Expression Of Endoplasmic Reticulum Stress Proteins And Glycosylated Protein Mucin2 In Mouse Colitis

Background: Inflammatory bowel disease(IBD) is a chronic nonspecific intestinal inflammatory disease, including ulcerative colitis(UC) and Crohn’s disease(CD). With the improvement of living conditions and the changes of environment recently, the cases of the disease reported were increased significantly in our country.Although the etiology and pathogenesis of inflammatory bowel disease remains unclear, it is believed that the disease is related to many factors, including heredity,immunity, environment and microorganism.Endoplasmic reticulum(ER) is an important organelle for the proteins’ synthesis,folding and secretion in eukaryotic cells. Many factors, such as lack of nutrition,oxidative stress, etc, can cause ER homeostasis disorder, declining the ability of ER,and resulting in the unfolded and misfolded proteins to accumulate in the ER which lesd to ER stress. When appears ER stress, a series of response are launched to adapt to the stress, this process is called “unfolded protein response(UPR)”. Through three signal pathways, UPR regulated sensor proteins assist protein folding, limits protein translation in order to reduce ER load, accelerate the degradation of misfolded proteins to restore the ER homeostasis. When ER stress last too long it will lead to cell apoptosis and tissue damage. As high secretory cells, intestinal epithelial cells updates constantly, and sensitive especially to ER stress.Recently, the ER stress was found to be involved in pathogenesis of varieties of diseases. Multiple pathways of the unfolded protein response(UPR) induced by ER stress are associated with the inflammatory process. ER stress was also believed to participate in the pathogenesis of IBD, but the mechanism of ER stress in IBD is unclear.Damage of intestinal mucosal protective barrier is a key factor of ulcerative colitis. The mucous layer which was constituted of a large number of glycosylated proteins is an important chemical barrier in intestinal epithelium surface. Mucin2(MUC2) produced by goblet cells is the main macromolecular structure of mucous layer. Studies have shown that abnormal expression of MUC2 is intimately linked with intestinal disease.This study used the dextran sulphate sodium(DSS) to induce mouse colitis model.The mice colon tissue was used to evaluate the protein and/or mRNA expression of ERstress chaperone molecules and a mucous protective glycosylated proteins, MUC2.The significance about the changes of these molecules protein and mRNA expression in the pathogenesis of IBD were analysed.Objective : To evaluate the expression of ER chaperone molecule GRP78,IRE1α, IRE1β, PERK, ATF6, XBP1 u, XBP1 s, CHOP and MUC2 in mice colitis induced by different concentrations of DSS.Methods:Eight-week-old C57BL/6 mice were randomly divided into three groups(n=10 for each group): 1.5%DSS group and 2.5%DSS group were given with drinking water dissolved with 1.5%(1.5g/100ml) and 2.5%DSS for 6 days respectively to establish acute mouse colitis; control group was given with only drinking water.Disease Activity Index(DAI), colon length, inflammatory pathological score of colon tissue were observed. Inflammatory cytokine, TNFα and IL-6, mRNAs were measured by quantitative real-time(RT) PCR. The protein expression of GRP78, IRE1β, PERK,ATF6 and MUC2 in colon tissue of mice were evaluated by immunohistochemistry.The mRNA expression of GRP78, IRE1α, IRE1β, PERK, ATF6, XBP1 u, XBP1 s,CHOP and MUC2 were detected by RT-PCR.Results:1. There was mild colitis in 1.5%DSS group and severe colitis in 2.5%DSS group,but no colitis in control group. TNFα and IL-6 mRNAs were increased in 1.5%DSS group, further increased in 2.5%DSS group(both P<0.05).2. Immunohistochemical results: GRP78, IRE1β, PERK, ATF6 and MUC2 in control group were found in colonic epithelial cells, the expression of protein IRE1β, PERK,ATF6 and MUC2 were decreased in 1.5%DSS group(all P<0.05); the expression of protein GRP78, IRE1β, PERK, ATF6 and MUC2 were all decreased significantly in2.5%DSS groups(all P<0.05); the difference of expression of IRE1β, PERK, ATF6 and MUC2 between 2.5%DSS group and 1.5%DSS groups is not significant.3. The mRNAs expression of GRP78, IRE1α, IRE1β, PERK, ATF6, XBP1 u, XBP1 s,CHOP and MUC2 were found in control group. The mRNAs expression of GRP78 and CHOP in 1.5%DSS group were no change, but were decreased significantly in2.5%DSS group(all P<0.05); XBP1 u mRNA was no difference among the three groups; IRE1α, IRE1β, PERK, ATF6, XBP1 s and MUC2 m RNAs were down-regulated in 1.5%DSS and 2.5%DSS group(all P<0.05), but shown no obvious difference in 2.5%DSS and 1.5%DSS group.Conclusions:1. ER stress chaperone molecule GRP78, IRE1α, IRE1β, PERK, ATF6, CHOP,XBP1 u and XBP1 s play important roles in maintaining of the physiological of colonic epithelium.2. Under inflammatory condition the decreased expression of GRP78, IRE1α, IRE1β,PERK, ATF6, CHOP and XBP1 s may lead to the attenuation of ER stress and unfolded protein response, and it may contribute to pathogenesis of mouse colitis.3. Damage of intestinal mucosa caused by the reduced expression of MUC2 contributes to the intestinal inflammation.