清金益气方治疗新型冠状病毒的药效研究及潜在作用机制

Pharmacodyamic study and potential mechanism of Qingjin Yiqi Decoction for the treatment of COVID-19

关键词:  清金益气方  新型冠状病毒  抗病毒  抗炎  网络药理学  机制

Key words:  Qingjin Yiqi Decoction  COVID-19  antiviral  anti-inflammatory  network pharmacology  mechanism

摘要:

[目的] 通过对清金益气方的体外药效研究和结合网络药理学探讨其对新型冠状病毒感染的潜在作用机制。[方法] 使用清金益气方干预Omicron BA.1、Omicron BA.5、Omicron XBB等不同严重急性呼吸综合征冠状病毒2(SARS-CoV-2)变异株感染非洲绿猴肾细胞(Vero E6)，通过细胞病变效应(CPE)实验和病毒空斑减少实验验证清金益气方的抗病毒作用；进一步构建SARS-CoV-2感染人肺腺癌细胞(Calu3)诱导宿主炎症紊乱模型，通过实时荧光定量聚合酶链反应(RT-qPCR)检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、干扰素诱导蛋白10(IP-10)和单核细胞趋化蛋白-1(MCP-1)的信使核糖核酸(mRNA)表达水平，验证清金益气方的抗SARS-CoV-2感染诱导炎症因子过度表达的影响。最后运用TCMSP、GeneCards、DrugBank等数据库进行网络药理学分析。[结果] 清金益气方可以抑制Omicron BA.1、Omicron BA.5、Omicron XBB等新型冠状病毒的复制，同时对SARS-CoV-2感染细胞诱导产生细胞空斑数量具有明显的抑制作用；且对SARS-CoV-2感染诱导的IL-6、TNF-α、MCP-1和IP-10等炎症因子mRNA水平的过度表达具有显著抑制作用。通过网络药理学分析，槲皮素(quercetin)、山柰酚(Kaempferol)、柚皮素(naringenin)、β-谷甾醇(beta-sitosterol)和黄芩素(wogonin)是清金益气方的核心成分；IL-6、TNF、蛋白激酶B (AKT1)、信号传导和转录激活因子3(STAT3)、白细胞介素-1β(IL-1β)和转录因子P53(TP53)等靶点是其治疗新型冠状病毒感染的关键靶点，KEGG富集通路主要涉及白细胞介素-17(IL-17)、TNF、磷脂酰肌醇-3-激酶-蛋白激酶B (PI3K-Akt)及丝裂原活化蛋白激酶(MAPK)等信号通路。[结论] 清金益气方具有广谱的抗SARS-CoV-2变异株作用，且对SARS-CoV-2变异株感染诱导的宿主炎症紊乱具有调控作用。其潜在的作用机制可能通过槲皮素、山柰酚、黄芩素和柚皮素等黄酮类成分与IL-6、TNF、AKT1及STAT3等核心靶点结合，调控病毒和炎症相关的TNF、PI3K-Akt及MAPK等信号通路，发挥防治新型冠状病毒感染的作用。说明了清金益气方抗新型冠状病毒感染的可行性和科学性，为临床应用及深入研究防治SARS-CoV-2感染的机制提供了依据。

Abstract:

[Objective] To explore the potential mechanism of action of Qingjin Yiqi Decoction against Coronavirus Disease 2019(COVID-19) through in vitro efficacy study and combined network pharmacology. [Methods] Firstly,Qingjin Yiqi Decoction was used to intervene in the infection of Vero E6 cells with different subtypes of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2) virus strains Omicron BA.1,Omicron BA.5 and Omicron XBB,and the antiviral effects of Qingjin Yiqi Decoction were verified by the cytopathic effect(CPE) assay and the plaque reduction assay；then,a model of host inflammatory disorder induced by Sars-CoV-2 virus of Calu3 cells was constructed,and the mRNA expression levels of interleukin 6(IL-6),tumor necrosis factor-α(TNF-α),interferon inducible protein-10(IP-10),and Monocyte chemotactic protein(MCP-1) were detected by Real-Time Quantitative PCR(RT-qPCR) to validate the anti-SARS-CoV-2-induced inflammatory effect of Qingjin Yiqi Decoction. Finally,TCMSP,GeneCards,DrugBank,and other databases were used for network pharmacological analysis. [Results] Qingjin Yiqi Decoction inhibited SARS-CoV-2 strains Omicron BA.1,Omicron BA.5 and Omicron XBB replication in Vero E6 cells,meanwhile,the treatment with Qingjin Yiqi Decoction following infection also had a dose-dependent inhibitory effect on plaque formation of the SARS-CoV-2 virus. Qingjin Yiqi Decoction markedly reduced pro-inflammatory cytokines(IL-6,TNF-α,MCP-1 and IP-10) production at the mRNA levels. By network pharmacological analysis,quercetin,kaempferol,naringenin,beta-sitosterol,and wogonin are the core components of Qingjin Yiqi Decoction；IL-6,TNF,protein kinase B(AKT1),signal transducer and activator of transcription 3(STAT3),interleukin 1β(IL-1β),and tumor protein 53(TP53) were the key targets for the treatment of COVID-19,and the KEGG-enriched pathway mainly involves interleukin 17(IL-17),TNF,phosphatidylinositol-3-kinase-protein kinase B(PI3K-Akt),mitogen-activated protein kinase(MAPK) and other signaling pathways. [Conclusion] Qingjin Yiqi Decoction has broad-spectrum anti-SARS-CoV-2 variant and anti-inflammatory effects. The potential mechanism of action may be through the binding of flavonoid components such as quercetin,kaempferol,wogonin and naringenin to core targets such as IL-6,TNF,AKT1 and STAT3,modulating the virus- and inflammation-associated TNF,the PI3K-Akt and the MAPK signaling pathways,exerting a role in the prevention and treatment of COVID-19. These findings suggest that Qingjin Yiqi Decoction prevents viral attack,making it a novel strategy for controlling COVID-19 disease.