蛇伤膏外敷治疗毒蛇咬伤火毒证的疗效观察与网络药理学机制研究

Observation of curative effect and study of network pharmacological mechanism of external application of snake wound ointment in treating fire-toxicity syndrome caused by venomous snake bites

目的:观察蛇伤膏外敷治疗毒蛇咬伤火毒证患者的临床疗效,运用网络药理学方法及分子对接技术分析蛇伤膏治疗毒蛇咬伤潜在的作用机制。方法:选取安徽中医药大学第一附属医院入诊的毒蛇咬伤火毒证患者60例,随机分为对照组和试验组,每组30例。对照组采用常规中医适宜技术治疗,试验组在对照组治疗基础上采用蛇伤膏外敷治疗。观察两组患者临床疗效、症状与体征积分、疼痛与肿胀程度评分、中性粒细胞百分比(NEUT%)、凝血酶原时间(PT)、C反应蛋白(CRP)、白细胞介素-6(IL-6)水平;基于药物与疾病数据库筛选蛇伤膏-毒蛇咬伤靶点及共有靶点,利用DAVID平台进行基因本体(GO)功能分析和京都基因与基因组百科全书(KEGG)通路富集分析,运用Cytoscape构建蛋白相互作用网络(PPI)并筛选关键基因与靶点,利用AutoDockPools1.5.7进行分子对接及PyMol可视化。结果:两组患者临床疗效有效率均为100%,试验组治愈率(67%,20/30例)高于对照组(27%,8/30例),差异有统计学意义(P<0.01);与治疗前比较,试验组治疗后症状与体征积分、疼痛与肿胀程度评分、NEUT、PT、CRP、IL-6水平均下降,差异有统计学意义(P<0.01);两组患者治疗后比较,试验组肿胀程度评分、NEUT均低于对照组,差异有统计学意义(P<0.05),临床疗效、疼痛评分、肢体症状与体征评分、PT、CRP、IL-6指标均明显低于对照组,差异有统计学意义(P<0.01);网络药理学分析显示,蛇伤膏-毒蛇咬伤的核心靶点为表皮生长因子(EGF)、表皮生长因子受体(EGFR)、纤连蛋白1(FN1)、白细胞介素-6(IL-6)、基质金属蛋白酶9(MMP9)、转化生长因子β1(TGF-β1)、肿瘤坏死因子(TNF),核心成分包括槲皮素、黄芩素、木犀草素、汉黄芩素等,主要涉及MAP激酶活性的正调节、细胞外空间、蛋白酶结合、AGE/RAGE通路、TNF等生物功能与信号通路,分子对接结果良好。结论:蛇伤膏治疗毒蛇咬伤火毒证效果显著;网络药理学初步探究了蛇伤膏通过多靶点、多通路途径治疗毒蛇咬伤,与临床相关指标具有联系性,为后续研究提供理论与依据。

Objective:To observe the curative effect of external application of snake wound ointment in treating firetoxicity syndrome caused by venomous snake bites,and to analyze the potential mechanism of snake wound ointment in treating venomous snake bites by using network pharmacology and molecular docking techniques.Methods:60 patients with fire-toxicity syndrome caused by venomous snake bites admitted to the First Affiliated Hospital of Anhui University of Chinese Medicine were randomly divided into control group and experimental group,with 30 cases in each group.The patients in the control group were treated with conventional traditional Chinese medicine appropriate technology,and the patients in the experimental group were treated with snake wound ointment on the treatment basis of the control group.The clinical efficacy,symptom and sign scores,pain and swelling degree scores,neutrophil percentage(NEUT%),prothrombin time(PT),C-reactive protein(CRP)and interleukin-6(IL-6)levels of the two groups were observed.Based on the Drug and Disease Database,the snake wound ointment and venomous snake bites targets and common targets were screened.The DAVID platform was used for gene ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Cytoscape was used to construct the protein-protein interaction network(PPI)and screen the key genes and targets.AutoDockPools 1.5.7 was used for molecular docking and PyMol visualization.Results:The clinical effective rates of the two groups were 100%,and the cure rate of the experimental group(67%,20/30 cases)was higher than that of the control group(27%,8/30 cases).The difference was statistically significant(P<0.01).Compared with those before treatment,the scores of symptoms and signs,pain and swelling degree,NEUT,PT,CRP and IL-6 levels in the experimental group after treatment were decreased,and the differences were statistically significant(P<0.01).After treatment,the swelling degree score and NEUT of the experimental group were lower than those of the control group,and the differences were statistically significant(P<0.05).The clinical efficacy,pain score,limb symptoms and signs score,PT,CRP,IL-6 index were significantly lower than those of the control group,and the differences were statistically significant(P<0.01).Network pharmacology analysis showed that the core targets of snake wound ointment-venomous snake bites were epidermal growth factor(EGF),epidermal growth factor receptor(EGFR),fibronectin 1(FN1),interleukin-6(IL-6),matrix metalloproteinase 9(MMP9),transforming growth factorβ1(TGF-β1),and tumor necrosis factor(TNF).The core components included quercetin,baicalein,luteolin,wogonin,etc.,mainly involved in the positive regulation of MAP kinase activity,extracellular space,protease binding,AGE/RAGE pathway,TNF and other biological functions and signaling pathways.The molecular docking results were good.Conclusion:Snake wound ointment has a significant effect on the treatment of fire-toxicity syndrome caused by venomous snake bites.Network pharmacology preliminary explores that venomous snake bites can be treated by snake wound ointment through multi-targets and multi-pathways,which is related to clinical related indicators and provides theory and basis for follow-up research.