多囊卵巢综合征中高雄激素诱导的颗粒细胞自噬激活作用

Testosterone induces autophagy activation of granulosa cells in polycystic ovary syndrome

目的探讨高雄激素睾酮(testosterone)对多囊卵巢综合征(polycystic ovary syndrome,PCOS)患者卵巢颗粒细胞自噬的影响。方法分离培养卵巢功能正常的不孕患者(对照组)及PCOS不孕患者(PCOS组)的卵巢颗粒细胞,分别用10μmol/L睾酮处理24 h;将人卵巢颗粒细胞株KGN培养至3×10^(5)个,分别进行小干扰RNA(small interfering RNA,siRNA)及小泛素样修饰物(small ubiquitin-like modifier,SUMO)特异肽酶3(SUMO specific peptidase 3,SENP3)质粒转染;RT-PCR法检测SENP3基因mRNA转录水平,Western blot法检测LC3Ⅱ/Ⅰ、P62及SENP3蛋白表达水平。结果与对照组比较,PCOS组卵巢颗粒细胞中LC3Ⅱ/Ⅰ蛋白表达水平显著升高(P <0.01),P62蛋白表达水平显著降低(P <0.01);两组患者卵巢颗粒细胞经睾酮处理后,LC3Ⅱ/Ⅰ蛋白表达水平显著升高(P均<0.05),P62蛋白表达水平显著降低(P均<0.05)。与对照组比较,PCOS组卵巢颗粒细胞中SENP3 mRNA转录及蛋白表达水平均显著降低(P均<0.05)。KGN细胞经siRNA干扰,LC3Ⅱ/Ⅰ蛋白表达水平上调(P <0.01),P62蛋白表达水平下降(P <0.05);KGN细胞经SENP3质粒转染,LC3Ⅱ/Ⅰ蛋白表达水平无明显变化(P> 0.05),P62蛋白表达水平上调(P <0.01)。PCOS组卵巢颗粒细胞经睾酮处理后,SENP3蛋白表达水平显著下降(P <0.05)。结论PCOS患者颗粒细胞有一定程度的自噬激活,且高雄激素参与了PCOS患者颗粒细胞的自噬激活过程,其作用机制可能与高雄激素抑制颗粒细胞中SENP3蛋白表达有关。

Objective To investigate the effect of testosterone on the autophagy of ovarian granulosa cells of patients with polycystic ovary syndrome(PCOS). Methods The ovarian granulosa cells of patients with PCOS were separated and treated with 10 μmol/L testosterone for 24 h,using those of infertile patients with normal ovarian function as control.Human ovarian granulosa cell strain KGN was cultured to a number of 3 × 10^(5) cells,and transfected with small interfering RNA(siRNA) and the plasmid containing small ubiquitin-like modifier(SUMO) specific peptidase 3(SENP3) gene.The transcription level of SENP3 mRNA was determined by RT-PCR,while the expression levels of LC3Ⅱ/Ⅰ,P62 and SENP3 proteins by Western blot. Results The expression level of LC3 Ⅱ/Ⅰ in PCOS group was significantly higher,while that of P62 protien was significantly lower,than those in control group(each P < 0. 01). After the ovarian granulose cells were treated with testosterone,the expression levels of LC3Ⅱ/Ⅰ in two groups increased significantly,while those of P62 protein decreased significantly(each P < 0. 05). Both the mRNA transcription and protein expression levels of SENP3 in ovarian granulose cells in PCOS group were significantly lower than those in control group(each P < 0. 05).The expression level of LC3Ⅱ/Ⅰ in KGN cells after siRNA interference increased significantly(P < 0. 01),while that of P62 protein decreased significantly(P < 0. 05). However,after KGN cells were transfected with plasmid containing SENP3 gene,the expression level of LC3 Ⅱ/Ⅰ showed no significant change(P > 0. 05),while that of P62 protein increased significantly(P < 0. 01). The expression level of SENP3 protein in ovarian granulose cells of PCOS group after treatment with testosterone decreased significantly(P < 0. 05). Conclusion The autophay of ovarian granulose cells of patients with PCOS were activated at a certain degree. Testosterone involved in the autophagy activation,of which the mechanism might be associated with the inhibition of SENP3 protein expression in granulosa cells.