Effects And Mechanisms Of GM-CSF On Endometrial Regeneration And Repair

Part I The effects of GM-CSF in endometrial repair and its potential mechanismsObjective: To explore the effects of GM-CSF on endometrial regeneration and repair,its biological effects and mechanisms on endometrial glandular epithelium and stromal cells,and to provide foundations for new treatments of clinically refractory thin endometrium.Materials and Methods: An endometrial injury model in mice was established by intrauterine infusion of 90% ethanol,GM-CSF was then injected intraperitoneally.Endometrial thickness,proliferation,and receptivity of mice were evaluated by HE staining,immunohistochemical expression of ki67 and the number of embryo implantion.For in vitro experiments in human primary endometrial glandular epithelium and stromal cells,Brd U assay was used to detect proliferation of endometrial glandular epithelial and stromal cells,Transwell assay was used to detect migration of stromal cells and to explore the effect of different concentrations of GM-CSF on the proliferation and migration capacity of endometrial cells.In endometrial glandular epithelial cells,p-Akt,c-Jun and p70S6 K protein molecules in the Akt signaling pathway were detected by Western Blot.Akt pathway inhibitor(LY294002)was used to inhibit the effect of GM-CSF on endometrial glandular epithelial cell.Results: Intrauterine infusion of ethanol can establish an effective and stable mice model of endometrial injury.Compared with the endometrium on the control side,the endometrial thickness of the injured side is thinner(342.1±30.2 ?m vs.215.1±32.4 ?m,p<0.05),decreased expression of Ki67 in glandular cells(IRS:11.7±0.2 vs.7.5±1.2,p<0.05)and reduced number of embryo implatation(7.7±0.8 vs.3.2±1.1,p<0.01).After GM-CSF intraperitoneal injection,endometrial thickness(350.2±21.3 ?m vs.356.0±22.4 ?m,p>0.05),Ki67 expression in glandular epithelial cells(IRS:7.5 ± 2.0 vs.7.1±1.8,p>0.05)and the number of embryo implantion(6.2 ± 0.5 vs.5.8±0.5,p<0.01)were not significantly different between control and injury side of mice endometrium,so that GM-CSF repaired injured endometrium.GM-CSF promotes proliferation of human primary endometrial glandular epithelial cell and migration of stromal cell.In endometrial glandular epithelial cells,GM-CSF activates p-Akt phosphorylation,increases the expression of p70S6 K and c-Jun protein molecules.The effects of GM-CSF on endometrial glandular epithelial cells can be inhibited by LY294002.Conclusion: GM-CSF can significantly promote the regeneration and repair of endometrial injury in mice model.It can promote proliferation of human primary glandular epithelial cells and migration of stromal cells and activate the Akt signaling pathway.Our findings provide new treatment ideas for thin endometrium caused by regeneration disorder.Part II Effects and mechanisms of GM-CSF on angiogenesisObjective: To explore the effects and mechanisms of GM-CSF on vascular regeneration,and to provide new insights of clinical treatment of vascular repair related to endometrial injury.Materials and Methods: To observe whether angiogenesis is promoted by GM-CSF,the expression of angiogenic factor CD31 was evaluted after injection of GM-CSF into mice model of endometrial injury.The effect of GM-CSF on promoting the repair and regeneration of zebrafish embryos model of intersegmental vascular injury induced by Sorafenib.Human umbilical vein vascular endothelial cells(HUVECs)were cultured in vitro,and the effects of different concentrations of GM-CSF on the regeneration of HUVECs were examined.Ed U assay was used to detect HUVECs proliferation.Matrigel angiogenesis experiment(Tube formation)was used to detect angiogenesis effect.Scratch repair and Transwell experiment were used to detect HUVECs migration.Real-time PCR and Western Blot technology was used to verify differentially expressed levels of m RNA and protein.Separation of cytoplasmic and nuclear protein of Western Blot was used to confirm protein location.Ch IP was used to verify protein regulated the expression of related DNA promoters after entering the nucleus.FAK inhibitor(PF573228)and STAT3 si RNA were used to verify whether the effect of GM-CSF on the biological behavior of HUVECs can be inhibited.Results: GM-CSF promoted the expression of angiogenic factor CD31 in the mice model of endometrial injury.GM-CSF can repair and regenerate the zebrafish embryos model of intersegmental vascular injury caused by Sorafenib.GM-CSF can promote HUVECs proliferation and migration,and significantly increase the formation of vascular-like network structures to promote angiogenesis of HUVECs.In GM-CSF treatment group,the m RNA expression of VEGF,MMP2,Ang1,Ang2 and Tie2 m RNA in HUVECs cells increased significantly;GM-CSF can activate the FAK/Src/ERK signaling pathway and increase phosphorylation expression levels of p-FAK,p-Src,p-ERK 1/2,p-STAT3,p-p38 MAPK,pc-Jun,p-CREB,p-Akt,and p-e NOS.And it can increase the expression of downstream VEGF and MMP2 proteins.GM-CSF treatment for 30 min can increase the expression of STAT3 and promote the translocation of STAT3 from cytoplasm to nucleus,thereby regulating the expression of VEGF and MMP2 downstream.The above effects of GM-CSF on the biological behavior of HUVECs can be inhibited by FAK inhibitors(PF573228)and STAT3 si RNA.Conclusion: GM-CSF can promote the vascular regeneration in mice model of endometrial injury and zebrafish model of vascular injury.GM-CSF may activate FAK/Src/ERK signaling pathway and promote STAT3 translocation into the nucleus,thereby exerting the effects of promoting proliferation,angiogenesis and migration of HUVECs.Our findings provide new ideas for clinical treatment of vascular abnormalities of endometrial injury.