三七总皂苷调控PI3K/Akt/mTOR通路对乳腺增生大鼠乳腺组织细胞凋亡的影响

Effect of Notoginseng Total Saponins on Apoptosis of Mammary Gland Cells in Rats with Mammary Gland Hyperplasia by Regulating PI3K/Akt/mTOR Pathway

目的:探究三七总皂苷对乳腺增生大鼠乳腺组织细胞凋亡的影响及调控机制。方法:将60只雌性未孕SD大鼠随机分为正常组、模型组、三七总皂苷低、中、高剂量(10、20、40 mg·kg^(-1))组及三苯氧胺组(1.8 mg·kg^(-1)),每组10只。采用肌肉注射苯甲酸雌二醇和黄体酮构建乳腺增生大鼠模型。按照实验分组剂量进行灌胃给药。每日1次,连续30 d。正常组和模型组大鼠给予每日等量的生理盐水灌胃。给药结束后,游标卡尺测量大鼠第2对乳头直径;留取组织标本,苏木素-伊红(HE)染色观察乳腺组织病理形态变化;免疫组织化学法观察细胞凋亡调控蛋白B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、Bcl-2表达;蛋白免疫印迹法(Western blot)检测磷酸化磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白表达。结果:与正常组比较,模型组大鼠第2对乳头直径明显增大(P<0.05),乳房小叶体积增大,腺泡数量增加,乳腺组织呈现弥散性增生,乳腺组织Bcl-2蛋白表达及磷酸化(p)-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR明显增加(P<0.05),Bax表达明显降低(P<0.05),与模型组比较,三七总皂苷低、中、高剂量组和三苯氧胺组大鼠第2对乳头直径明显减小(P<0.05),乳腺小叶数量、腺泡数、分泌物量均较少,乳腺增生减轻,乳腺组织Bcl-2蛋白表达及p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR明显降低(P<0.05),Bax表达明显升高(P<0.05)。结论:三七总皂苷能够改善大鼠乳腺增生,其机制与调控PI3K/Akt/mTOR通路,促进乳腺组织细胞凋亡有关。

Objective:To explore the effect and regulatory mechanism of notoginseng total saponins on apoptosis of mammary gland cells in rats with mammary gland hyperplasia.Method:Sixty female non-pregnant SD rats were randomly divided into control group,model group,Notoginseng total saponins low-,medium-and high-dose(10,20,40 mg·kg^(-1),respectively)groups and tamoxifen group(1.8 mg·kg^(-1)),10 rats per group.Rat model of mammary gland hyperplasia was established by intramuscular injection of estradiol benzoate and progesterone.Oral administration was performed according to the experimental dose of each group,once a day for 30 consecutive days.The rats in the control group and the model group were given equal volume of normal saline by gavage every day.After administration,the diameter of the second pair of nipples of the rats was measured with vernier calipers,and breast tissue samples were collected and stained with hematoxylin and eosin(HE)to observe the pathological changes.Immunohistochemistry was used to determine the expression of apoptosis regulators B-cell lymphoma-2(Bcl-2)-associated X(Bax)and Bcl-2,and Western blot was conducted to detect the expression of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway-related proteins.Result:Compared with the control group,the model group had increased diameter of the second pair of nipples(P<0.05),elevated volume of mammary lobules and number of acinus,diffuse mammary gland hyperplasia,and up-regulated expression of Bcl-2 protein,increased ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR(P<0.05)and decreased expression of Bax in the mammary gland(P<0.05).Compared with the conditions in the model group,the diameter of the second pair of nipples of the rats in the notoginseng total saponins low-,medium-and high-dose groups and tamoxifen group was decreased(P<0.05),and the number of mammary lobules and acinus and the amount of secretions were reduced.In addition,the mammary gland hyperplasia was alleviated,and a decrease was observed in the expression of Bcl-2 protein and the ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR(P<0.05),and an increase in the expression of Bax(P<0.05).Conclusion:Notoginseng total saponins could improve mammary gland hyperplasia in rats,and its mechanism was related to regulating PI3K/Akt/mTOR pathway and promoting apoptosis of mammary gland cells.