Effect Of G Protein-coupled Estrogen Receptor(GPER) On Ulcerative Colitis And Its Mechanism

ObjectivesInflammatory bowel disease(IBD)includes Ulcerative Colitis(UC)and Crohn's Disease(CD).UC is a mucosal/submucosal chronic non-specific intestinal inflammatory disease with bloody diarrhea as the main clinical manifestation.Various factors such as genetics,environment,infection and immune factors participate in the occurrence and development of UC.With the changes of environment and living habits,the incidence rate in China has increased significantly in recent years,but the mechanism is still unclear.The G protein-coupled estrogen receptor(GPER,also known as GPR30)is a specific membranous estrogen receptor,participate in the functional regulation of the body under physiological and multiple pathological conditions.One clinical research have suggested that GPER may be involved in the development of IBD.However,until now there is no direct evidence to confirm the effect of GPER on UC.We designed this study to explore the role of GPER in UC and its potential mechanism.We look forward to providing experimental and theoretical basis for the discovery of new therapeutic targets for UC.Methods1.Establishment of UC model:Colitis was induced by feeding C57BL/6 male mice with 2.5%dextran-sulfate sodium(DSS)which was dissolved in the animals'drinking water for 7days.Control animals received only normal tap water.Experiment groups In vivo:According to the experimental requirements,animals were divided into four groups(control group,DSS group,DSS+ selective GPER agonist G-1 treatment group,DSS+G-1+ selective GPER blocker G15 treatment group)or three groups(control group,DSS group,DS S+G-1 group)2.Cell culture and treatment in vitro:The cells included Caco-2 cells(human cloned colon adenocarcinoma cells)and CCD841 cells(human normal colonic epithelial cells).Cells were cultured with high glucose DMEM containing 10%fetal bovine serum,streptomycin(100 ?g/ml)in a humidified culture incubator at 37? and 5%C02.Caco-2 cells were treated with 2.5%DSS to observe the expression of tight junction proteins,which were divided into three groups:control group,DSS group,DSS+G-1 treatment group.CCD841 cells were stimulated with thapsigargin(TG)to establish endoplasmic reticulum stress(ERS)model.The CCD841 cells were divided into three groups:control group,TG treatment group,TG+G-1 treatment group.3.Experiment in vivo:Mice were monitored daily for weight loss and stool consistency,fecal bleeding to analysis the disease active index(DAI).Colon length changes was evaluation on day 7.Hematoxylin-eosin staining was used to detect of intestinal histological changes.Detection of tight junction protein JAM-1,Occludin expression was tested with immunohistochemical method.Immunohistochemical detection of MUC-2 and PAS staining observed changes in goblet cells and mucus barrier of colonic epithelium.Four hours following intragastric administration of FITC-dextran,the colonic mucosal penneability was evaluated by plasma FITC-dextran concentration.The expression of Cleaved-caspase-3 in colonic epithelial cells was detected by immunohistochemistry.Apoptosis was also evaluated using the TUNEL assay.The expressions of apoptosis-related proteins Cleaved-caspase-3,Bcl-2 and Bax were detected by Western Blotting.The number of Ki67 and Brdu positive cells in crypt was recorded to evaluate the colonic crypt cell proliferation.Cell cycle protein CyclinD1,CyclinB1 expression was detected by Western Blotting.We also used Western blotting to detect the expression of ERS proteins GRP78,CHOP as well as the expression of three proteins sensors on the ER membrane(PERK,IRE1 ?,and ATF-6)and their active forms(p-PERK and p-IREla)in UPR.Immunofluorescence double-labeling technique was used to locate the expression of GPER in the colonic crypt.4.Cell culture in vitro:Immunofluorescence staining was used to detect the expression of JAM-1 in Caco-2 cells of different treatment groups in order to observe the changes of epithelial barrier in each group.We used Western Blotting to detect the content of endoplasmic reticulum stress-related proteins GRP78,CHOP,as well as the expression of the three proteins sensors on the ER membrane(PERK,IRE1 a,and ATF-6)and their active forms(p-PERK and p-IRE1 a))in UPR.The effect of G-1 treatment on TG-induced apoptosis in CCD841 cells was detected by flow cytometry.The proliferation of CCD841 cells was detected by CCK8 and EDU kits.Results1.GPER was expressed in colon crypts.Both the crypt transit amplifying(TA)region cells and the Lgr5+ stem cells expressed GPER.2.The DSS induced UC mice showed obvious bloody stools,body weight loss,colon length became shorter and increased disease activity index(DAI),whereas selective GPER agonist G-1 alleviated all these symptoms significantly colonic mucosal injury score and crypt score were significantly increased in UC model,which also could be ameliorated by G-1 treatment.The selective GPER blocker G15 treatment reversed all the protective effect of G-1 on UC.3.The expression of tight junction proteins JAM-1 and Occludin decreased in mice with UC.Colonic epithelial mucus barrier was impaired which indentified by PAS glycogen expression and decreased MUC-2 positive goblet cells.In line with the result from epithelial barrier the colonic mucosal permeability increased significantly in UC model.Selective GPER agonist G-1 treatment could improve colonic epithelial barrier injury significantly in mice with UC.G-1 treatment also protected Caco-2 cells in vitro from abnormal expression of tight junction protein JAM-1 induced by DSS.4.In mice with UC the expression of apoptosis-related protein Cleaved-caspase-3 was up-regulated and the ratio of Bcl-2/Bax was down-regulated.Immunohistochemistry showed that the number of Cleaved-caspase-3 positive cells and TUNEL positive cells increased significantly in colonic epithelial cells including crypts following DSS treatment.Intraperitoneal injection of G-1 could inhibited cell apoptosis induced by DSS in intestinal epithelial.5.In the DSS induced UC mice,the number of Brdu+ cells and Ki67+ cells was significantly decreased in crypt.The expression of CyclinD1 and CyclinB1 was decreased.These results suggested the proliferation ability of colonic crypt cells was impaired in UC mice.G-1 treatment ameliorated the proliferation of colonic crypt cells in mice with UC significantly,as well as the expression of CyclinD1 and CyclinB 1.6.In the DSS induced UC mice the expression of ERS marker GRP78,CHOP and UPR pathway protein ATF6 was up-regulated.The active forms of PERK,eIF2?,and IREla were significantly increased,too.Intraperitoneal injection of G-1 inhibited UPR in UC mice and relieved ERS.7.TG treatment induced obvious ERS in CCD841 cells.The expression of ERS proteins GRP78,CHOP and UPR pathway protein ATF6 was up-regulated,as well as the activities of PERK,eIF2a and IRE1?.G-1 treatment could down-regulated the UPR and inhibited ERS induced by TG.8.G-1 treatment could inhibit cell apoptosis induced by ERS in CCD841 cells.9.TG treatment inhibited the proliferation of CCD841 cells significantly.G-1 treatment improved proliferation in CCD841 cell induced by ERS.Conclusion1.GPER is expressed in colonic epithelium,cells in crypt of TA region and Lgr5+stem cells express GPER.2.GPER activation protected UC by protecting colonic epithelial barrier and improving intestinal mucosal permeability.The protective effect of GPER on UC was related to its anti-apoptosis of intestinal epithelial,promotion of colonic crypt cell proliferation and colonic epithelial regeneration.3.GPER activation down-regulated the UPR and inhibited over-activated ERS in UC.GPER helped to restore the balance of colonic epithelial apoptosis and regeneration via inhibiting ERS.