基于“味性化味”理论加权网络与AlphaFold2反向对接的藏药晶珠肝泰舒治疗肝炎物质基础及分子机制研究

Study on the material basis and molecular mechanism of Tibetan medicine Jingzhu Gantaishu in treating hepatitis based on the "Ro Nus Zhurjes" weighted network and AlphaFold2-based reverse docking

关键词:  藏药  晶珠肝泰舒  加权网络  AlphaFold2反向对接  分子动力学模拟

Key words:  Tibetan medicine  Gantaishu capsules  Weighted Network  AlphaFold2-based Reverse Docking  Molecular dynamics simulation

摘要:

目的 本研究将“味性化味”理论参数化并赋予药味权重值，构建加权网络，联合基于AlphaFold2的反向对接、分子动力学模拟及体外实验，探究晶珠肝泰舒治疗肝炎的物质基础及分子机制。方法 文献检索和TCMSP数据库收集晶珠肝泰舒所含化学成分，SwissTargetPrediction预测其潜在靶点。从OMIM、GeneCards、DrugBank、TTD数据库收集肝炎相关靶点。将藏医“味性化味”理论参数化并赋予药味权重值，采用邻接矩阵（Adjacency Matrix，AM）方法构建“药味-成分-靶点”加权网络，直观关联成分与靶点，显示多成分多靶点协同特征，并在此基础上筛选度值大于中位数的核心成分与核心靶点进行基于AlphaFold2的反向对接，确定结合能力最优的核心代表性成分，通过分子动力学模拟探究成分-靶点动态互作机制。体外验证采用CCK-8法检测细胞活力，Hoechst染色法、流式细胞术检测细胞凋亡，JC-10染色法检测线粒体膜电位。结果 加权网络清晰展示了晶珠肝泰舒成分与靶点的多对多关联，揭示了复方多成分多靶点的协同作用特征，由此筛选出度值大于中位数的关键潜在活性化学成分和靶点各80个。反向对接发现甘草酚与硬脂酰辅酶A去饱和酶（Stearoyl-CoA Desaturase，SCD）的对接打分最高，为结合能力最强的核心代表性成分，其通过氢键、π相互作用与疏水相互作用稳定嵌入SCD的活性口袋；分子动力学模拟证实其与SCD的关键残基His-171形成氢键和π-π相互作用，可能通过影响活性中心的构象干扰酶的正常催化功能，进而抑制SCD的活性。体外实验证实，甘草酚可提高LPS诱导的肝星形细胞活力（P < 0.05），抑制细胞凋亡（P < 0.05）并上调线粒体膜电位。结论 本研究基于“味性化味”理论，通过加权网络揭示了晶珠肝泰舒多成分多靶点的整体作用特征；预测甘草酚可能通过靶向SCD发挥抗肝炎活性，并初步验证了其对肝星形细胞损伤的保护效果。该研究策略为藏药复方物质基础研究和藏医理论现代化提供了新范式。

Abstract:

OBJECTIVE To parameterize the “Ro Nus Zhurjes” theory and assign medicinal flavor weights, construct a weighted network to reveal the multi-component, multi-target synergistic features of Jingzhu Gantaishu capsules in treating hepatitis, and further integrate AlphaFold2-based reverse docking, molecular dynamics simulations, and in vitro experiments to elucidate the material basis and molecular mechanism. METHODS Chemical constituents of Jingzhu Gantaishu capsules were collected through literature retrieval and the TCMSP database, and their potential targets were predicted using SwissTargetPrediction. Hepatitis?related targets were retrieved from OMIM, GeneCards, DrugBank, and TTD databases. The Tibetan medicine theory of “Ro Nus Zhurjes” was parameterized and assigned medicinal flavor weights, and a “medicinal flavor–component–target” weighted network was constructed using the adjacency matrix method to intuitively correlate components with targets and display multi-component, multi-target synergistic characteristics. Based on this network, core components and core targets with degree values exceeding the median were screened and subjected to AlphaFold2-based reverse docking with hepatitis targets. The component with the best binding capacity was identified, and molecular dynamics simulations were performed to explore the dynamic interaction mechanisms. In vitro, CCK-8 assay was used to detect cell viability, Hoechst staining and flow cytometry were employed to observe and quantify apoptosis, and JC?10 staining was applied to measure mitochondrial membrane potential. RESULTS The weighted network clearly displayed many-to-many associations between components and targets of Jingzhu Gantaishu, revealing the multi-component, multi-target synergistic characteristics, through which 80 potential active components and 80 corresponding targets with degree values above the median were screened out. Reverse docking identified glycyrol as having the highest docking score with stearoyl?CoA desaturase (SCD), representing the core component with the strongest binding capacity. Glycyrol was stably embedded in the active pocket of SCD through the synergistic effects of hydrogen bonds, π interactions, and hydrophobic interactions. Molecular dynamics simulations confirmed that glycyrol formed hydrogen bonds and π–π stacking with the key residue His?171 of SCD, which may interfere with the normal catalytic function by affecting the active?site conformation, thereby inhibiting SCD activity. In vitro experiments demonstrated that glycyrol increased the viability of LPS?induced HSC?T6 cells (P < 0.05), inhibited apoptosis (P < 0.05), and up?regulated mitochondrial membrane potential. CONCLUSION Based on the “Ro Nus Zhurjes” theory, the weighted network revealed the overall multi-component, multi-target action characteristics of Jingzhu Gantaishu; it predicted that glycyrrhizin may exert its anti-hepatitis activity by targeting SCD, and preliminarily validated its protective effect against damage to hepatic astrocytes. This strategy provides a new paradigm for investigating the material basis of Tibetan medicine formulas and modernizing Tibetan medicine theory.