A Study Of The Mechanism By Which G Protein-Coupled Estrogen Receptor Signaling Inhibits Intestinal Inflammation

Objective（s）:Inflammatory bowel disease（IBD）is a kind of chronic inflammatory lesion in the intestinal tract,including ulcerative colitis（UC）and Crohn’s disease（CD）.It is characterized by mucosal barrier damage and mucosal immune abnormalities,and its incidence is increasing worldwide every year,but its etiology is unknown.IBD has significant gender differences in its incidence,clinical manifestations and outcomes.The results of previous studies in our laboratory showed that macrophage subsets of the lamina propria in the intestinal mucosa expressed G protein-coupled estrogen receptor（GPER）.The expression of tight junction proteins（such as ZO-1,claudin-3,occludin,etc.）in intestinal epithelium of GPER knockout mice decreased.There were GPER⁺macrophage subpopulations in intestinal mucosa of CD patients.These suggest that the role of GPER in the regulation of intestinal mucosal barrier and immune inflammatory response is crucial,but the specific regulatory pathway is unclear.This study aims to futher elucidate the effect of GPER on intestinal inflammation and mucosal barrier,and the mechanism through which GPER is involved in the intestinal immune regulatory response,so as to provide new ideas for the targeted management of IBD-related diseases.Methods:Firstly,GPER fluorescence reporter mice（GPER-cre-td Tomato）were used to construct acute enteritis model with 3%DSS,then the distribution and changes of GPER⁺cells were observed.The changes of GPER protein and gene expression were detected after the wild type（WT）mice were modeled with DSS.GPER expression in intestinal tissue of UC patients were detected by immunofluorescence,Western blot and qPCR.Then,GPER gene knockout mice(GPER^-/-mice)and WT mice with/without DSS-induced acute colitis were used to detect intestinal phenotype and histological changes,and also the gene expression of proinflammatory factors（TNF-α,IL-6,IL-17）and anti-inflammatory factor（IL-10）.The distribution and proliferation of bacteria in WT mice and GPER knockout mice were observed by fluorescent in situ hybridization（FISH/RNAscope）.The changes of intestinal mucosal permeability after inflammation modeling were observed by FITC-dextran,and the changes of various inflammatory factors were detected by qPCR.The changes of inflammation condition after GPER agonist（G1）or antagonist（G15）treatment were also observed to determine the effect of GPER on intestinal inflammation and mucosal barrier.The concentration of estradiol,dehydroepiandrosterone and the expression of CYP1A1 and CYP11A1 proteins in colon of WT mice and GPER knockout mice were detected.The expression of Shh were detected after administration of G1 or G15 to GPER^-/-mice and WT mice.GPER-cre-td Tomato mice were used for immunofluorescence detection to observe the distribution and expression of Shh,Ptch and F4/80.The gene and protein expression of Shh,CYP1A1,CYP11A1,IL-6 and IL-10 were detected.Results:GPER⁺cells were increased and concentrated in the severe damaged mucosal side and lamina propria of intestine after the enteritis model was established in GPER fluorescence reporter mice.The expressions of GPER protein and gene were increased in WT mice with enteritis.The distribution area of GPER⁺cells and GPER expression in UC patients were consistent with those in mice.In the model of acute colitis induced by 3%DSS,GPER^-/-mice showed the most severe intestinal damage,increased intestinal mucosal permeability,excessive proliferation of intestinal bacteria,and increased expression of pro-inflammatory factors and decreased expression of pro-inflammatory factors by qPCR.Moreover,the intestinal inflammation of DSS mice was relieved after the agonist administration,while the intestinal inflammation was aggravated after the antagonist administration.Although GPER expression was up-regulated during inflammation,the synthesis of estradiol and dehydroepiandrosterone was limited and the concentration was decreased.Shh gene and protein expressions in WT mice were increased after G1 administration while decreased after G15administration.Immunofluorescence staining of colon samples from GPER fluorescence reporter mice revealed that Shh was distributed in the lamina propria of the intestinal mucosa.GPER⁺macrophages were found to express Shh.Furthermore,Shh⁺cells were found to be in close proximity to F4/80^high macrophages.Ptch⁺cells were observed in the intestinal mucosal lamina propria and muscular area,and were close to GPER⁺cells.Conclusions（s）:GPER deficiency will lead to aggravation of intestinal inflammation,the damage of intestinal barrier,the bacteria overgrowth and translocation to the intestinal wall.GPER activation can effectively inhibit intestinal inflammation.Although GPER expression is up-regulated following intestinal inflammation,GPER signaling might be compromised due to impaired production of endogenous ligands such as estradiol.GPER may regulate the barrier and inflammation through Shh signal pathway.