目的 基于网络药理学、分子对接及体内实验验证探讨霍山石斛对非酒精性脂肪肝（non-alcoholic fatty liver disease，NAFLD）的疗效及作用机制。方法 利用文献检索霍山石斛活性成分，采用SwissTargetPrediction数据库预测其潜在靶点，采用DisGeNET和GeneCards数据库筛选NAFLD靶点，并将药物靶点和疾病靶点取交集，构建蛋白质-蛋白质相互作用网络，借助DAVID数据库进行基因本体功能和京都基因与基因组百科全书通路富集分析，构建“活性成分-靶点-疾病-通路”网络，并采用AutoDock等软件对关键活性成分和核心靶点进行分子对接验证。采用高脂高糖饮食诱导NAFLD小鼠模型，设置对照组、模型组、吡格列酮（10 mg/kg）组和霍山石斛低、中、高剂量（200、400、600 mg/kg）组，连续给药8周。检测血清三酰甘油（triglyceride，TG）、总胆固醇（total cholesterol，TC）、低密度脂蛋白胆固醇（low density lipoprotein cholesterol，LDL-C）、高密度脂蛋白胆固醇（high density lipoprotein cholesterol，HDL-C）、丙氨酸氨基转移酶（alanine aminotransferase，ALT）及天冬氨酸氨基转移酶（aspartate aminotransferase，AST）的含量；ELISA检测肝脏中TC、TG、白细胞介素-6（interleukin-6，IL-6）、IL-1β及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；油红O染色和苏木素-伊红（HE）染色检测肝组织病理变化；Western blotting检测肝组织中胰岛素受体（insulin receptor，InsR）、磷脂酰肌醇-3-激酶（phosphatidylinositol-3-kinase，PI3K）、蛋白激酶B（protein kinase B，Akt）、糖原合成酶激酶-3β（glycogen synthase kinase-3β，GSK-3β）蛋白表达。结果 霍山石斛中筛选出石斛酚、铁皮石斛素A、4,4′-dihydroxy-3,5-dimethoxydihydrostilbene、柚皮素等38个活性成分，与NAFLD共同靶点有155个，作用于胰岛素、AKT1、白蛋白、TNF、IL-6、血管内皮生长因子A、过氧化物酶体增殖物激活受体γ、IL-1β等19个核心靶点，主要涉及癌症途径、PI3K/Akt通路、晚期糖基化终产物及其受体信号通路、缺氧诱导因子信号通路、TNF信号通路等，分子对接显示前5位核心成分与前5位核心靶点具有较好的接合能力。动物实验结果证实成功构建小鼠NAFLD模型，与模型组比较，霍山石斛组小鼠体质量、肝脏指数以及血清TG、TC、ALT、AST、LDL-C和肝脏TC、TG水平显著降低（P<0.05、0.01），血清HDL-C水平显著升高（P<0.01），改善肝脏脂肪蓄积和变性，肝脏InsR、PI3K、p-Akt/Akt蛋白表达水平显著增加（P<0.05、0.01），p-GSK-3β/GSK-3β蛋白表达显著减少（P<0.01）。结论 霍山石斛对NAFLD具有保护作用，其机制可能与激活PI3K/Akt通路、改善胰岛素抵抗和减少炎症反应有关。

Objective To explore the therapeutic effect and mechanism of Dendrobium huoshanense on non-alcoholic fatty liver disease (NAFLD) based on network pharmacology, molecular docking and in vivo experimental verification. Methods The active components of D. huoshanense were searched by literature, the potential targets were predicted by SwissTargetPrediction database, the targets of NAFLD were screened by DisGeNET and GeneCards databases, drug targets and disease targets were intersected to construct protein-protein interaction network. Gene ontology function and Kyoto encyclopedia of genes and genomes pathway enrichment analysis were carried out by DAVID database, and “active ingredient-target-disease-pathway” network was constructed, molecular docking verification of key active ingredients and core targets was carried out by AutoDock and other software. NAFLD mouse model was induced by high-fat and high-sugar diet, control group, model group, pioglitazone group (10 mg/kg) and D. huoshanense low-, medium-and high-dose (200, 400, 600 mg/kg) groups were set up, drugs were continuously given for eight weeks. Levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected; The levels of TC, TG, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in liver were detected by ELISA. The pathological changes of liver tissue were detected by oil red O staining and hematoxylin-eosin (HE) staining. Western blotting was used to detect the expressions of insulin receptor (InsR)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway related proteins in liver tissue. Results A total of 38 active components were screened from D. huoshanense, including moscatilin, dendrobixin A, 4,4′-dihydroxy-3,5-dimethohydroxystilbene, naringin, and 155 common targets with NAFLD, acting on 19 core targets such as insulin, protein kinase B1 (AKT1), albumin, tumor necrosis factor (TNF), interleukin-6 (IL-6), vascular endothelial growth factor A, peroxisome proliferator-activated receptor γ, IL-1β, mainly involving cancer pathway, PI3K/Akt pathway, advanced glycation end products-receptor for advanced glycation end products signaling pathway, hypoxia inducible factor signaling pathway and TNF signaling pathway. Molecular docking showed that the top five core components have good binding ability with the top five core targets. The animal experiment results confirmed that the mouse NAFLD model was successfully established. Compared with model group, body weight, liver index, levels of TG, TC, ALT, AST, LDL-C in serum and TC, TG in liver of D. huoshanense group were significantly decreased (P < 0.05, 0.01), and HDL-C level in serum was significantly increased (P < 0.01), improved the accumulation and degeneration of liver fat. And expression levels of InsR, PI3K and p-Akt/Akt proteins in liver were significantly increased (P < 0.05, 0.01), while the expression of p-GSK-3β/GSK-3β protein was significantly decreased (P < 0.01). Conclusion D. huoshanense has protective effect on NAFLD, and its mechanism may be related to activating PI3K/Akt pathway, improving insulin resistance and reducing inflammatory reaction.

R285.5

国家自然科学基金联合基金项目（U19A2009）；省部共建安徽道地中药材品质提升协同创新中心项目（教科信厅函[2022]4号）；安徽省教育厅项目（皖教秘科[2014]44号）