基于网络药理学和实验验证探究疏清颗粒治疗甲型H1N1流感的作用机制^*

Exploring the mechanism of Shuqing Granules in the treatment of influenza A (H1N1) based on network pharmacology and experimental verification

摘要

目的 探究疏清颗粒治疗甲型H1N1流感的潜在作用靶点及机制。方法 通过TCMSP数据库筛选疏清颗粒活性成分及靶点,结合TTD数据库获取甲型流感相关靶点,对共有靶点进行京都基因与基因组百科全书通路富集分析。构建甲型H1N1流感病毒感染A549细胞模型,CCK8法检测疏清颗粒对细胞活力影响及抗感染的效果;ELISA法检测疏清颗粒干预后分泌干扰素(IFN)的含量;Western blot和实时荧光定量PCR检测丝裂原活化蛋白激酶(MAPK)通路蛋白磷酸化水平和干扰素刺激基因(ISGs)mRNA表达。结果 共筛选出疏清颗粒活性成分89个,流感相关靶基因10个;富集分析显示MAPK信号通路为疏清颗粒抗流感作用的核心通路;疏清颗粒浓度为25.78 mg/mL、作用48 h时,抗流感效果最佳,治疗指数为4.51;疏清颗粒可降低细胞外信号调节激酶、p38蛋白磷酸化水平,促进IFN-β释放;上调ISGs(PKR、MxA、OAS1)mRNA表达。结论 疏清颗粒可显著抑制甲型H1N1流感,其机制可能与调节MAPK通路蛋白磷酸化、诱导IFN-β分泌及上调下游ISGs表达有关。

Abstract

Objective To explore the potential targets and mechanisms of Shuqing Granules in the treatment of influenza A (H1N1). Methods The active components and targets of Shuqing Granules were screened through the TCMSP, and the targets related to influenza A were obtained in combination with the TTD. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted on overlapping targets. A cellular model of influenza A (H1N1) virus infection was established using A549 cells, and the effect of Shuqing Granules on cell viability and anti-infection was detected by the CCK8 method. The content of interferon (IFN) secreted after Shuqing Granules intervention was detected by ELISA. The phosphorylation levels of mitogen-activated protein kinase(MAPK) pathway proteins and the mRNA expression of interferon-stimulated genes (ISGs) were detected by Western blot and real-time quantitative polymerase chain reaction. Results A total of 89 active components of Shuqing Granules and 10 influenza-related target genes were identified. MAPK signaling pathway was the core anti-influenza pathway of Shuqing Granules. Shuqing Granules showed an optimal anti-influenza effect at 25.78 mg/mL for 48 h (therapeutic index was 4.51). Shuqing Granules could reduce the phosphorylation levels of extracellular signalregulated kinase and p38 proteins, promote the release of IFN-β, and up-regulated the mRNA expressions of PKR, MxA and OAS1 in ISGs. Conclusion Shuqing Granules exert a significant inhibitory effect against influenza A (H1N1). The mechanism may be related to regulating the phosphorylation of MAPK pathway proteins, inducing IFN-β secretion and up-regulating the expression of downstream ISGs.

关键词

网络药理学 / 甲型H1N1流感病毒 / 疏清颗粒 / 丝裂原活化蛋白激酶信号通路 / 干扰素

Key words

Network pharmacology / Influenza A (H1N1) virus / Shuqing Granules / Mitogen-activated protein kinase signaling pathway / Interferon