羟基γ-异山椒素抑制人体卵巢颗粒细胞雌激素生物合成的机制研究

Mechanism of hydroxy-γ-isosanshool on the inhibition of estrogen biosynthesis in human ovarian granulosa cells

目的:探讨羟基γ-异山椒素抑制人体卵巢颗粒细胞雌激素合成的相关机制。方法:利用CCK-8检测4种山椒素对人卵巢颗粒细胞系(KGN细胞株)增殖的影响;Elisa法检测4种山椒素对KGN细胞雌二醇合成的影响;RT-qPCR检测KGN细胞芳香化酶基因;Western blot法检测芳香化酶蛋白及相关通路蛋白。结果:羟基γ-异山椒素抑制雌二醇生物合成效果最佳[半抑制浓度(IC50)=(16.63±0.39)μmol·L^(-1)],并且在该浓度下对KGN细胞的增殖无明显抑制作用。在羟基γ-异山椒素干预后,芳香化酶基因和蛋白表达水平显著下降(P<0.05),且芳香化酶的活性未受到明显影响;同时,羟基γ-异山椒素能显著降低P-CERB,P-AKT,P-p38蛋白的表达(P<0.05)。结论:羟基γ-异山椒素可能通过抑制p38/MAPK和PI3K/AKT通路的激活,降低CREB的磷酸化,抑制芳香化酶转录,最终抑制KGN细胞雌激素生物合成。

Objective:To investigate the mechanism of inhibitory effect of hydroxy-γ-isosanshool on estrogen synthesis in human ovarian granulosa cells.Methods:The effects of four kinds of sanshool on the proliferation of human ovarian granulosa cell line(KGN cell line)were detected by CCK-8.The effects of four kinds of sanshool on estradiol synthesis in KGN cells were detected by Elisa.Aromatase gene in KGN cells was detected by RTqPCR,and aromatase protein and related pathway proteins were detected by Western Blot.Results:Hydroxy-γ-isosanshool had the strongest inhibitory effect on estradiol biosynthesis[IC50=(16.63±0.39)μmol·L^(-1)],while had no significant inhibitory effect on the proliferation of KGN cells at the same concentration.The expression levels of aromatase gene and protein decreased significantly after the intervention of hydroxy-γ-isosanshool(P<0.05),and the activity of aromatase was not significantly affected.Hydroxy-γ-isosanshool significantly reduced the protein expressions of P-CERB,P-AKT and P-p38(P<0.05).Conclusion:Hydroxy-γ-isosanshool may inhibit aromatase transcription and estrogen biosynthesis in KGN cells by inhibiting the activation of p38/MAPK and PI3K/AKT pathways and reducing the level of CREB phosphorylation,inhibit aromatase transcription,and ultimately inhibit the estrogen biosynthesis of KGN cells.