新疆医科大学学报

目的 通过网络药理学及动物实验探讨复方首乌颗粒治疗高脂血症的作用机制。方法 通过TCMSP、TCMID和Herb数据库筛选复方首乌颗粒的有效成分；经SwissTargetPrediction数据库预测复方首乌颗粒的作用靶点；采用GeneCards和OMIM数据库预测高脂血症的作用靶点；通过Venny 2.1平台对复方首乌颗粒作用靶点和高脂血症作用靶点取交集并绘制韦恩图；通过String数据库构建蛋白质-蛋白质互作(Protein-protein interaction, PPI)网络图；通过Cytoscape 3.9.1软件构建“复方首乌颗粒-活性成分-靶点”可视化网络图；将交集靶点导入DAVID数据库进行基因本体(Gene ontology, GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路富集分析；通过分子对接技术检验复方首乌颗粒有效成分与核心靶点的结合活性。取48只昆明小鼠随机选取40只建立高脂血症模型，余下8只为正常对照组，通过喂养高脂饲料建立高脂血症模型，通过测定小鼠血清中三酰甘油(Triglyceride, TG)、总胆固醇(Total cholesterol, TC)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol, LDL-C)水平进行模型验证。造模成功后，随机分为模型组、复方首乌颗粒低、中、高剂量组、血脂康组，每组8只。正常对照组与模型组均以0.9%生理盐水灌胃，其余各组均以相应药物灌胃。4 w后，检测血清中TG、TC、LDL-C、高密度脂蛋白胆固醇(High-density lipoprotein cholesterol, HDL-C)及肝功能的水平。采用苏木精-伊红染色及油红O染色观察小鼠肝脏病理变化。应用逆转录实时定量聚合酶链反应和蛋白免疫印迹法检测肝组织PI3K/AKT/mTOR信号通路中基因和蛋白表达水平。结果 通过数据库筛选得到复方首乌颗粒有效成分119个，主要有效成分6个，有效成分作用靶点815个；高脂血症作用靶点1 785个，复方首乌颗粒治疗高脂血症交集靶点234个；PPI网络分析获得16个核心靶点；GO功能富集分析结果显示，复方首乌颗粒主要参与脂质代谢，细胞生物调节等生物过程。KEGG富集分析结果显示，复方首乌颗粒通过PI3K/AKT信号通路发挥治疗高脂血症的关键作用。分子对接结果显示，芒柄花苷，毛蕊异黄酮葡萄糖苷分别与AKT1,mTOR具有良好的结合活性。动物实验结果表明，与正常对照组相比，模型组小鼠血清中TG、TC、LDL-C、碱性磷酸酶(Alkaline phosphatas, ALP)、谷丙转氨酶(Alanine, ALT)、谷草转氨酶(Aspartate, AST)的水平升高，HDL-C水平降低(P<0.05);肝脏中p-PI3K、p-AKT1、p-mTOR蛋白表达水平升高(P<0.05)。与模型组相比，复方首乌颗粒中、高剂量组与血脂康组小鼠血清中TG、TC、LDL-C、ALP、ALT、AST的水平均降低，HDL-C水平升高(P<0.05);复方首乌颗粒中、高剂量组与血脂康组肝脏中p-PI3K、p-AKT1、p-mTOR蛋白表达水平降低(P<0.05)。HE染色结果显示，模型组肝脏有明显的脂肪变性，并有大量脂质空泡的形成，复方首乌颗粒中、高剂量组和血脂康组泡性脂变明显减少；油红O染色显示，模型组橘红色脂滴面积增多，复方首乌颗粒中、高剂量组小鼠给药后，脂质沉积改善明显且呈剂量依赖性。结论 复方首乌颗粒对高脂血症小鼠具有显著的治疗作用，同时可以改善高脂血症引起的肝损伤，其调脂作用机制与PI3K/AKT/mTOR信号通路有关。

Objective To explore the mechanism of action of Fufang Shouwu Granules in the treatment of hyperlipidemia(HLP) through network pharmacology and animal experiments. Methods The effective components of Fufang Shouwu Granules were screened through the TCMSP, TCMID and Herb databases. The target sites of Fufang Shouwu Granules were predicted by SwissTarget Prediction database. The GeneCards and OMIM databases were used to predict the target sites of HLP. The intersection of the action targets of Fufang Shouwu Granules and HLP was taken through the Venny 2.1 platform, and the Venny diagram was drawn.The protein-protein interaction(PPI) network diagram was constructed through the String database. The visual network diagram of "Fufang Shouwu Granules-Active Ingredients-Targets" was constructed through Cytoscape 3.9.1 software. The intersection targets were imported into the DAVID database for Gene ontology(GO) function and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis. The binding activity of the active ingredients of Fufang Shouwu Granules to the core target was examined by molecular docking technology. 48 Kunming mice were randomly selected. 40 of them were used to establish the hyperlipidemia model, and the remaining 8 were normal controls. The HLP model was established by feeding high-fat diet. The triglyceride(TG) and total cholesterol in the serum of the mice were determined. Model validation was conducted using TC and low-density lipoprotein cholesterol(LDL-C) levels. After successful modeling, the patients were randomly divided into the model group, the low-, medium-and high-dose groups of Fufang Shouwu Granules, and the Xuezhikang group, with 8 rats in each group. Both of normal control group and model group were given 0.9% normal saline by gavage, while the other groups were given corresponding drugs by gavage. 4 weeks later, the levels of TG, TC, LDL-C, high-density lipoprotein cholesterol(HDL-C) and liver function in the serum were detected. The pathological changes of the liver in mice were observed by hematoxylin-eosin staining and oil red O staining. The expression levels of genes and proteins in the PI3K/AKT/mTOR signaling pathway in liver tissue were detected by reverse transcription quantitative real-time polymerase chain reaction and Western blot. Results Through database screening, 119 active ingredients, including 6 major effective components and 815 active ingredient action targets of Fufang Shouwu Granules were obtained. There were 1 785 targets for HLP, and 234 intersection targets for the treatment of HLP with Fufang Shouwu Granules. PPI network analysis identified 16 core targets. The results of GO functional enrichment analysis showed that Fufang Shouwu Granules mainly participated in biological processes such as lipid metabolism and cellular biological regulation. The KEGG enrichment analysis results showed that Fufang Shouwu Granules played a key role in the treatment of HLP through the PI3K/AKT signaling pathway. The results of molecular docking showed that miscanthus glycosides and vermiculone glucosides had good binding activities with AKT1 and mTOR, respectively. The results of animal experiments showed that compared with the normal control group, the levels of TG, TC, LDL-C, alkaline phosphatase(ALP), alanine aminotransferase(ALT) and aspartate aminotransferase in the serum of mice in the model group.The level of AST was increased, and the level of HDL-C was decreased(P<0.05). The protein expression levels of p-PI3K, p-AKT1 and p-mTOR in the liver were increased(P<0.05). Compared with model group, the levels of TG, TC, LDL-C, ALP, ALT and AST in the serum of mice in the medium and high-dose groups of Fufang Shouwu Granules and the Xuezhikang group were all decreased, while the level of HDL-C was increased(P<0.05). The medium and high-dose groups of Fufang Shouwu Granules and Xuezhikang group was decreased the protein expressions of p-PI3K, p-AKT1 and p-mTOR in the liver(P<0.05). The results of HE staining showed that the liver in the model group had obvious steatosis and the formation of a large number of lipid vacuoles. The bubble-like lipidosis was significantly reduced in the medium and high-dose groups of Fufang Shouwu Granules and Xuezhikang group. Oil red O staining showed that the area of orange-red lipid droplets in the model group was increased. After administration to the medium and high-dose groups of Fufang Shouwu Granules in mice, lipid deposition improved significantly and in a dose-dependent manner. Conclusion Fufang Shouwu Granules have a significant therapeutic effect on mice with HLP and can also improve liver damage caused by HLP. Its lipid-regulating mechanism is related to the PI3K/AKT/mTOR signaling pathway.