背景:中风是一种高发病率的衰弱状态,残疾,和死亡率显著影响患者的生活质量。在中国,温阳扶源方广泛用于治疗缺血性中风。然而,潜在的机制仍然未知,因此,探索该公式的潜在作用机制对中风治疗具有重要的现实意义。
目的:本研究采用网络药理学,分子对接,和体内实验来澄清活性成分,潜在目标,温阳复元方在脑缺血再灌注损伤中的分子机制,以期为该配方的后续研究提供坚实的科学基础。
方法:利用中药系统药理学数据库和分析平台,对温阳福源方的有效成分进行筛选。采用网络药理学方法探讨温阳复元方治疗脑缺血再灌注损伤的潜在靶点和作用机制。制备大脑中动脉阻塞/再灌注2hSD大鼠模型,应用TTC染色和改良的神经严重程度评分来检查大鼠的神经功能缺损。HE染色和Nissl染色观察大鼠的病理变化。免疫荧光标记和Elisa分析用于检测某些蛋白质和相关因子的表达水平。同时应用qRT-PCR和Westernblotting检测疾病相关信号通路中连锁蛋白和mRNA的表达水平。
结果:我们确定了文阳富源配方中的62种关键活性成分,具有222个高度重要的I/R目标,形成138对药物成分和成分目标,前五名是槲皮素,山奈酚,木犀草素,β-谷甾醇,和豆甾醇。关键目标包括TP53、RELA、TNF,STAT1和MAPK14(p38MAPK)。与脑缺血再灌注损伤相关的靶点富含化学反应,酶结合,内膜系统,虽然丰富的途径包括脂质和动脉粥样硬化,流体剪切应力和动脉粥样硬化,糖尿病并发症中的AGE-RAGE信号传导。此外,文阳富源配方中的5种主要活性成分和靶标表现出较高的结合亲和力(如豆甾醇和MAPK14,总能量<-10.5Kcal/mol)。在动物实验中,文阳富源方减少了脑组织损伤,增加了存活神经元的数量,并下调S100β和RAGE蛋白的表达。此外,温阳复元方组TP53、RELA和p38MAPKmRNA的相对表达水平显著下调,血清IL-6和TNF-a因子水平降低。经过温阳扶源配方治疗,AGE-RAGE信号通路和下游NF-kB/p38MAPK信号通路相关蛋白被显著调节。
结论:本研究利用网络药理学,分子对接,和动物实验,以确定温阳扶源方的潜在机制,这可能与AGE-RAGE信号通路的调节以及下游NF-kB/p38MAPK通路中靶蛋白和mRNA的抑制有关。
BACKGROUND: Stroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment.
OBJECTIVE: This study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe.
METHODS: Active ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways.
RESULTS: We identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, β-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <-10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100β and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated.
CONCLUSIONS: This study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.