in CD patients intestinal mucosa were significantly higher than control group,and E-cadherin mRNA transcript levels were significantly lower than the control group;IL-17A,vimentin and snail protein levels in CD patients intestinal mucosa were significantly higher than control group,and E-cadherin protein levels were significantly lower than control group.α-SMA protein level in CD patients intestinal mucosa was also significantly higher than control group.Correlation analysis between IL-17A mRNA transcript levels and E-cadherin,vimentin and snai mRNA transcript levels showed that IL-17A and E-cadherin mRNA levels were negatively correlated,and IL-17A and vimentin transcript levels were positively correlated.2.Stimulated IEC-6 by TGF-β1（10ng/ml）,and IL-17A（10ng/ml,50ng/ml and 100ng/ml）after 72h,the shape of cells turned to like mesenchymal cell.Immunofluorescence detected the expression of E-cadherin of IEC-6 cells after simulated,found that the expression of E-cadherin was significantly reduced compared with the negative control group.Western blot examination revealed that IL-17A can induce the expression of vimentin,snail,FSP1 andα-SMA,while inhibiting the expression of E-cadhenrin,wherein the effect of 100ng/ml IL-17A were the most significant effect in inducing EMT.3.mouse model of experimental colitis TNBS compared with the model group and treatment group IgG isotype,anti-IL-17A neutralizing antibodies in mice with experimental colitis symptoms most obvious,DAI score a minimum;anti-IL-17A treatment mice colon tissue pathology scores were significantly lower than the control group and the alcohol group;anti-IL-17A treatment group was significantly lower than the colon fibrosis score TNBS model group and IgG isotype control group;compared with the control group and the alcohol group,TNBS model group and the isotype IgG treated group Hyp significantly increased,while the anti-IL-17A antibody treatment group Hyp was significantly lower than TNBS model group and the isotype IgG antibody treatment group;compared with,the expression of E-cadherin in anti-IL-17A antibody treatment group was significantly higher than TNBS model group and isotype IgG treated group,while the expression vimentin,snail,andα-SMA in anti-IL-17A antibody treatment group was significantly lower than TNBS model group and isotype IgG treated group.Conclusion:1.IL-17A was involved in the development of CD-related intestinal fibrosis2.IL-17A promote the development of intestinal fibrosis possibly by inducing EMT...