苍附导痰丸含药血清调控大鼠卵巢颗粒细胞自噬的作用

目的:研究苍附导痰丸含药血清调控大鼠卵巢颗粒细胞(GCs)自噬的作用。方法:将3月龄SD大鼠分为生理盐水组(生理盐水,灌胃)、卵巢刺激素(FSH)注射组(10.71 IU/kg,皮下注射)和苍附导痰丸高、中、低剂量灌胃组0.5、1、2 mg/g(以生药量计),灌胃],每组6只,每天皮下注射/灌胃给药1次,连续3天;末次给药后,于腹主动脉采血,获取各组大鼠的含药血清。将大鼠GCs分为空白对照组、模型组、FSH组(阳性对照)和苍附导痰丸高、中、低剂量组,除空白对照组直接加入生理盐水组大鼠血清100μL外,其余各组均先给予丙酸睾丸酮诱导以复制自噬模型,然后模型组加入生理盐水组大鼠血清100μL,各给药组分别加入对应药物组含药血清100μL。采用酶联免疫吸附法(ELISA)检测各组细胞上清液中雌二醇(E2)、孕激素(P)的含量,采用Western blot法检测各组GCs中磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)蛋白的相对表达量;采用实时荧光定量聚合酶链式反应法检测各组细胞中PI3K、Akt、mTOR和自噬相关因子Beclin 1、LC3Ⅰ、LC3Ⅱ、p62 mRNA的相对表达量。结果:与空白对照组比较,模型组细胞上清液中E2含量以及细胞中PI3K、Akt、mTOR mRNA和蛋白的相对表达量均显著降低(P<0.01),Beclin 1、LC3Ⅰ、LC3ⅡmRNA的相对表达量均显著升高(P<0.01)。与模型组比较,FSH组和苍附导痰丸中、高剂量组细胞上清液中E2含量显著升高,细胞中Beclin 1、LC3Ⅰ、LC3Ⅱ、p62 mRNA的相对表达量均显著降低(P<0.05或P<0.01);各给药组细胞中PI3K、Akt、mTOR mRNA和蛋白的相对表达量均显著升高(P<0.05或P<0.01)。结论:苍附导痰丸可能是通过激活PI3K/Akt/mTOR通路相关蛋白及其mRNA的表达,从而抑制GCs的自噬。

Effect of Cangfu Daotan Pill Containing Serum on Autophagy of Ovarian Granulosa Cells of Rats

OBJECTIVE:To study the effect of Cangfu daotan pill(CDP)containing serum on autophagy of ovarian granulosa cells(GCs)of rat.METHODS:Three-month-old SD rat were divided into normal saline group(normal saline,ig),FSH injection group(10.71 IU/kg,ih),CDP irrigation high-dose,medium-dose and low-dse groups0.5,1,2 mg/g(by crude drug),ig],with 6 rats in each group.They were given relevant medicine subcutaneously/intragastrically,once a day,for consecutive 3 days.After last medication,blood sample was collected from the abdominal aorta to obtain drug-containing serum.GCs of rat were divided into blank control group,model group,FSH group(positive control)and CDP high-dose,medium-dose and low-dose groups.The autophagy model was induced by giving testosterone propionate,except that the blank control group was directly added with 100μL serum of normal saline group.Then model group was given 100μL serum of normal saline group,and administration groups were given 100μL drug-containing serum of corresponding drug group.The contents of estradiol(E2)and progesterone(P)in supernatant of cells were determined by ELISA.Western blot assay was used to detect protein expression of PI3K,Akt and mTOR in cells.The mRNA expression of PI3K,Akt,mTOR,Beclin 1,LC3Ⅰ,LC3Ⅱand p62 were detected by RT-PCR.RESULTS:Compared with blank control group,the content of E2 in supernatant,relative mRNA and protein expression of PI3K,Akt and mTOR were decreased significantly in model group(P<0.01),while relative mRNA expression of Beclin 1,LC3Ⅰand LC3Ⅱwere increased significantly(P<0.01).Compared with model group,the content of E2 in supernatant were significantly increased in FSH group,CDP medium-dose and high-dose groups,while relative mRNA expression of Beclin 1,LC3Ⅰ,LC3Ⅱand p62 were decreased significantly(P<0.05 or P<0.01);relative mRNA and protein expression of PI3K,Akt and mTOR were increased significantly in administration groups(P<0.05 or P<0.01).CONCLUSIONS:CDP can inhibit autophagy of GCs by activating related protein and mRNA expression of PI3K/Akt/mTOR signaling pathway.