Establishment Of Mouse Model Of DSS-induced Inflammatory Bowel Disease And Exploration Of Its Mechanisms

Inflammatory bowel diseases（IBD）,including Crohn’s disease（CD）and ulcerative colitis（UC）,are characterized by chronic intestinal inflammation and tissue damage.IBD are common in worldwide and have a natural course characterized by alternating periods of remission and relapse.Significantly,patients with IBD have an increased risk of colorectal cancer（CRC）in the future.The etiology of IBD ramains unknown,and there is still no effective way to treat IBD.However,recent studies have implicated the impaired intestinal epithelium which caused by the imblance between pro-and anti-inflammatory cytokines maybe the important pathogenesis of inflammatory bowel disease.Here,we established the mouse model of DSS-induced inflammatory bowel disease and observed the pathological injuries in IBD,which would help to find the pathogenesis of inflammatory bowel disease.Objective:To observe the changes of pro-and anti-inflammatory cytokines and the damage of intestinal epithelium and Lgr5⁺intestinal crypt stem cells by establishing the mouse model of DSS-induced inflammatory bowel disease.Methods:The male heterogyzous mouse of B6.129P2-Lgr5^{tm1（cre/ERT2）Cle}/J were fed3%（wt/vol）DSS dissolved in distilled water ad libitum from day 0 to 7 to induce inflammatory bowel disease.Animal body weight loss,fecal consistency,blood in the stool and the resultant IBD disease activity index were monitored daily during DSS induction stage.Mouse were sacrificed on the 7th day after administeration of DSS.The changes of intestinal morphology were observed and the length of intestinal were measured.The middle of the small intestine and the whole colon were removed and fixed in freshly prepared 4%（w/v）paraformaldehyde,processed,embedded in OCT and cut into sections;The pathological changes such as structure of intestinal crypt-villus,length of villus were analyzed by hematoxylin-eosin staining.Levels of IL-6,TNF-α,IFN-γ,IL-12p70,MCP-1and IL-10 in serum were measured by Cytometric Bead Array.The histopathological technique and laser scanning confocal microscopy were used to analyze changes of Lgr5⁺intestinal crypt stem cells in the colon.The changes of Lgr5⁺intestinal crypt stem cells in the suspension of single cell of colon were measured by Flow Cytometry.Results:Compared with the normal mouse,DSS-treated mouse were obvious weight loss,diarrhea,blood in the stool and the DAI socre also significantly increased with morphological changes such as surface erosion,ulceration,congestion.The length of small intestine was shorter（28.17±2.62）cm in DSS-treated mouse than（32.08±2.18）cm in normal mouse（P<0.05）.The colonic length was shorter（3.72±0.68）cm in DSS-treated mouse than（5.60±0.37）cm in normal mouse（P<0.01）.Hematoxylin-eosin staining revealed that DSS treatment caused injuries to the intestinal tissues,which resulted in the deformation of crypt architecture,infiltration of inflammatory cells and disorganization of villus.The length of villus was significantly shorter（196.80±39.44）μm in DSS-treated mouse than（358.40±24.50）μm in normal mouse（P<0.01）.The serum TNF-αlevels was higher（18.52±3.08）pg/ml in DSS-treated mouse than（10.88±2.18）pg/ml in normal mouse（P<0.01）.The serum IL-6 levels was higher（17.44±6.85）pg/ml in DSS-treated mouse than（5.40±1.86）pg/ml in normal mouse（P<0.01）.The serum IL-12p70 levels was lower（8.62±2.65）pg/ml in DSS-treated mouse than（30.67±19.92）pg/ml in normal mouse（P<0.05）.The serum IL-10 levels was lower（21.27±6.77）pg/ml in DSS-treated mouse than（93.29±60.28）pg/ml in normal mouse（P<0.05）.The serum IFN-γlevels was（3.11±0.49）pg/ml in normal mouse and（3.68±0.38）pg/ml in DSS-treated mouse,and there was no significant change（P=0.051>0.05）.The serum MCP-1 levels was（74.55±11.59）pg/ml in normal mouse and（62.82±7.39）pg/ml in DSS-treated mouse,and there was no significant change（P=0.06>0.05）.Histological evaluation revealed that the number of Lgr5⁺intestinal crypt stem cells in colonic crypt was less（1.0±1.0）/HP in DSS-treated mouse than（4.5±1.0）/HP in normal mouse（P<0.05）.The results of Flow Cytometry suggested that the proportion of Lgr5⁺intestinal crypt stem cells in the suspension of single cell of colon was lower（1.96%）in DSS-treated mouse than（4.10%）in normal mouse.Conclusion:We confirmed the imbalance between pro-and anti-inflammatory cytokines and the damage of intestinal epithelium in IBD,and further to find that Lgr5⁺intestinal crypt stem cells are seriously damaged or even deleted during IBD pathology,which provide a clue to investigate the pathogenesis and the effective treatment of IBD.