齐墩果酸对衰老睾丸间质细胞(Leydig细胞)的作用以及EGF/EGFR表达的影响

The effect of oleanolic acid on senescent Leydig cells and the expression of EGF/EGFR

目的观察齐墩果酸对衰老睾丸间质细胞(Leydig细胞)的影响以及对表皮生长因子(epidermal growth factor,EGF)和表皮生长因子受体(epidermal growth factor receptor,EGFR)的调控作用。方法原代培养大鼠Leydig细胞,将其分为正常组、模型组和齐墩果酸组。模型组和齐墩果酸组采用H2O2(300μmol/L)和FeSO4(100μmol/L)每日处理2 h,持续4 d,造细胞衰老模型。之后正常组和模型组在DMEM/F12培养液培养3 d,齐墩果酸组在齐墩果酸终浓度为20μmol/L的DMEM/F12培养液中培养3 d。ELISA法检测细胞睾酮分泌情况;BrdU免疫荧光染色检测细胞增殖率;DAPI荧光染色检测细胞凋亡率;qRT-PCR检测细胞EGF和EGFR mRNA表达。结果模型组睾酮分泌水平、细胞增殖率、EGF和EGFR mRNA表达水平均低于正常组(P<0.05),凋亡率均高于正常组(P<0.05)。齐墩果酸可抑制Leydig细胞凋亡(P<0.05),并提高细胞睾酮分泌水平、细胞增殖水平、EGF和EGFR mRNA表达水平(P<0.05)。结论齐墩果酸可促进衰老Leydig细胞睾酮分泌及细胞增殖、抑制细胞凋亡,其机制与调控EGF和EGFR mRNA表达有关。

Objective To observe the effect of oleanolic acid on senescent Leydig cells and the regulation of EGF and EGFR.Methods Primary cultured rat Leydig cells were divided into normal group,model group and oleanolic acid group.The model group and the neat fruit acid group were treated with H2O2(300μmol/L)and FeSO4(100μmol/L)daily for 2 h for 4 d.Thereafter,the normal and model groups were cultured in DMEM/F12 medium for 3 d,and the oleanolic acid group was cultured in DMEM/F12 medium with a final concentration of 20μmol/L.Testosterone secretion was measured by ELISA,proliferation by immunofluorescence staining by BrdU,apoptosis by DAPI staining,and EGF and EGFR mRNA expression by qRT-PCR.Results Compared with the normal group,the testosterone secretion level,cell proliferation rate,EGF and EGFR mRNA expression level were significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05).Oleanolic acid significantly inhibited apoptosis in the model group(P<0.05)and significantly increased testosterone secretion,cell proliferation levels,and EGF and EGFR mRNA expression levels(P<0.05).Conclusion Oleanolic acid can promote testosterone secretion and cell proliferation and inhibit cell apoptosis,and its mechanism may be related to the regulation of EGF and EGFR mRNA expression.