榆栀止血颗粒干预多囊卵巢综合征大鼠的机制研究

Mechanism of Yuzhi Zhixue Granules in treating polycystic ovary syndrome in rats

探讨榆栀止血颗粒对多囊卵巢综合征(polycystic ovary syndrome,PCOS)模型大鼠的治疗作用并运用代谢组学技术探究其作用机制。将大鼠随机分为正常组,模型组,榆栀止血颗粒低、中、高(0.5、1.5、4.5 g·kg^(-1)·d^(-1))剂量组,阳性药组(二甲双胍0.2 g·kg^(-1)·d^(-1))。除正常组外,其余各组灌胃1 mg·kg^(-1)·d^(-1)来曲唑并饲喂高糖高脂饲料,连续造模40 d后,模型组继续灌胃来曲唑和蒸馏水,给药组灌胃来曲唑和不同剂量榆栀止血颗粒,阳性药组灌胃来曲唑和二甲双胍,正常组灌胃同等体积蒸馏水,每日1次,连续50 d。采用血糖仪检测口服糖耐量;酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测大鼠血清胰岛素水平;放射免疫法检测大鼠血清中卵泡刺激素(follicle-stimulating hormone,FSH)、黄体生成素(luteinizing hormone,LH)、雌二醇(estradiol,E_(2))和睾酮(testosterone,T)水平;全自动生化分析仪检测血清中高密度脂蛋白胆固醇(high density lipoprotein-cholesterol,HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein-cholesterol,LDL-C)、甘油三酯(triglyceride,TG)水平;苏木精-伊红(hematoxylin-eosin,HE)染色观察大鼠卵巢组织病理改变;蛋白免疫印迹(Western blot)法检测大鼠卵巢组织中磷脂酰肌醇3-激酶(phosphatidylinositide 3-kinases,PI3K)、蛋白激酶B(protein kinase B,Akt)蛋白及其磷酸化水平;超高效液相色谱-串联质谱(LC-MS/MS)检测PCOS大鼠粪便及血清代谢产物。结果表明,与模型组相比,给药组大鼠糖耐量受损改善,胰岛素敏感性增强;血清HDL-C、E_(2)水平升高,TG、T水平下降;卵巢组织病理多囊样状态改善;卵巢组织p-PI3K、p-Akt蛋白表达水平升高;粪便代谢筛选出46个差异代谢物和10条代谢通路,主要涉及不饱和脂肪酸的生物合成、酪氨酸代谢等;血清代谢筛选出34个差异代谢物和15条代谢通路,主要涉及不饱和脂肪酸的生物合成、氨酰基tRNA生物合成等。综上所述,榆栀止血颗粒可改善PCOS糖脂、性激素紊乱,调节卵巢状态,其机制可能与影响粪便及血清代谢,激活PI3K/Akt通路有关。

This study aims to explore the therapeutic effect of Yuzhi Zhixue Granules on polycystic ovary syndrome(PCOS)in rats and explain the underlying mechanism by metabolomics.Rats were randomized into normal,model,low-,medium-,and high-dose(0.5,1.5,and 4.5 g·kg^(-1)·d^(-1))Yuzhi Zhixue Granules,and positive control(metformin,0.2 g·k^(-1)·d^(-1))groups.The rats in other groups except the normal group were administrated with 1 mg·kg^(-1)·d^(-1) letrozole and fed with a high-sugar and high-fat diet for the modeling of PCOS.After 40 days of modeling,the normal and model groups received distilled water and letrozole+distilled water,respectively,and other groups received letrozole and corresponding drugs,once a day for 50 days.The oral glucose tolerance test(OGTT)was carried out and enzyme-linked immunosorbent assay(ELISA)was conducted to detect insulin release,and the radioimmunoassay was employed to measure the serum levels of follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E_(2)),and testosterone(T).The serum levels of high density lipoprotein-cholesterol(HDL-C),low density lipoprotein-cholesterol(LDL-C),and triglyceride(TG)were determined by an automatic biochemical analyzer.The pathological changes in the ovary were observed by hematoxylin-eosin(HE)staining.The protein levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),and their phosphorylated forms in the ovary were determined by Western blot.Ultra-high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)was employed to study the fecal and serum metabolites in the rat model of PCOS.The results showed that compared with the model group,drug administration repaired the impaired glucose tolerance,enhanced the insulin sensitivity,elevated the serum levels of HDL-C and E_(2),lowered the serum levels of TG and T,ameliorated the pathological changes in the ovarian tissue,and up-regulated the protein levels of p-PI3K and p-Akt in the ovarian tissue.A total of 46 differential metabolites and 10 metabolic pathways in the fecal samples were screened out,which were mainly related to the biosynthesis of unsaturated fatty acids and tyrosine metabolism.In terms of the serum metabolism,34 differential metabolites and 15 metabolic pathways were screened out,mainly related to the biosynthesis of unsaturated fatty acids and aminoacyl-tRNA.In conclusion,Yuzhi Zhixue Granules can alleviate the disorders of glucose,lipids,and sex hormones and improve the ovarian status in the rat model of PCOS by regulating the serum and fecal metabolism and activating the PI3K/Akt pathway.