MIF通过诱导自噬促进卵巢颗粒细胞的胰岛素抵抗

MIF promotes insulin resistance in ovarian granulosa cells by inducing autophagy

目的在多囊卵巢综合征(PCOS)中探究巨噬细胞迁移抑制因子(MIF)的表达及其对卵巢颗粒细胞的自噬与胰岛素抵抗(IR)的影响。方法收集40例PCOS患者(n=40)及20例对照女性(对照组,n=20)的卵泡液与卵巢颗粒细胞,其中PCOS包含伴IR者(PCOS伴IR组,n=20)与不伴IR者(PCOS不伴IR组,n=20)。采用ELISA法检测三组受试者卵泡液中MIF的表达,透射电子显微镜与Western blot法观察三组受试者颗粒细胞中的自噬空泡与自噬标志物微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)/微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)比值;体外培养人卵巢颗粒细胞系KNG,CCK-8法检测不同浓度MIF对颗粒细胞活性的影响;将KNG细胞分为4组:正常培养组(NC组)、MIF组、自噬抑制剂氯喹组(CQ组)和MIF+CQ组,Western blot法检测MIF对各组颗粒细胞中自噬相关蛋白LC3、自噬相关基因7(Atg7)、泛素结合蛋白(p62)及胰岛素信号通路中胰岛素受体底物-1(IRS-1)与葡萄糖转运蛋白4(GLUT4)表达及蛋白激酶B(Akt)蛋白磷酸化的影响,葡萄糖摄取实验检测MIF对各组颗粒细胞摄取葡萄糖能力的变化。结果与对照组或PCOS不伴IR组相比,PCOS伴IR组患者的卵泡液中MIF的表达水平及颗粒细胞的自噬水平增加(P<0.05,P<0.01);体外实验显示,MIF能够以浓度依赖性抑制颗粒细胞KNG的增殖活性;与NC组相比,MIF组与MIF+CQ组颗粒中LC3Ⅱ/LC3Ⅰ比值,Atg7蛋白表达增加(P<0.05),而p62蛋白、IRS-1蛋白、Akt蛋白磷酸化水平和GLUT4蛋白表达水平及葡萄糖摄取能力均降低(P<0.05),而与MIF组相比,MIF+CQ组细胞中LC3Ⅱ/LC3Ⅰ比值与Atg7蛋白表达水平均降低(P<0.05),而p62、IRS-1蛋白、Akt蛋白磷酸化水平和GLUT4蛋白表达水平及葡萄糖摄取能力均增加(P<0.05)。结论MIF可能通过上调颗粒细胞的自噬水平促进PCOS患者的IR发展,而抑制颗粒细胞的自噬则能改善MIF诱导IR。

Objective To investigate the expression of macrophage migration inhibitory factoR(MIF)and its effect on autophagy and insulin resistance of ovarian granulosa cells in polycystic ovary syndrome(PCOS).Methods FolliculaRfluid and ovarian granulosa cells were collected from 40 PCOS patients(n=40)and non PCOS patients(control group,n=20).PCOS included patients with IR(PCOS with IR,n=20)and patients without IR(PCOS without IR,n=20).The expression of MIF in folliculaRfluid was detected by ELISA.The ratio of autophagy vacuoles to microtubule-associated protein light chainⅡ(LC3Ⅱ)/microtubule-associated protein light chainⅠ(LC3Ⅰ)in granulosa cells was observed by transmission electron microscopy and Western blot.Human ovarian granule cell line KNG was cultured in vitro and CCK-8 was used to detect the effects of different concentrations of MIF on granule cell activity.KNG cells were divided into fouRgroups:normal culture group(NC group),MIF group,chloroquine group(CQ group)and MIF+CQ group.The effects of MIF on the expression of autophagy related proteins LC3,autophagy related genes 7(Atg7),ubiquitin binding protein(p62),and insulin signaling pathway related proteins insulin receptoRsubstrate-1(ISR-1),glucose transporteR4(GLUT4),protein kinase B(Akt)phosphorylation were observed by Western blot,and the effect of MIF on glucose uptake ability of granulosa cells was detected by glucose uptake test.Results Compared with the control group oRPCOS without IRgroup,MIF expression in folliculaRfluid and autophagy level of granulosa cells in PCOS with IRgroup increased(P<0.05 oRP<0.01);In vitro experiments showed that MIF could significantly inhibit the cellulaRactivity of KNG in granulocytes in a concentration-dependent manner,in which 100 ng/ml MIF was selected foRsubsequent relevant experiments;Compared with NC group,LC3Ⅱ/LC3I ratio and Atg7 protein in MIF group and MIF+CQ group increased(P<0.05),while p62 protein,IRS-1 protein,Akt phosphorylation level,GLUT4 protein expression level and glucose uptake ability decreased(P<0.05),while the above autophagy markers in MIF+CQ group were significantly higheRthan those in MIF group(P<0.05),and the protein related to insulin signal transduction and glucose uptake increased(P<0.05).Conclusion MIF may promote IRdevelopment in PCOS patients by up-regulating the autophagy level of granulosa cells,while inhibiting the autophagy of granulosa cells can improve MIF-induced IR.