The Protective Effect Of G Protein Coupled Estrogen Receptor (GPER) In Intestinal Ischemia Reperfusion Injury And Its Mechanism

Background and aimIntestinal ischemia reperfusion(I/R)injury is a common pathophysiological process in many severe diseases.The previous studies have found that estrogen has a protective effect on the intestinal I/R injury.However,the mechanisms are unclear so far.In this study,we proposed to define the type of receptor that mediates the protective effects of estrogen on intestinal I/R injury,and further to explore the potential mechanisms.We aimed to provide theoretical evidences for the new therapeutic targets at the intestinal I/R injury.Part oneMethods and contentHealthy male adult C57BL/6 mice were used for all animal experiments.After 45 minutes of intestinal ischemia via superior mesenteric artery clamping,the clip was removed to initiate blood reperfusion,followed by 2 hours,4 hours,24 hours and 72 hours reperfusion.Hematoxylin-eosin staining was used to evaluate the changes of jejunal intestinal mucosal injury at different time points.The estrogen was applied during the ischemic phase(when the intestine was been ischemia about 30 minutes)and then the effect of acute estrogen administration on the intestinal I/R was observed by HE staining and immunohistochemistry analysis.In order to determine the potential receptor that mediates estrogen's effect,both ICI 182,780,a selective blocker of estrogen nuclear receptor and G15,a selective blocker of GPER,were used.Following pre-treated with different blocker the protective effect of was evaluated.ResultsI.The intestinal I/R caused jejunal mucosal injury significantly.The top of the villi was damaged,the subepidermal space at the top of the villi was expanded,and the epithelium was separated with lamina propria on both sides of the villi,with neutrophil infiltrating and capillary congesting.The jejunal mucosa was severely damaged at reperfusion 2 hours and 4 hours,as evidenced by a significant decrease in the Chiu score compared with I/R vehicle group(n=4,p<0.05,vs Sham group).At 24 hours and 72 hours after reperfusion,jejunal mucosa was repaired and the damage score of jejunal mucosal is higher than the sham group,but there is no statistical significance.2.17?-estradiol(E2)applied during the ischemic period showed a tency to protect the jejunal mucosal injury induced by 2 hours reperfusion,while,the chiu score has no statistical significance compared with the I/R vehicle group.The treatment of E2 had a protective effect on jejunum mucosal injury induced by 4 h reperfusion,as evidenced by decresased Chiu score compared with the I/R vehicle group(n=4,p<0.05,vs I/R vehicle group).Thus,we choosed the mice undergoing 45 minutes ischemia followed by 4hours reperfusion as the I/R injury model in the subsequent experiments.3.Pretreatment with ICI 182,780,a selective blocker of estrogen nuclear receptor,did not affect the protective effect of E2 on jejunal mucosal injury in the intestinal I/R injury model.On the contrary,pretreatment of G15,a selective blocker of GPER,blocked completely the protective effect of E2 on the jejunal mucosal injury in the intestinal I/R injury model,the damage score of jejunal mucosal is significantly higher compared with I/R+E2 group(n=4,p<0.05,vs I/R+E2 group).4.After the intestinal I/R injury,the continuity of the linear expression of the zonal tight junction protein ZO-1 in the jejunal epithelium was interrupted or absent.Application of E2 during the ischemic phase significantly improved the abnormal expression and distribution of ZO-1 in the jejunal epithelium caused by I/R injury.The protective effect of E2 could be blocked by G15.In contrast,pretreatment with ICI182,780 did not affect the protective effect of E2 on the expression of ZO-1 in the intestinal I/R injury model.5.In the intestinal I/R injury model,the continuity and regularity of Occludin,a protein of tight junction protein,was lost.Application of E2 during the ischemic phase significantly improved the disorder distribution of Occludin in the jejunal epithelium caused by I/R injury.The protective effect of E2 could be blocked by G15,while ICI182,780 did not affect the protective effect of E2.Part twoMethods and contentIn health adult male C57BL/6 mice,the superior mesenteric artery was clamped for 45 minutes and reperfusion was performed for 4 hours to establish an animal model of intestinal I/R injury.Animals were divided into three groups:sham group,I/R vehicle group,and I/R+G-1 group(G-1,a selective agonist of GPER).The effect of the selective activation of GPER on the intestinal I/R injury was observed by HE staining,transmission electron microscopy,the vitality of myeloperoxidase(MPO),the assay of permeability of intestinal mucosal,expression of jejunal tight junction protein,The expression and localization of GPER in jejunal was examined by RT-PCR,western blot and immunofluorescence.HE staining and immune-histochemistry were applied to detect the effect of G-1 treatment on the proliferation of jejunal crypt stem cells in the intestinal I/R injury model.The effect of G-1 on the proliferation of jejunal crypt stem cells in the intestinal I/R injury model were detected by HE staining and immunohistochemical.Besides,by using immunofluorescence and western blot,we detected the effect of GPER activation on iNOS expression in intestinal I/R injury model.Then we further explored effect of iNOS inhibition on the proliferation of jejunal stem cells in the intestinal I/R injury model.In the end,the expression of endoplasmic reticulum stress-related protein GRP78 and CHOP was tested following pret-treated with G-1 in intestinal I/R injury model.Results1.G-1 treatment significantly improved the injury of jejunal mucosal induced by I/R(n=6,p<0.05,vs I/R vehicle group).Transmission electron microscopy showed that in intestinal I/R injury the boundaries between jejunal epithelial cells being unclear,lots of vacuolar-like changes in the cytoplasm and heterochromatin in the nucleus.Selective activation of GPER improved the damage caused by I/R injury.2.The activity of MPO in jejunum tissue increased significantly in I/R injury compared with the sham group(n=5,p<0.05,vs Sham group).G-1 treatment significantly inhibited increasing of MPO activity induced by I/R(n=5,p<0.05,vs I/R vehicle group).3.After intestinal I/R injury the expression of tight junction protein,ZO-1 and Occludin in jejunum epithelium was significantly impaired,in line with the increasing of intestinal epithelial permeability.G-1 treatment significantly improved I/R-induced jejunum intestinal mucosal barrier disruption induced by I/R.4.GPER was expressed in the jejunal epithelium,villous stroma and submucosa.Importantly,GPER was predominantly expressed in the jejunal crypts,in LGR5+ stem cells.5.Followed I/R injury,the ability of jejunal stem cell proliferation and epithelium regeneration were significantly inhibited.G-1 treatment significantly improved the downregulation of jejunal crypt stem cell proliferation and jejunal epithelial regeneration induced by I/R.6.Expression of iNOS in the jejunal crypts was significantly increased after I/R injury in the small intestine.G-1 treatment significantly decreased the expression of iNOS induced by I/R.The 1400w,a selective inhibitor of iNOS,treatment during the ischemic period significantly improved jejunal crypt stem cell proliferation and up-regulated epithelial regeneration and repair in I/R imjury model,which indicated that GPER might promote the stem cell proliferation by regulating iNOS expression.7.In intestinal I/R injury model,G-1 treatment significantly inhibited the high expression of GRP78 and CHOP induced by I/R injury in the jejunum,indicating that GPER activation alleviated I/R-induced endoplasmic reticulum stress.Conclusions1.45 minutes ischemia followed by 2 hours and 4 hours reperfusion caused siginificant intestinal mucosal injury.The estrogen played protective effect at the intestinal I/R injury via activating GPER.2.GPER showed obvious dominant expression in the jejunal crypts,including LGR5+ stem cells.GPER activation played a protective role against the intestinal I/R injury through inhibiting the inflammatory responses,protectiving jejunal mucosal barrier and improving the permeability of intestinal mucosa.The mechanism that GPER activation protected the I/R injury might be related to down-regulatiion of iNOS expression in crypt cell and inhibition of endoplasmic reticulum stress,which helped to enhance the proliferative capacity of the intestinal crypt stem cells and promot the intestinal epithelium regeneration and repair.