热毒宁注射液抗流感病毒作用及机制研究  

Effect and mechanism of Reduning injection on influenza A virus

作　　者：颜海燕 王萌 王辉强 李玉环 YAN Hai-yan;WANG Meng;WANG Hui-qiang;LI Yu-huan(CAMS Key Laboratory of Antiviral Drug Research,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院、北京协和医学院医药生物技术研究所,中国医学科学院抗病毒药物研究重点实验室,北京100050

出　　处：《药学学报》2023年第6期1596-1602,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金青年科学基金资助项目(8220131872);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-048)。

摘　　要：为了探究热毒宁注射液(Reduning injection, RI)抗甲型流感病毒的作用及其机制,本研究在A549和MDCK细胞上采用CCK-8 (cell counting kit-8)法检测药物细胞毒性;采用Western blot和CPE (cytopathic effect)方法检测RI对病毒蛋白、细胞病变和病毒毒力的影响评价其药效;Western blot法检测血红素加氧酶1 (heme oxygenase 1, HO-1)的蛋白水平、转录因子NF-E2相关因子2 (nuclear factor erythroid 2-related factor 2, Nrf2)核转运、P38 MAPK (mitogen-activated protein kinases)和ERK1/2 (extracellular signal-regulated kinases 1/2)磷酸化;实时荧光定量PCR检测IFN-α/β (interferon-α/β) mRNA含量;双荧光素酶报告基因检测Nrf2的转录活性。结果显示, RI剂量依赖性地抑制流感病毒诱导的MDCK细胞病变,降低流感病毒M2蛋白水平和病毒毒力,具有明确的抗流感病毒作用;促进P38 MAPK和ERK1/2的磷酸化并激活Nrf2核转录因子活性,促进细胞核内Nrf2蛋白表达,从而上调HO-1蛋白表达,最终增加IFN-α/β mRNA水平。综上,本研究表明, RI通过激活MAPK/Nrf2/HO-1信号通路发挥抗流感病毒作用,揭示了RI抗流感病毒新的作用机制,为其临床治疗流感病毒提供了理论依据。The purpose of this study is to investigate the effect of Reduning injection(RI)on influenza A virus(IAV)and its mechanism.We evaluated the cytotoxicity of RI in A549 and MDCK cells by cell counting kit-8(CCK-8)assay.Western blot and cytopathic effect(CPE)assays were applied to test the effects of RI on viral protein,CPE and virus virulence to evaluate its inhibitory effect.The proteins level of heme oxygenase 1(HO-1),nuclear factor erythroid 2-related factor 2(Nrf2),phosphorylation of P38 mitogen-activated protein kinases(MAPK)and extracellular signal-regulated kinases 1/2(ERK1/2)were detected by Western blot.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the RNA expression of interferon-α/β(IFN-α/β).The relative luciferase reporter assay was used to analyze the promoter activity and transcriptional regulation of Nrf2.The results indicated that RI inhibited IAV-induced MDCK cytopathies in a dose-dependent manner,decreased M2 protein of influenza virus and viral titer,indicating that it has definite effect on inhibiting IAV.RI promotes the phosphorylation of P38 MAPK and ERK1/2,activates the activity of Nrf2 nuclear transcription factor,increases the expression of Nrf2 protein in the nucleus,thus up-regulates the expression of HO-1 protein,and ultimately increases the IFN-α/βmRNA level.In summary,our results demonstrated that RI inhibits the replication of IAV by activating MAPK/Nrf2/HO-1 signaling pathway,revealing a new mechanism of RI against influenza virus,and providing theoretical basis for clinical treatment of influenza virus.

关 键 词：热毒宁注射液 流感病毒 血红素加氧酶1 干扰素 抗流感药物 

分 类 号：R966[医药卫生—药理学]