基于PI3K/AKT/mTOR信号通路研究补肾法对体外培养小鼠卵母细胞成熟的促进作用

Promotion of kidney-tonifying therapy on maturation of mouse oocyte in vitro based on PI3K/AKT/mTOR signaling pathway

目的研究补肾法对体外培养小鼠卵母细胞成熟的促进作用及其与磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白(PI3K/AKT/mTOR)信号通路的关系。方法给6~8周龄雌性大鼠灌服补肾调经Ⅲ号方制备含药血清。将3~4周龄雌性小鼠随机分为A、B组,A组灌胃蒸馏水,B组序贯灌胃补肾调经系列方(Ⅱ号方和Ⅲ号方),各灌胃12 d。第11天腹腔注射孕马血清促性腺激素(5 IU/只),48 h后剥离卵巢,取出卵丘-卵母细胞复合体(COCs)进行体外培养。将A组COCs分为空白对照组和抑制剂组(25μmol/L LY294002),B组分为补肾组(10%补肾调经Ⅲ号方含药血清)和补肾加抑制剂组(10%补肾调经Ⅲ号方含药血清+25μmol/L LY294002)。体外培养18 h后,在显微镜下统计各组第一极体排出量并计算排出率。收集COCs, RT-PCR法检测各组COCs中Bcl-2、Bax、PI3K、AKT、mTOR mRNA的表达,免疫荧光染色法检测各组COCs中Bcl-2、Bax、PI3K、AKT、mTOR、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)、磷酸化雷帕霉素靶蛋白(p-mTOR)的蛋白表达。结果第一极体排出率补肾组高于空白对照组(P<0.05),抑制剂组低于空白对照组(P<0.05),补肾加抑制剂组低于补肾组(P<0.05)。RT-PCR结果显示,与空白对照组比较,补肾组COCs中Bcl-2、PI3K、AKT、mTOR mRNA表达及Bcl-2/Bax值均升高(P<0.05),Bax mRNA表达降低(P<0.05);抑制剂组COCs中Bcl-2、PI3K、AKT、mTOR mRNA表达及Bcl-2/Bax值均下降(P<0.05),Bax mRNA表达升高(P<0.05)。与补肾组比较,补肾加抑制剂组COCs中Bcl-2、PI3K、AKT、mTOR mRNA表达及Bcl-2/Bax值均降低(P<0.05),Bax mRNA表达升高(P<0.05)。免疫荧光染色结果显示,各组间PI3K、AKT、mTOR蛋白表达差异无统计学意义(P>0.05);与空白对照组比较,补肾组COCs中Bcl-2蛋白表达、p-PI3K/PI3K、p-AKT/AKT升高(P<0.05),Bax蛋白表达降低(P<0.05);抑制剂组COCs中Bcl-2蛋白表达、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR降低(P<0.05),Bax蛋白表达升高(P<0.05)。与补肾组比较,补肾加抑制剂组COCs中Bcl-2蛋白表达、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR降低(P<0.05),Bax蛋白表达升高(P<0.05)。结论补肾调经系列方可通过调控PI3K/AKT/mTOR信号通路、上调Bcl-2和Bax mRNA及蛋白表达的比值、抑制COCs凋亡从而促进小鼠卵母细胞成熟。

Objective To explore the promotion of kidney-tonifying therapy on maturation of oocyte in vitro and its relationship with PI3 K/AKT/mTOR signaling pathways. Methods Female rats(6-8 weeks old) were given Bushen Tiaojing Fang Ⅲ(Kidney-tonifying Menstruation-regulating Formulas Ⅲ, BSTJF Ⅲ) to prepare serum containing BSTJF Ⅲ. Female mice(3-4 weeks old) were randomly divided into two groups: Group A and Group B. The two groups were given distilled water and a series of BSTJFs(BSTJF Ⅱ and BSTJF Ⅲ) by gavage respectively for 12 days. On the 11 th day, all the mice were intraperitoneally injected with 5 IU of gonadotropin each and 48 hours later, the ovaries were removed and cumulus-oocyte complexes(COCs) were taken out for in vitro culture. COCs of Group A were divided into the blank control group and the inhibitor group(25 μmol/L of LY294002), and those of Group B were divided into the kidney-tonifying group(serum containing 10% of BSTJF Ⅲ) and kidney-tonifying + inhibitor group(serum containing 10% of BSTJF Ⅲ+25 μmol/L of LY294002). After 18 hours of culture in vitro, the number of discharged first polar bodies of the two groups were counted under microscope and the discharge rates were calculated. Real-time fluorescence quantitative PCR(RT-PCR) was used to detect the expressions of mRNA of Bcl-2, Bax, PI3 K, AKT and mTOR in COCs of all groups. Immunofluorescence staining was used to detect the expressions of Bcl-2, Bax, PI3 K, AKT, mTOR, p-PI3 K, p-AKT and p-mTOR proteins in COCs of all groups. Results The first polar body discharge rate in the kidney-tonifying group was higher than that in the blank control group(P<0.05), that of the inhibitor group was lower than that of the blank control group(P<0.05), and that of the kidney-tonifying+inhibitor group was lower than that of the kidney-tonifying group(P<0.05). RT-PCR results showed that compared with the blank control group, the expressions of mRNA of Bcl-2, PI3 K, AKT, and mTOR and Bcl-2/Bax of COCs increased(P<0.05) and the expressions of Bax mRNA decreased(P<0.05) in the kidney-tonifying group and the expressions of mRNA of Bcl-2, PI3 K, AKT, and mTOR and Bcl-2/Bax of COCs decreased(P<0.05) and the expressions of Bax mRNA increased(P<0.05) in the inhibitor group. Compared with the kidney-tonifying group, the expressions of mRNA of Bcl-2, PI3 K, AKT, and mTOR and Bcl-2/Bax of COCs decreased(P<0.05), and the expressions of Bax mRNA increased(P<0.05) in the kidney-tonifying+inhibitor group. Immunofluorescence staining results showed that there was no significant difference in the expressions of total protein of PI3 K, AKT, and mTOR between the groups(P>0.05). Compared with the blank control group, the expression of Bcl-2 protein, p-PI3 K/PI3 K, and p-AKT/AKT of COCs increased(P<0.05) while the expression of Bax protein was decreased(P<0.05) in the kidney-tonifying group, and the expression of Bcl-2 protein, p-PI3 K/PI3 K, p-AKT/AKT, and p-mTOR/mTOR of COCs decreased(P<0.05) while the expression of Bax protein was increased(P<0.05) in the inhibitor group. Compared with the kidney-tonifying group, the expression of Bcl-2 protein, p-PI3 K/PI3 K, p-AKT/AKT, and p-mTOR/mTOR of COCs decreased(P<0.05) and Bax protein expression increased(P<0.05) in the kidney-tonifying+inhibitor group. Conclusion BSTJFs can promote mouse oocyte maturation by regulating the PI3 K/AKT/mTOR signaling pathways, up-regulating the mRNA and ratio of protein expressions of Bcl-2 and Bax, and inhibiting granulosa cell apoptosis.