GRIM19 Induced Macrophages Pyroptosis Through NLRP3 Pathway In Adenomyosis

ObjectiveAdenomyosis is a common benign gynecological disease.It is clinically characterized by abnormal uterine bleeding,progressive dysmenorrhea,dyspareunia,fertility disorders and infertility.The endometrium of adenomyosis is infiltrated by a large number of macrophages,and secretion of interleukin 1β(IL-1β)is significantly increased.It is unclear the role of pyroptosis as one of the ways to release IL-1β in adenomyosis.As an upstream molecule inducing pyroptosis,NOD-Like receptor pyrin domain-containing 3(NLRP3)can be regulated the activation by reactive oxygen species(ROS)which is significantly enhanced because of Gene associated with retinoid-interferon-induced mortality 19(GRIM 19)deficiency.But it remains to be further investigated whether GRIM 19 deficiency affects NLRP3 expression and pyroptosis.Thus,the aim of this study is to investigate the correlations with GRIM19 expression and pyroptosis mediated by NLRP3,and to explain whether the release of IL-1β resulted by pyroptosis is a relevant factor in the regulation of adenomyosis progression.Methods1、Endometrial tissues were collected from patients with(n=12)and without(n=12)adenomyosis,respectively as the experimental and control group.They were stored at-80℃to be tested.2、GRIM 19 expression of adenomyosis tissues was analyzed by western blot and qRT-PCR.3、THP-1 were cultured in vitro and induced to differentiation into M0 macrophages.4、Knockdown the expression of GRIM 19 in THP-1 derived M0 macrophages by transfection technology,the protein and mRNA expression of NLRP3,caspase-1,ASC,GSDMD were measured by western blot and qRT-PCR.IL-1β and IL-18 in the culture supernatant were detected by ELISA.5、The HESC were co-cultured with THP-1 derived M0 macrophages,or GRIM19-depleted M0 macrophages.CCK-8 assay was used to measure HESC proliferation ability;Transwell cell migration assay and wound healing assay were used to measure HESC migration ability;and Annexin V-FITC/PI staining was used to measure HESC apoptotic levels.6、The HESC were co-cultured with THP1 derived GRIM 19-knockdown macrophages in the presence of anti-IL-1 β mouse IgG1 neutralize antibodies,or mouse IgG1 isotype control antibodies as the control group.CCK-8 assay was used to measure HESC proliferation ability;Transwell cell migration assay and wound healing assay were used to measure HESC migration ability;and Annexin V-FITC/PI staining was used to measure HESC apoptotic levels.Results1、The protein expression level(P=0.0002)and the mRNA level(P=0.0005)of GRIM 19 in patients with adenomyosis was significantly lower than the control group.2、In THP-1-derived M0 macrophages,western blot showed that the protein expression of NLRP3(P<0.0001),ASC(P=0.0176),caspase-1(P=0.0368),GSDMD(P=0.0453)are increased after downregulation of GRIM19.Increased mRNA expression of NLRP3(P=0.0039),ASC(P=0.0126),caspase-1(P=0.0218),GSDMD(P=0.0198),IL-1β(P=0.0208)in GRIM 19-depleted macrophages was also confirmed by qRT-PCR.3、Enzyme-linked immunosorbent assay analysis showed that GRIM 19 knockdown induced the release of IL-1β(P=0.0195)in THP-1-derived macrophages.4、The results of flow cytometry analysis showed that the apoptosis level of HESC co-cultured with GRIM 19 knockdown macrophages was significantly decreased(P<0.0001),CCK-8 showed that the proliferation ability(P=0.0254)was enhanced,Transwell(P=0.0002)and Wound healing assay showed(P<0.0001)that migration ability was markedly promoted.5、When existence of IL-1β neutralizing antibody in supernatants of HESC co-cultured with GRIM19 knockdown macrophages,flow cytometry analysis showed that the apoptosis level was significantly improved(P<0.0001),CCK-8 showed that the proliferation ability(P<0.0001)was inhibited,Transwell(P<0.0001)and Wound healing assay showed(P<0.0001)that migration ability was also markedly suppressed.ConclusionsGRIM19 downregulation induces pyroptosis of macrophages through NLRP3 pathway,increases the secretion of IL-1β and promotes adenomyosis progression.