基于网络药理学探究金银花-连翘治疗奶牛乳房炎的作用机制及体外验证

Exploring the mechanism of Honeysuckle-Forsythia in the treatment of dairy cow mastitis and in vitro validation based on network pharmacology

为了探究金银花-连翘治疗奶牛乳房炎的作用机制,试验采用中药系统药理学分析(TCMSP)数据库获得金银花-连翘的活性成分及相关作用靶点蛋白,并将作用靶点蛋白进一步转化为靶点基因;利用DisGeNET数据库、GeneCards数据库和NCBI数据库检索奶牛乳房炎相关基因靶点;通过String数据库和Cystoscape v3.7.1软件构建金银花-连翘的有效活性成分靶点与奶牛乳房炎疾病靶点蛋白互作(PPI)网络图;采用David数据库进行金银花-连翘与奶牛乳房炎交集靶点所涉及的GO功能注释与KEGG信号通路富集分析,进而探究金银花-连翘治疗奶牛乳房炎的作用机制;建立奶牛乳腺上皮细胞炎症模型,采用ELISA检测金银花-连翘水提物对相关细胞因子表达的影响。结果表明:金银花-连翘主要活性成分包括β-谷甾醇、山奈酚、木犀草素、槲皮素等;共筛选获得239个药物活性成分主要涉及基因靶点及296个奶牛乳房炎基因靶点,其中交集靶点38个,以肿瘤坏死因子(TNF)、白细胞介素-6(IL-6)、白蛋白(ALB)、白细胞介素-1β(IL-1β)、白细胞介素-8(CXCL8)等为主。GO功能注释到的生物进程主要包括MAP激酶活性的正调控、炎症反应、一氧化氮生物合成过程的正调控等;细胞组分主要包括细胞核、细胞质、细胞表面等;分子功能主要包括细胞因子活性、转录因子活性等。KEGG信号通路主要富集在脂质与动脉粥样硬化、IL-17信号通路等;金银花-连翘水提物在体外可显著降低IL-6、IL-1β和CXCL8水平(P<0.05),发挥抗炎作用。说明金银花-连翘可能通过β-谷甾醇、山奈酚、木犀草素、槲皮素等活性成分调节IL-17信号通路,并作用于IL-6、IL-1β、CXCL8等靶点来治疗奶牛乳房炎。

In order to explore the mechanism of Honeysuckle-Forsythia in the treatment of dairy cowmastitis,in the experiment,the Traditional Chinese Medicine System Pharmacology(TCMSP) database was used to obtain the effective active ingredients and the related target proteins of honeysuckle-forsythia,and the target proteins were further converted into target genes.DisGeNET database,GeneCards database and NCBI database were used to search for dairy cow mastitis-related gene targets.The protein-protein interaction(PPI) network diagram of the effective active ingredient targets of Honeysuckle-Forsythia and the targets of dairy cow mastitis was constructed by String database and Cystoscape v3.7.1 software.The David database was used to analyze the GO function annotation and KEGG signaling pathway involved in the intersection targets of Honeysuckle-Forsythia and dairy cow mastitis,and then to explore the mechanism of Honeysuckle-Forsythia in the treatment of dairy cow mastitis.A model of dairy cow mammary epithelial cell inflammation was established,and ELISA was used to detect the effect of aqueous extract of Honeysuckle-Forsythia on the expression of related cytokines.The results showed that the main active ingredients of Honeysuckle-Forsythia included β-sitosterol,kaempferol,luteolin,quercetin,etc.A total of 239 active drug ingredients were screened,mainly involving gene targets and 296 gene targets for dairy cow mastitis,including 38 intersecting targets,mainly tumor necrosis factor(TNF),interleukin-6(IL-6),albumin(ALB),interleukin-1β(IL-1β),interleukin-8(CXCL8),etc.GO function enrichment showed that the biological processes mainly included the positive regulation of MAP kinase activity,inflammatory response,and nitric oxide biosynthesis process,etc.;the cell components mainly included nucleus,cytoplasm and cell surface;the molecular functions mainly included cytokine activity transcription factor activity,etc.;the KEGG pathway enrichment results showed that the main lipids involved in the pathway,atherosclerosis,and IL-17 signaling pathway,etc.;in vitro validation test showed that the aqueous extract of Honeysuckle-Forsythia could significantly reduce IL-6,IL-1β and CXCL-8 levels(P<0.05),which exerted anti-inflammatory effects.The results suggested that Honeysuckle-Forsythia might regulate the IL-17 signaling pathway through active ingredients such as β-sitosterol,kaempferol,luteolin,and quercetin,and act on target genes such as IL-6,IL-1β,and CXCL-8 to treat dairy cow mastitis.