金荞麦水提物通过调控RIG-Ⅰ信号通路与细胞自噬抑制甲型流感病毒H1N1的复制  

Aqueous Extract from Fagopyrum dibotrys Inhibits Influenza Virus A/H1N1 Replication by Regulating RIG-ⅠSignaling Pathway and Autophagy

作　　者：张哲雯 冯燕[2] 徐昌平[2] 高见[2] 吴巧凤[1] Zhang Zhewen;Feng Yan;Xu Changping;Gao Jian;Wu Qiaofeng(College of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053;Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310057)

机构地区：[1]浙江中医药大学药学院,杭州310053 [2]浙江省疾病预防控制中心,杭州310057

出　　处：《中药药理与临床》2022年第1期103-110,共8页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金项目(编号:81473335/H2803);浙江省医药卫生科技计划项目(编号:2018KY334)

摘　　要：目的:探讨金荞麦水提物体外抗甲型流感病毒H1N1的效果及其作用机制。方法:考察金荞麦水提物抗H1N1感染犬肾(Madin-Darby canine kidney,MDCK)细胞的活性及作用方式;转录组测序(RNA-sequencing,RNA-seq)分析金荞麦水提物处理后感染细胞的基因差异表达,筛选与抗病毒相关的信号通路;蛋白质印迹法(Western blot,WB)检测LC3Ⅱ的表达水平,荧光显微镜观察自噬流,分析金荞麦水提物对感染细胞自噬水平的影响。结果:金荞麦水提物具有良好的抑制细胞内H1N1复制的活性,细胞对MDCK 1.25 mg/mL抑制率为51.02%,5 mg/mL抑制率为98.22%。与模型对照组比较,金荞麦水提物处理感染细胞后,引起2682个基因差异表达。KEGG通路富集分析表明,差异表达基因富集在RIG-Ⅰ和细胞自噬信号通路。金荞麦水提物1.25、2.5、5 mg/mL处理的感染细胞中,LC3Ⅱ蛋白表达及LC3Ⅱ/LC3Ⅰ比值显著低于模型对照组(P<0.01),且出现自噬溶酶体。结论:金荞麦水提物具有显著体外抗H1N1的活性,其作用机制与对宿主细胞RIG-Ⅰ信号通路的调控,以及抑制感染诱导的细胞自噬相关。Objective:To investigate the in vitro antiviral activity and the underlying mechanism of aqueous extract of Fagopyrum dibotrys(AEFD)against influenza virus A/H1N1.Methods:The activity of AEFD against H1N1 infection in Madin-Darby canine kidney(MDCK)cells was evaluated.RNA-sequencing(RNA-seq)was performed to mine the differentially expressed genes(DEGs)in infected cells after AEFD treatment.The antiviral-related signaling pathways were identified by KEGG pathway enrichment.The expression level of LC3Ⅱwas determined by western blotting,and the autophagy flow was observed under a fluorescence microscope.Results:AEFD showed good inhibitory activity against H1 N1 replication,with the inhibition rates of 51.02%(1.25 mg/mL)and 98.22%(5 g/mL)in MDCK cells.Compared with the infected cells,the AEFD-treated cells showed 2682 DEGs,which were mainly involved in RIG-Ⅰand autophagy signaling pathways.The expression levels of LC3Ⅱin the cells treated with 1.25,2.5,and 5 mg/mL AEFD were significantly lower than that in the model group(P<0.01).Meanwhile,autophagolysosome were observed in the AEFD-treated cells.Conclusion:AEFD may exerted the activity against H1N1 in vitro by regulating the RIG-Ⅰsignaling pathway and inhibiting the H1N1-induced autophagy.

关 键 词：金荞麦水提物 甲型流感病毒 转录组测序 差异表达基因 RIG-Ⅰ信号通路 细胞自噬 

分 类 号：R285[医药卫生—中药学]