基于Akt/mTOR信号通路探究芍药甘草汤和生麦芽对高泌乳素血症的作用机制

To Explore the Mechanism of Shaoyao Gancao Decoction(芍药甘草汤)and Raw Maiya(Hordei Fructus Germinatus)on Hyperprolactinemia Based on Akt/mTOR Signaling Pathway

目的:分别考察生麦芽、芍药甘草汤对高泌乳素血症(HPRL)大鼠血清泌乳素相关激素水平及乳腺组织病理形态的影响,以及对乳腺组织p-Akt、p-mTOR、LC3等蛋白表达水平的影响。方法:随机取8只大鼠作为正常组,32只大鼠皮下注射甲氧氯普胺制备高泌乳素血症模型。造模成功的32只大鼠随机分为模型组、芍药甘草汤组、生麦芽组和溴隐亭组,每组8只。各药物组大鼠灌胃给予相应药物,正常组和模型组大鼠给予等体积蒸馏水,连续给药30 d。采用酶联免疫吸附法(ELISA)测定大鼠血清中泌乳素(PRL)、促黄体生成素(LH)、雌二醇(E2)、孕酮(Pg)、促卵泡素(FSH)含量;采用苏木精-伊红(HE)染色观察大鼠乳腺组织病理形态变化;蛋白免疫印迹法(Western blotting)及免疫组化(IHC)检测大鼠乳腺组织p-Akt、p-mTOR、LC3等蛋白表达水平。结果:模型组大鼠血清PRL水平明显高于正常组(P<0.01),血清LH、FSH、Pg和E2水平明显低于正常组(P<0.01);芍药甘草汤组、生麦芽组、溴隐亭组大鼠血清PRL水平明显低于模型组(P<0.01);芍药甘草汤组大鼠血清FSH、Pg水平明显高于模型组(P<0.05或P<0.01);生麦芽组大鼠血清FSH、Pg和E2水平明显高于模型组(P<0.05或P<0.01);溴隐亭组大鼠血清LH、FSH、Pg和E2水平明显高于模型组(P<0.05或P<0.01)。HE染色结果显示模型组大鼠乳腺增生,小叶数量、腺泡数量明显多于正常组,小叶体积大于正常组,并出现腺泡和导管扩张;芍药甘草汤组、生麦芽组和溴隐亭组大鼠乳腺增生情况均得到改善,乳腺小叶数量、腺泡和导管增生明显少于模型组。Western blotting检测结果显示,模型组大鼠乳腺组织中p-Akt和p-mTOR蛋白相对表达量及LC3-Ⅱ/Ⅰ高于正常组(P<0.01);芍药甘草汤组、生麦芽组和溴隐亭组大鼠乳腺组织中p-Akt和p-mTOR蛋白相对表达量及LC3-Ⅱ/Ⅰ均低于模型组(P<0.01)。IHC检测结果显示,模型组大鼠乳腺组织中Akt、p-Akt、mTOR、p-mTOR、LC3蛋白表达水平明显高于正常组(P<0.05或P<0.01);芍药甘草汤组、生麦芽组、溴隐亭组大鼠乳腺组织中p-Akt、p-mTOR、LC3蛋白表达水平明显低于模型组(P<0.05或P<0.01);芍药甘草汤组、溴隐亭组大鼠乳腺组织中mTOR蛋白表达水平明显低于模型组(P<0.05)。结论:芍药甘草汤、生麦芽能够有效降低高泌乳素血症大鼠血清泌乳素水平,抑制乳腺组织增生,其机制可能与减轻大鼠乳腺组织中自噬作用有关。

Objective:To investigate the effects of raw Maiya(Hordei Fructus Germinatus)and Shaoyao Gancao Decoction on serum prolactin-related hormone levels and pathological morphology of breast tissue in rats with hyperprolactinemia(HPRL),as well as the expression levels of p-Akt,p-mTOR,LC3 and other proteins in breast tissue.Methods:Eight rats were randomly selected as the normal group,and 32 rats were injected subcuta neously with metoclopramide to prepare a hyperprolactinemia model.The 32 successfully modeled rats were randomly divided into model group,Shaoyao Gancao Decoction group,raw Maiya(Hordei Fructus Germinatus)group and bromocriptine group,with 8 rats in each group.The rats in each drug group were given corresponding drugs by gavage,and the rats in the normal group and the model group were given equal volume of distilled water for 30 days.The contents of prolactin(PRL),luteinizing hormone(LH),estradiol(E2),progesterone(Pg)and follicle stimulating hormone(FSH)in serum of rats were determined by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining was used to observe the pathological changes of rat breast tissue.The protein expression levels of p-Akt,p-mTOR and LC3 in rat breast tissue were detected by Western blotting and immunohistochemistry(IHC).Results:The serum PRL level in the model group was significantly higher than that in the normal group(P<0.01),and the serum LH,FSH,Pg and E2 levels were significantly lower than those in the normal group(P<0.01).The serum PRL levels in Shaoyao Gancao Decoction group,raw Maiya(Hordei Fructus Germinatus)group and bromocriptine group were significantly lower than those in the model group(P<0.01).The levels of serum FSH and Pg in the Shaoyao Gancao Decoction group were significantly higher than those in the model group(P<0.05 or P<0.01).The levels of serum FSH,Pg and E2 in the raw Maiya(Hordei Fructus Germinatus)group were significantly higher than those in the model group(P<0.05 or P<0.01).The levels of serum LH,FSH,Pg and E2 in bromocriptine group were significantly higher than those in model group(P<0.05 or P<0.01).The results of HE staining showed that the number of lobules and acinar cells in the model group was significantly higher than that in the normal group,the volume of lobules was larger than that in the normal group,and acinar and duct dilatation appeared.The hyperplasia of mammary glands in Shaoyao Gancao Decoction group,raw Maiya(Hordei Fructus Germinatus)group and bromocriptine group was improved,and the number of mammary lobules,acinar and ductal hyperplasia were significantly less than those in the model group.The results of Western blotting showed that the relative expression levels of p-Akt and p-mTOR proteins and LC3Ⅱ/Ⅰin the breast tissues of rats in the model group were higher than those in the normal group(P<0.01).The relative expression levels of p-Akt and p-mTOR proteins and LC3Ⅱ/Ⅰin breast tissues of rats in Shaoyao Gancao Decoction group,raw Maiya(Hordei Fructus Germinatus)group and bromocriptine group were lower than those in model group(P<0.01).The results of IHC showed that the expression levels of Akt,p-Akt,mTOR,p-mTOR and LC3 protein in the breast tissue of the model group were significantly higher than those in the normal group(P<0.05 or P<0.01).The expression levels of p-Akt,p-mTOR and LC3 protein in breast tissue of rats in Shaoyao Gancao Decoction group,raw Maiya(Hordei Fructus Germinatus)group and bromocriptine group were significantly lower than those in model group(P<0.05 or P<0.01).The expression level of mTOR protein in breast tissue of rats in Shaoyao Gancao Decoction group and bromocriptine group was significantly lower than that in model group(P<0.05).Conclusion:Shaoyao Gancao Decoction and raw Maiya(Hordei Fructus Germinatus)can effectively reduce the level of serum prolactin in rats with hyperprolactinemia and inhibit the hyperplasia of breast tissue.The mechanism may be related to the reduction of autophagy in rat breast tissue.