基于网络药理学研究消肿止痛合剂治疗皮瓣缺血再灌注损伤的作用机制＊

目的 基于网络药理学方法探究消肿止痛合剂对皮瓣缺血再灌注损伤（Ischemia/Reperfusion,I/R）的作用机制。方法 通过TCMSP在线数据库分析平台获取消肿止痛合剂的潜在活性成分及作用靶点,利用GeneCards、TTD、OMIM、GAD数据库检索与皮瓣I/R相关靶点,将上述二者共有的靶点输入STRING数据库,获取PPI网络;通过Cytoscape 3.2.1软件中CytoNCA插件筛选潜在核心靶点,并构建“活性成分-核心靶点-疾病”网络;对关键节点进行基因本体（GO）和通路富集（KEGG）分析;将SD大鼠随机分为假手术组、模型组、消肿组,分别在给药5天,10天,15天,20天统计皮瓣成活率,同时在给药7天后透明硫酸纸描绘皮瓣成活情况,采集标本应用HE染色技术进行组织病理形态观察,应用Western Blot方法对关键靶点进行验证。结果 通过TCMSP数据库共检索出消肿止痛合剂药物主要有效成分所对应的靶点进行去重后,共获得238个潜在作用靶点,筛选得到消肿止痛合剂治疗皮瓣I/R的潜在靶点94个。GO功能富集分析发现消肿止痛合剂对防治皮瓣I/R主要与氧化应激反应、细胞对化学应激的反应、细胞对氧化应激的反应等相关。KEGG通路富集分析发现消肿止痛合剂防治皮瓣I/R主要与脂质、动脉粥样硬化与流体剪应力等相关。应用Western Blot技术验证排名靠前的PTGS2、PPARG、IL1B、AKT1、CXCL8五个关键核心靶点,结果提示消肿止痛合剂可减低皮瓣组织中PTGS2、IL1B、AKT1、CXCL8的蛋白表达。HE染色观察发现,消肿组大鼠皮瓣水肿明显减轻,组织间隙炎症细胞浸润减少。结论 消肿止痛合剂通过多成分、多靶点、多途径的作用方式,从氧化应激反应、细胞对化学应激的反应、细胞对氧化应激的反应等方面发挥防治皮瓣I/R的作用,为后续深入研究提供理论基础。

Study on the Mechanism of Xiaozhongzhitong Mixture on Ischemia/Reperfusion Injury of Skin Flap Based on Network Pharmacology

Objective To explore the mechanism of Xiaozhuzhitong mixture on Ischemia/Reperfusion (I/R) injury in flap based on online pharmacological methods.Methods The potential active components and their action targets of the mixture were obtained through the online database analysis platform TCMSP, and the targets related to flap I/R were searched using GeneCards, TTD, OMIM and GAD databases. The targets shared by the two were input into the STRING database to obtain PPI network. Potential core targets were screened by the CytoNCA plug-in in Cytoscape 3.2.1 software, and an "active component-core target-disease" network was constructed. The key nodes were analyzed by gene ontology (GO) and pathway enrichment (KEGG). SD rats were randomly divided into control group, model group, detumescence, respectively in 5 days, 10 days, 15 days, 20 days of statistics the survival rate of skin flap, respectively for 7 days after the transparent vegetable parchment depicting flap survival situation, application samples HE staining techniques for histopathologic morphology observation, using Western Blot method to verify this key targets.Results A total of 238 potential action targets were obtained after deweighting the main active components of detumor-analgesic mixture (Detumor-analgesic mixture) through TCMSP database, and 94 potential targets were obtained for detumor-analgesic mixture treatment of flap I/R. GO function enrichment analysis showed that the effect of Xiaozhuzhitong mixture on preventing and treating flap I/R was mainly related to oxidative stress response, cell response to chemical stress, cell response to oxidative stress and so on. KEGG pathway enrichment analysis showed that the prevention and treatment of flap I/R by Xiaozhongzhitong mixture was mainly related to lipids, fluid shear stress and atherosclerosis, etc. Finally, the top PTGS2, PPARG, IL1B, AKT1 and CXCL8 were determined as the key core targets. HE staining results showed that the edema of rat flaps in detumescence group was significantly reduced, and the infiltration of inflammatory cells in the interstitial space was reduced. Western Blot results indicated that detumor-analgesic mixture could reduce the protein expression of PTGS2, IL1B, AKT1 and CXCL8 in the flap tissue, thus protecting the I/R of the flap.Conclusion Through multi-component, multi-target and multi-pathway action, Xiaozhuzhitong mixture can improve the oxidative stress response, cell response to chemical stress.