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目的探讨逍遥丸提高体外卵母细胞质量的可能作用机制。方法分离58只昆明小鼠腔前卵泡,分别以10%正常大鼠血清,10%逍遥丸低、中、高剂量大鼠含药血清,10%逍遥丸高剂量大鼠含药血清+10μmol/L K02288孵育,设为对照组,疏肝低、中、高剂量组,抑制剂组,培养6天。实时荧光定量PCR法检测卵母细胞中Smad1、Smad5、Smad8 mRNA表达,细胞免疫荧光检测Smad1、Smad5、Smad8和磷酸化的Smad1/5/8(p-Smad1/5/8)蛋白表达。结果疏肝高剂量组Smad1 mRNA和蛋白高于对照组(P<0.05);疏肝各剂量组Smad5、Smad8 mRNA和蛋白及p-Smad1/5/8蛋白均高于对照组(P<0.05),其中疏肝高、中剂量组Smad5 mRNA和蛋白比较差异无统计学意义(P>0.05),均高于低剂量组(P<0.05),Smad8 mRNA和蛋白及p-Smad1/5/8蛋白以疏肝高剂量组最高(P<0.05);抑制剂组p-Smad1/5/8蛋白低于疏肝高剂量组(P<0.05)。结论逍遥丸提高体外卵母细胞质量的作用可能与上调卵母细胞中Smad1、Smad5、Smad8 mRNA和蛋白及p-Smad1/5/8蛋白表达有关,以高剂量组效果更佳。
Objective To explore the possible mechanism of Xiaoyao Pill in improving oocyte quality in vitro. Methods Fifty-eight Kunming mouse preantral follicles were isolated and divided into three groups: 10% normal rat serum, 10% Xiaoyao Pill low, medium and high dose rat serum, 10% Xiaoyao Pill high dose rat serum containing + 10μmol / L K02288, set as control group, low, medium and high dose group, inhibitor group, cultured for 6 days. The expression of Smad1, Smad5 and Smad8 in oocytes was detected by real-time fluorescence quantitative PCR. The expressions of Smad1, Smad5, Smad8 and phosphorylated Smad1 / 5/8 protein were detected by immunofluorescence. Results The mRNA and protein expressions of Smad1 in high dose Shugan group were significantly higher than those in control group (P <0.05). The mRNA and protein expressions of Smad5, Smad8 and p-Smad1 / 5/8 in Shugan group were higher than those in control group (P <0.05) (P <0.05). There was no significant difference in Smad5 mRNA and protein between Shugan and medium dose groups (P> 0.05), which were significantly higher than those in low dose group (P <0.05). Smad8 mRNA and protein and p-Smad1 / 5 / The content of p-Smad1 / 5/8 in the inhibitor group was lower than that in the high-dose group (P <0.05). Conclusion The effect of Xiaoyao Pill in enhancing oocyte quality in vitro may be related to up-regulating the expression of Smad1, Smad5, Smad8 mRNA and protein and p-Smad1 / 5/8 protein in oocytes, and the effect of Xiaoyao Pill is better in high-dose group.