Role And Mechanism Of G Protein Coupled Estrogen Receptor (GPER) In The Maintenance Of Intestinal Stem Cells Homeostasis

ObjectivesIntestinal epithelium is indispensable for the maintenance of balance between internal and external environment,which is often exposed to the damage brought by chemical regimens,microbia and immunological factors.Intestinal epithelial regeneration relies on the self-renew,proliferation and differentiation of intestinal stem cells(ISCs).The homeostasis of intestinal stem cells is closely related to intestinal disease.The dysfunction of stem cells can induce intestinal epithelial repair disorders,and excessive activation may induce colorectal tumors.Currently,how to preserve the homeostasis of intestinal stem cells has been increasingly focused.The proliferation of colonic epithelial cells in female mouse has been confirmed to be influenced by physiological estrous cycle,demonstrating that estrogen might be involved in the regulation of intestinal stem cells proliferation,while its underlying mechanism has remained unknown.There are gender differences in the occurrence and development of intestinal diseases such as inflammatory bowel disease and colorectal tumors.it is necessary to study the physiological/pathological effects of estrogen and its receptors in the intestines of different gender groups.G protein coupled estrogen receptor(GPER,also known as GPR30),is a specific membranous estrogen receptor.Many signaling pathways could be recruited after GPER activation,modulating cell proliferation in ubiquitous tissue and system.Our previous study has demonstrated that GPER activation exerted protective effect on crypt cells proliferation after injury,suggesting the significant role of GPER in intestinal stem cells proliferation.However,it has remained unknown on whether GPER mediate intestinal stem cells homeostasis under physiological condition.In our study,we used ovariectomized mic to concentrated on the role of GPER in the maintenance of intestinal stem cell homeostasis in female mice.Methods1.Grouping and treatment of experimental animals:Healthy female adult C57BL/6 mice were anesthetized with isoflurane,then bilateral ovarian castration was performed under aseptic conditions.The experiment would begin two weeks after operation.Grouping:the animals were randomly divided into two groups,control group was intraperitoneally injected with normal saline 0.1ml at 8 am daily;the G-1 group was injected with selective GPER agonist G-1(0.03 mg/kg)solution 0.1ml;the MEK Inhibitor group was injected with PD0325901(1mg/kg)solution 0.1ml.The animals in G-1 group were sacrificed after 14 or 28 days,and that in PD group were sacrificed after 10 days.A section of ileum and colon was routinely prepared for paraffin section and detected by HE staining,immunohistochemistry and immunofluorescence staining,or frozen in liquid nitrogen for QRT-PCR or Western blot detection.2.Enteritis model:Ovariectomized mice was intraperitoneally injected with normal saline or G-1 solution 0.1ml at 8 am daily for 28 days.on the morning of the 29th day,lipopolysaccharide(LPS 0111:B4)from Escherichia coli was injected intraperitoneally,and the animals were sacrificed 4 hours later.A segment of ileum was routinely prepared for paraffin section for HE staining and immunohistochemical detection,frozen in liquid nitrogen for Western blot detection.3.Radiation-induced intestinal injury mode:Ovariectomized mice was intraperitoneally injected with normal saline or G-1 solution 0.1ml at 8am daily for 28 days.on the morning of the 29th day,the mice were exposed to whole body radiation(10GY)by 6MV-X linear accelerator.The animals were weighed at 9am daily.And were sacrificed after 3 days..A segment of ileum was routinely prepared and paraffin sections were prepared for HE staining and immunohistochemical detection,or frozen in liquid nitrogen for Western blot detection.4.Organoids culture:After euthanizing the mice,the abdominal cavity was quickly dissected and perfused with ice-cold PBS.Ileum was separated(10cm from ileocecal)about 15cm.Cut it longitudinally,wash the small intestine thoroughly with cold PBS,and then scrape down the villi using a coverslip or scalpel and wash in cold PBS.The small intestine was cut into pieces and transferred to a 50ml centrifuge tube.Add 30 ml PBS,discard the supernatant after blowing and repeat three times.Then add 30 ml ice-cold 2 mmol EDTA/PBS and shake at 4? for 30 min.Next,centrifuge at 100 rpm and remove the supernatant.Add 20 ml cold PBS,pipette and let it stand.Take the supernatant and sieve it into a new 50 ml centrifuge tube with a 70 ?m cell sieve,and centrifuge at 300 rpm for 5 min at 4?.The supernatant was removed,crypt cells were collected,isolated and cultured,and cultured in a 37-degree,5%CO2 cell incubator.After 24 hours of culture,the cells were treated with different drugs according to the needs of the study(G1:10-7M;Wnt3a:5ng/ml;IWP2:5?M).After 4-5 days of culture,the cells were collected for EdU staining,QRT-PCR or Western blot detection.Research contents1.In vivo experiment:Immunofluorescence staining of GPER and GFP(labeled Lgr5+)was used to detect the expression and localization of GPER in ileal;and immunofluorescence staining p-ERK1/2 was used to detect the expression and localization of p-ERK1/2 in ileum.The crypt depth and crypt depth of ileum were detected by HE staining;the proliferation of crypt cells was detected by immunohistochemical Ki67 and Brdu staining;the expression of goblet cells was detected by immunohistochemical MUC-2 staining and PAS(Periodic Acid-Schiff stain)glycogen staining;and Paneth cells were counted by immunohistochemical staining with lysozyme(lysozyme,Lyz).Western blot was used to detect the expression of Lgr5,cyclin CyclinD1 and CyclinB1,lysozyme,defensin ?1(Defensin alpha1,Defal)and Wnt3,Wnt signal pathway target proteins ?-catenin and c-Myc;Wnt signal pathway,p-ERK1/2 and ERK1/2 expression.Expression of iNOS,COX2 and TNF-? in inflammatory mediators.QRT-PCR was used to detect the expression of paneth cells maturation-related genes MMP7 and SOX9 mRNA,and the expression of downstream target genes ?-catenin and c-Myc of Wnt.2.Organoids culture:Observe and record the organoid area and budding;The EdU kit was used to detect the proliferation of organoids.Western blot was used to detect the expression of lysozyme,defensin ?1 and Wnt3;the expression of core protein?-catenin of Wnt signal pathway;and the expression of stem cell marker.QRT-PCR was used to detect the expression of Paneth cell maturation related genes MMP7 and SOX9mRNA.Results1.GPER was expressed at both Lgr5+stem cells and Paneth cells.2.Effect and mechanism of GPER activation on stem cell proliferation2.1 Compared with saline group,Selective GPER agonist G-1 increased the crypt depth,the number of Ki67 and Brdu positive cells in crypt,and the expression of stem cell markers Lgr5,cyclin CyclinD1,CyclinB1.After G-1 treatment for 2 weeks,the number of Ki67 positive cells in ileal increased,but the expression of Lgr5 remained unchanged.Organoid cultured in vitro,the area and buds were significantly increased,the number of EdU-positive cells increased,the expression of stem cell markert Lgr5 remained unchanged.2.2 G-1 treatment for 4 weeks augmented the expression of Wnt3(the ligand for Wnt signaling pathway)in,Paneth cells.The downstream of Wnt signaling pathway,including ?-catenin and c-Myc,were both up-regulated in mRNA and protein level.G-1 treatment for 2 weeks up-regulated the expression of Wnt3 and its target gene?-catenin.Crypt organoid culture in vitro showed that G-1 treatment for 5 days up-regulated the expression of Wnt3 and ?-catenin.The administration of IWP2 blocked the secretion of Wnt3 and inhibited the expression of ?-catenin despite the upregulation of Wnt3.It is suggested that the promotion of stem cell proliferation by GPER activation is related to the synthesis and secretion of Wnt3 in Paneth cells.3.The effect of GPER activation on paneth cells and goblet cells3.1 G-1 treatment for 4 weeks decreased the number of Paneth cells(lysozyme positive)and up-regulated the expression of lysozyme and defensin al in ileum.The mRNA level of MMP7 and SOX9 were also elevated,which suggested the increase in maturation of Paneth cells.Crypt organoid culture in vitro showed that G-1 treatment for 5 days exerted no effect on the expression of lysozyme and defensin ?1.The mRNA level of MMP7 and SOX9 also remained unchanged.3.2 G-1 treatment for 2 weeks exerted no effect on the number of goblet cells(PAS positive).G-1 treatment for 4 weeks reduced the number of PAS+ goblet cells and MUC-2+goblet cells.3.3 Immunofluorescence demonstrated that p-ERK1/2 was located at crypt-based columnar cells(CBCs)and transit amplifying cells(TACs).G-1 treatment for four weeks exerted no effect on the expression of p-ERK1/2 in ileum.G-1 treatment for four weeks up-regulated p-ERK1/2 expression.MEK specific inhibitor PD0325901 down-regulated the expression of p-ERK1/2 and increased PAS+goblet cells number,but exerted no effect on the expression of Wnt3as well as lysozyme,and lysozyme+Paneth cells number remained unchanged.3.4 G-1 treatment for four weeks up-regulated p-ERK1/2 expression in colon;PAS+goblet cells were decreased,but the expression of Wnt3 remained unchanged.PD0325901 inhibited the activation of ERK1/2 and increased number in goblet cells,with Wnt3 expression remaining unchanged.4.The protective effect of GPER activation on stem cell injury in the model of chemotherapy-induced intestinal injury.Radiation-induced intestinal injury model,the body weight of mice decreased significantly,and the scores of epithelial injury and crypt injury increased significantly,the number of ki67 positive cells decreased.G-1 pretreatment significantly inhibited radiation-induced weight loss,improved intestinal epithelial and crypt injury,and up-regulated the number of Ki67-positive cells,suggesting that GPER activation can protect stem cells from radiation-induced injury.5.Anti-inflammatory effect of GPER activation in enteritis model.In the model of acute enteritis induced by LPS,ileal edema and mucosal epithelial injury were obvious in mice.G-1 pretreatment could improve the symptoms of enteritis,the expression of inflammation-related factors iNOS,COX2 and Tnf-?were down-regulated,and the expression of Paneth cells antimicrobial peptide defensin ?1 was up-regulated,suggesting that G-1 treatment alleviated the inflammatory response of enteritis induced by LPS.Conclusions1.GPER was expressed in Lgr5+stem cells and paneth cells in ileum crypt.2.The activation of GPER of paneth cells up-regulated the expression of Wnt3,activated the Wnt/catenin signaling pathway of stem cells by paracrine mechanism,promoted the self-renewal and proliferation of stem cells,G-1 pretreatment played a protective role on the stem cell injury in the model of radio-induced intestinal injury.3.Wnt3 derived from Paneth cells also induced the maturation of paneth cells by autocrine mechanism,increased the secretion of antimicrobial peptides in paneth cells,G-1 pretreatment increased the secretion of antimicrobial peptides inhibited the intestinal inflammation induced by LPS.4.GPER activation of stem cells inhibits differentiation of stem cells into goblet cells,which is related to the activation of ERK1/2 induced by GPER.