更年汤含药血清对大鼠卵巢颗粒细胞PI3K/Akt/m TOR通路的影响

目的研究更年汤对围绝经期模型大鼠颗粒细胞磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素蛋白(m TOR)信号通路的影响。方法 10～14月龄雌性SD大鼠150只,筛选出围绝经期模型大鼠,随机分为模型组、替勃龙组(0.22 mg/kg)和更年汤低、中、高剂量(1.0、2.0、4.0 g/kg)组,连续ig给药15 d,1次/d,第15天后采血,制备大鼠含药血清;3～4月龄雌性SD大鼠120只,筛选出动情前期老鼠,剔出卵巢行体外颗粒细胞原代培养,分别用各组含药血清培养液培养颗粒细胞,72～120h后用实时荧光定量PCR(qRT-PCR)检测颗粒细胞中PI3K、Akt、m TOR、生长分化因子9(GDF9)、翼状螺旋转录因子2(FOXL2)mRNA表达水平;Western blotting法检测PI3K、Akt、mTOR、GDF9、FOXL2蛋白表达。结果与模型组比较,替勃龙组和更年汤各剂量组卵巢颗粒细胞中PI3K、m TORm RNA和蛋白表达水平均显著升高(P0.05、0.01),Akt mRNA和蛋白表达水平显著降低(P0.05、0.01),GDF9、FOXL2 mRNA和蛋白表达水平均显著降低(P0.01)。结论更年汤含药血清在临床剂量上可提高卵巢颗粒细胞PI3K、mTOR的表达,抑制Akt及其下游的GDF9、FOXL2表达,从而推测更年汤可调节卵泡的生长发育,抑制卵泡闭锁、改善卵巢功能。

Effect of drug-containing serum of Gengnian Decoction on signal pathway of PI3K/Akt/mTOR in oval cells of rats

Objective To study the effect of Gengnian Decoction on rat granulocyte in the signal pathway of PI3K/Akt/mTOR of perimenopausal model. Methods A total of 150 female SD rats aged from 10 to 14 months were screened out of the perimenopausal model rats and randomly divided into five groups:model group, tibolone group and Gengnian Decoction low, medium and high dose groups, continuous stomach ig administration for 15 d, once daily. The blood was collected to prepare the rat serum containing drugs 15 d later; A total of 120 female SD rats aged from 3 to 4 months were screened for pro-estrus mice, and ovarian granulosa cells were cultured in vitro; Granular cells were cultured with drug-containing serum of each group. After 72-120 h, mRNA and protein expression of PI3K, Akt, mTOR, GDF9, and FOXL2 levels were detected by qRT-PCR, and Western blotting, respectively. Results Compared with the model group, the mRNA and protein expression levels of PI3K and mTOR in ovarian granulosa cells of tibolone group and dose decoction groups were significantly increased (P<0.05, 0.01), while the expression levels of Akt mRNA and protein were significantly decreased (P<0.05, 0.01), the mRNA and protein expression levels of GDF9 and Foxl2 were significantly decreased (P<0.01). Conclusion Gengnian Decoction serum can increase the expression of PI3K and mTOR in ovarian granulosa cells and inhibit the expression of Akt and its downstream GDF9 and Foxl2, so it was predicted that Gengnian Decoction can regulate the growth and development of ovarian follicles, inhibit follicles latching, and improve ovarian function.