Table 3_Umbelliferone as an effective component of Rhodiola for protecting the cerebral microvascular endothelial barrier in cSVD.xlsx

ObjectiveRhodiola is a common Chinese herb in the treatment of cerebral small vessel disease (cSVD). Umbelliferone, one of the effective components of Rhodiola, can protect the endothelial barrier. But its mechanisms are still unclear. Therefore, this study is aimed to explore mechanisms of umbelliferone of an effective component of Rhodiola in protecting the cerebral microvascular endothelial barrier in cSVD.
MethodsFirstly, ETCM, SwissTargetPrediction and literatures were used to screen components and targets of Rhodiola. GeneCards was used to obtain targets of cSVD. STRING and Cytoscape were utilized for building the PPI and C-T network. Metascape was utilized to construct GO and KEGG enrichment analysis. Then, molecular docking was employed to evaluate the binding ability of the compounds for their respective target molecules. Ultimately, the endothelial cell damage caused by OGD was employed to explore the protective impact of umbelliferone, a bioactive constituent of Rhodiola, on the endothelial barrier. Endothelial cell leakage and migration assays were used to assess the permeability and migration ability of endothelial cells. IF and WB techniques were employed to ascertain the expression of endothelial tight junction protein. The major target proteins and related pathways were validated by WB.
ResultsSix effective components and 106 potential targets were identified and 1885 targets of cSVD were obtained. Nine key targets were selected. GO and KEGG enrichment analysis suggested that effects of Rhodiola in cSVD were associated with PI3K-Akt, Ras, Rap1 and MAPK signal pathways. Molecular docking results showed good binding ability between 28 pairs of key proteins and compounds. Umbelliferone of an effective component of Rhodiola can protect tight junction proteins and improve the permeability and migration ability of endothelial cells damaged by OGD through MMP9, MMP2, CCND1, PTGS2 and PI3K-Akt, Ras, Rap1 signaling pathways.
ConclusionOur study systematically clarified mechanisms of Rhodiola in treating cSVD by network pharmacology and molecular docking, characterized by its multi-component, multi-target and multi-pathway effects. This finding was validated through in vitro tests, which demonstrated that umbelliferone of an effective component in Rhodiola can protect the brain microvascular endothelial barrier. It provided valuable ideas and references for additional research.

红景天（Rhodiola）是治疗脑小血管病（cerebral small vessel disease, cSVD）的常用中药。伞形酮（Umbelliferone）作为红景天的有效成分之一，可保护内皮屏障，但其具体作用机制尚未明确。为此，本研究旨在探讨红景天有效成分伞形酮保护脑微血管内皮屏障以干预cSVD的作用机制。
方法 首先，通过ETCM、SwissTargetPrediction及相关文献筛选红景天的活性成分与对应靶点；借助GeneCards数据库获取cSVD相关靶点；利用STRING与Cytoscape软件构建蛋白相互作用（Protein-Protein Interaction, PPI）网络及成分-靶点（C-T）网络；采用Metascape平台开展基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。随后，通过分子对接实验评估化合物与其对应靶点分子的结合能力。最终，采用氧糖剥夺（Oxygen Glucose Deprivation, OGD）诱导的内皮细胞损伤模型，探究红景天活性成分伞形酮对内皮屏障的保护作用。通过内皮细胞渗漏实验与迁移实验评估内皮细胞的通透性与迁移能力；采用免疫荧光（Immunofluorescence, IF）与蛋白质印迹（Western Blot, WB）技术检测内皮紧密连接蛋白的表达水平；通过WB实验验证核心靶点蛋白及相关信号通路的表达情况。
结果 本研究共筛选得到6种有效成分及106个潜在靶点，同时获取1885个cSVD相关靶点，最终筛选出9个核心靶点。GO与KEGG富集分析结果显示，红景天治疗cSVD的作用机制与PI3K-Akt、Ras、Rap1及丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）信号通路密切相关。分子对接结果表明，28对核心蛋白与化合物间呈现出良好的结合活性。红景天有效成分伞形酮可通过调控基质金属蛋白酶9（MMP9）、基质金属蛋白酶2（MMP2）、细胞周期蛋白D1（CCND1）、前列腺素内过氧化物合酶2（PTGS2）及PI3K-Akt、Ras、Rap1信号通路，改善OGD诱导的内皮细胞损伤，保护紧密连接蛋白，提升内皮细胞的通透性与迁移能力。
结论 本研究通过网络药理学与分子对接技术，系统阐明了红景天治疗cSVD的多成分、多靶点、多通路作用特点，并通过体外实验验证了红景天有效成分伞形酮对脑微血管内皮屏障的保护作用，为后续相关研究提供了极具价值的思路与参考依据。