The Mechanism Of M2 Macrophages Involved In Adenomyosis By Regulating The Expression Of GRIM-19

ObjectiveAdenomyosis is a nonneoplastic condition characterized by the benign invasion of ectopic endometrium into the myometrium.The hyperplastic endometrial tissue has the characteristics of new blood vessel formation,increasing proliferation,decreasing apoptosis,increased adhesion and invasion ability and other characteristics of malignant tumors.The gene associated with retinoid-interferon(IFN)-induced mortality 19(GRIM-19)could promotes apoptosis to some extent.Previous study confirmed the expression of GRIM-19 reduced in endometrium of patients with adenomyosis.The purpose of this study was to investigate the causes of decreased GRIM-19 expression and the role of M2 macrophages in the pathogenesis of adenomyosis.MethodsIn vivo study:Normal endometrium was collected as control group(15 cases in secretory phase)and ectopic endometrium of adenomyosis as experimental group(15 cases in secretory phase).Immunohistochemical method was used to detect the location and expression of GRIM-19 and toll like receptor 4(TLR4)both in the experimental group and the control group.TUNEL was used to analyze the level of apoptosis;Western blot was used to analyze the protein expression of GRIM-19,TLR4 and caspase-3 in the control group and the eutopic tissue of adenomyosis.In vitro study:(1)Low expression of GRIM-19 in hESC cells were expressed by GRIM-19-siRNA technique.Flow cytometry and TUNEL fluorescence staining were used to detect the change of apoptosis;CCK-8 assay was used to detect the level of cell proliferation;TRANSWELL cell migration assay was used to detect the change of cell migration ability;Western blotting was used to detect the protein level of GRIM-19 and caspase-3.(2)In this experiment,M2 macrophages derived from THP-1 were co-cultured with hESC cells.Flow cytometry and TUNEL fluorescence staining were used to detect the change of apoptosis;CCK-8 assay was used to detect the level of cell proliferation;TRANS WELL cell migration assay was used to detect the change of cell migration ability;Western blotting was used to detect the protein level of GRIM-19,TLR4 and caspase-3;Real-time RCR was used to detect the mRNA level of GRIM-19 and TLR4.(3)hESC cells were stimulated by TLR4 activators and inhibitors and the protein expression of GRIM-19 was detected by Western blot.Results(1)GRIM-19 positivity in endometrial glandular cells of the Normal endometrium and ectopic endometrium in patients with adenomyosis.The protein level of GRIM-19 in the endometrium of adenomyosis was significantly lower than that of the control group(P<0.001).(2)TUNEL staining showed that the apoptosis level of endometrium in adenomyosis was lower than that in the control group(P<0.01).(3)GRIM-19 was successfully knocked down in hESC cells.The results of flow cytometry showed an decrease in the proportion of apoptotic cells(the lower right quadrant)(P<0.01),TUNEL staining showed the number of apoptotic cells decreased(P<0.01),and CCK-8 showed that the cell proliferation ability was enhanced(P<0.001),TRANS WELL showed that the cell migration ability was improved(P<0.01),and Western blot results showed that the level of caspase-3 protein in the experimental group cells was reduced(P<0.05).(4)When hESC cells were co cultured with M2 macrophages,the Western blot results showed that the GRIM-19 protein level in the experimental group decreased(P<0.05),and the real time PCR results showed that the GRIM-19 mRNA level in the experimental group decreased(P<0.001).(5)hESC cells were co-cultured with M2 macrophages.The results of flow cytometry showed an decrease in the proportion of apoptotic cells(the lower right quadrant)(P<0.001),TUNEL staining showed the number of apoptotic cells decreased(P<0.01),and CCK-8 showed that the cell proliferation ability was enhanced(P<0.001),TRANS WELL showed that the cell migration ability was improved(P<0.001),and Western blot results showed that the level of caspase-3 protein in the experimental group cells was reduced(P<0.01).(6)When hESC cells were co-cultured with M2 macrophages,western blot showed that the protein level of TLR4 in the experimental group was decreased(P<0.01),and real time PCR showed that the level of TLR4 mRNA in the experimental group was decreased(P<0.05).(7)When TLR4 was activated in hESC cells,western blot showed that the protein level of GRIM-19 increased(P<0.05).Then the inhibitors were added,western blot showed that the protein level of GRIM-19 decreased(P<0.05).(8)The results of immunohistochemistry showed that the level of TLR4 in ectopic tissue of adenomyosis was significantly lower than that in the control group(P<0.001),and western blot showed that the protein level of TLR4 decreased(P<0.05).ConclusionsM2 macrophages may regulate the expression of GRIM-19 through TLR4 in endometrial adenomyosis which will influence the ability of apoptosis,proliferation and migration of endometrial cells.