健脾益肾化浊方对多囊卵巢综合征模型大鼠卵巢颗粒细胞凋亡蛋白及PI3K/AKT/mTOR通路的影响  

Effects of Jianpi Yishen Huazhuo Formula(健脾益肾化浊方)on Ovarian Granulosa Cell Apoptosis Protein and PI3K/AKT/mTOR Pathway in Polycystic Ovary Syndrome Model Rats

作　　者：陈苗 马会明 冯亚宏 张晓静 陈冬梅[5] 罗嘉琦 杜小利[1,2] CHEN Miao;MA Huiming;FENG Yahong;ZHANG Xiaojing;CHEN Dongmei;LUO Jiaqi;DU Xiaoli(School of Traditional Chinese Medicine,Ningxia Medical University,Yinchuan,750004;Key Laboratory of Integrated Tradi-tional Chinese and Western Medicine Research on High Incidence Disease,Ningxia Regional;Key Laboratory of Fertility Mainte-nance of Ministry of Education,Ningxia Medical University;Ningxia Chinese Medicine Research Center;Ningxia Key Laboratory of Stem Cell and Regenerative Medicine,General Hospital of Ningxia Medical University)

机构地区：[1]宁夏医科大学中医学院,宁夏回族自治区银川市750004 [2]宁夏区域高发病中西医结合防治研究重点实验室 [3]宁夏医科大学生育力保持教育部重点实验室 [4]宁夏中医医院暨中医研究院 [5]宁夏医科大学总医院宁夏干细胞与再生医学重点实验室

出　　处：《中医杂志》2023年第15期1585-1592,共8页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81660813);宁夏自然科学基金(2020AAC03430,2022AAC03216);宁夏少数民族医药现代化教育部重点实验室/宁夏区域高发病中西医结合防治研究重点实验室开放课题(311/20020502)。

摘　　要：目的 探讨健脾益肾化浊方治疗多囊卵巢综合征(PCOS)的可能作用机制。方法 70只SD雌性大鼠随机分为空白组和模型组各15只,二甲双胍组,健脾益肾化浊方低、中、高剂量组各10只。除空白组外,其余各组均采用来曲唑灌胃联合高脂饲料喂养21天构建PCOS模型。造模成功后,空白组和模型组用10 ml/(kg·d)生理盐水灌胃;二甲双胍组用盐酸二甲双胍片100 mg/(kg·d)灌胃;健脾益肾化浊方低、中、高剂量组分别用健脾益肾化浊方药物混悬液1.275、2.55、5.10 g/(kg·d)灌胃,干预30天。将卵巢称重并计算卵巢指数;ELISA法测定大鼠血清促卵泡生成素(FSH)、促黄体生成素(LH)、睾酮(T)、抗苗勒管激素(AMH)和雌二醇(E2)水平;HE染色观察卵巢组织形态改变;免疫组织化学染色法检测大鼠卵巢组织半胱氨酸天冬氨酸酶3 (Caspase-3)、半胱氨酸天冬氨酸酶9 (Caspase-9)和磷脂酰肌醇-3激酶(PI3K)、蛋白激酶B (AKT)、哺乳动物雷帕霉素靶蛋(mTOR)蛋白阳性表达;Western blot法检测大鼠卵巢组织促凋亡蛋白(BAX)、抗凋亡蛋白(Bcl-2)、Caspase-9、细胞色素C (Cytochrome-C)和PI3K、AKT、mTOR蛋白相对表达。结果 与空白组比较,模型组大鼠卵巢组织以囊性扩张卵泡为主,颗粒细胞层减少,间质增生明显;卵巢指数、血清T、LH、AMH、卵巢组织Caspase-3、Caspase-9平均光密度和BAX/Bcl-2值、Cytochrome-C、Caspase-9蛋白相对表达量明显升高,血清E2、FSH及卵巢组织PI3K、AKT、mTOR平均光密度和蛋白表达显著降低(P<0.05或P<0.01)。与模型组比较,健脾益肾化浊方各剂量组和二甲双胍组卵巢组织多囊样改变得到改善,可见各级卵泡和黄体,颗粒细胞层增厚,卵巢指数、血清LH、AMH、卵巢组织Caspase-9平均光密度和蛋白相对表达明显降低,血清E2、FSH,卵巢组织PI3K和AKT平均光密度,PI3K、AKT和mTOR蛋白相对表达显著升高(P<0.05或P<0.01);二甲双胍组和健脾益肾化浊方中、高�Objective To explore the possible mechanism of Jianpi Yishen Huazhuo Formula(健脾益肾化浊方,JYHF)in treating polycystic ovary syndrome(PCOS).Methods Seventy SD female rats were randomly divided into blank group and model group with 15 rats each,metformin group,JYHF low-,medium-and high-dose groups with 10 rats each.Except for the blank group,all the other groups were fed with letrozole orally combined with high-fat diet for 21 days to establish the PCOS model.After successful modeling,the blank group and the model group were intragastrically adminis⁃tered with 10 ml/(kg·d)of normal saline,while the metformin group was administered with 100 mg/(kg·d)of metformin hy⁃drochloride tablets by gavage,and the JYHF low-,medium-and high-dose groups were administered intragastrically with 1.275,2.55,and 5.10 g/(kg·d)of JYHF suspension respectively,all for 30 days.The ovaries were weighed and the ova-rian index was calculated,and serum follicle-stimulating hormone(FSH),luteinizing hormone(LH),testosterone(T),anti-Müllerian hormone(AMH)and estradiol(E 2)levels were measured through ELISA.HE staining was used to observe the morphological changes of ovarian tissue.Immunohistochemical staining was used to detect caspase-3,caspase-9 and phosphatidylinositol-3 kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR)protein positive expression.Western blot was performed to detect relative expressions of rat ovarian tissue pro-apoptotic protein(BAX),anti-apoptotic protein(Bcl-2),Caspase-9,Cytochrome-C,PI3K,AKT,and mTOR proteins.Results Compared to those in the blank group,the ovarian tissue of the rats in the model group was dominated by cystic dilated follicles,and the granulosa cell layer was reduced,with obvious interstitial hyperplasia;the ovarian index,serum T,LH,AMH,as well as Caspase-3 and Caspase-9 average optical density,BAX/Bcl-2 value,the relative expression of Cytochrome-C and Caspase-9 proteins significantly increased,while serum E 2 and FSH levels,average optical density and protein express

关 键 词：多囊卵巢综合征 健脾益肾化浊方 颗粒细胞 细胞凋亡 PI3K/AKT/mTOR信号通路 

分 类 号：R285.5[医药卫生—中药学]