论文部分内容阅读
目的通过丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路探讨养精种玉汤正丁醇(ZDC)及乙酸乙酯(YSYZ)提取物降低猪卵巢颗粒细胞雄激素水平的作用机制。方法分离并培养猪卵巢颗粒细胞,将细胞按不同浓度的MAPK抑制剂PD98059孵育,分为0(空白对照)、1、3、10、25μmol/L共5组,培养24 h后,采用实时荧光定量PCR(Real-time PCR)法检测17α-羟化酶/C17,20-裂解酶(cytochrome P450c17a,CYP17)mRNA表达水平,应用放射免疫测定法(RIA)检测细胞上清液雄激素(睾酮)含量,筛选最佳的PD98059作用浓度;采用10μmol/L PD98059干预卵巢颗粒细胞24 h后,将细胞培养液更换成含或不含有不同浓度(0、1、5、25、50 mg/m L)的养精种玉汤有效成分提取物ZDC及YSYZ干预不同的时间(3、6、18、24 h)后,采用Western blot法检测各组磷酸化的细胞外信号调节激酶(p-ERK1/2)、c-Fos及CYP17蛋白表达水平,RIA法检测细胞上清液睾酮含量。结果 10μmol/L PD98059可明显降低猪卵巢颗粒细胞p-ERK1/2蛋白表达,增加CYP17 mRNA表达,并可增加细胞上清液睾酮含量(P<0.05)。当养精种玉汤ZDC、YSYZ提取物浓度为25 ng/m L、作用时间为6 h时,可增加猪卵巢颗粒细胞p-ERK1/2、c-Fos蛋白水平并降低CYP17蛋白的表达,降低细胞上清液睾酮含量(P<0.05)。结论养精种玉汤有效成分通过增加MAPK的活性从而降低猪卵巢颗粒细胞的雄激素生成。
OBJECTIVE: To investigate the mechanism of androgen deprivation in porcine ovarian granulosa cells (ZOU) by using the mitogen-activated protein kinase (MAPK) signaling pathway. Methods Porcine ovarian granulosa cells were isolated and cultured. The cells were incubated with different concentrations of MAPK inhibitor PD98059 and divided into 0 (blank control), 1, 3, 10, 25μmol / L for 5 days. After cultured for 24 hours, The mRNA expression of 17α-hydroxylase / C17,20-lyase (CYP17) was detected by Real-time PCR. Radioimmunoassay (RIA) was used to detect the expression of androgen (testosterone) , And the optimal concentration of PD98059 was screened. After intervention of ovarian granulosa cells with 10μmol / L PD98059 for 24 hours, the cell culture medium was replaced with or without different concentration (0,1,5,25,50 mg / m L) (3, 6, 18, 24 h) of ZDC and YSYZ were used to detect the phosphorylation of extracellular signal-regulated kinase (p-ERK1 / 2 ), C-Fos and CYP17 protein levels, RIA assay of cell supernatant testosterone content. Results 10μmol / L PD98059 could significantly decrease the expression of p-ERK1 / 2 and increase the expression of CYP17 mRNA in porcine ovarian granulosa cells, and increase the content of testosterone in supernatant of cells (P <0.05). When the concentration of ZDC and YSYZ extract was 25 ng / m L and the effect time was 6 h, the levels of p-ERK1 / 2 and c-Fos in porcine ovarian granulosa cells and the expression of CYP17 protein in porcine ovarian granulosa cells were increased, Reduce the testosterone content of cell supernatant (P <0.05). CONCLUSION: The active ingredients of Yangjing Zhongyu Decoction can reduce the androgen production of porcine ovarian granulosa cells by increasing the activity of MAPK.