Wei Wen-Yang, Wan Hai-Tong, Yu Li, Lu Yi-Yu, He Yu
Zhejiang Chinese Medical University, Hangzhou 310053, China.
Zhejiang Center for Disease Control and Prevention, Hangzhou 310053, China.
Zhongguo Zhong Yao Za Zhi. 2018 Feb;43(3):563-570. doi: 10.19540/j.cnki.cjcmm.20171113.010.
To study the effect and underlying mechanism of Mahuang Tang against influenza A virus , the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods, and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER), median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4, TLR7, MyD88 and TRAF6 in MDCK cells at 24, 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92,1.59 g·L⁻¹ respectively, the TI values were 12.53, 38.78 respectively, and when the concentration of Mahuang Tang was 5.00 g·L⁻¹, ER values were 50.21%, 98.41% respectively, showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile, as compared with the virus group, the virus load was significantly inhibited in Mahuang Tang groups, and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4, TLR7,MyD88 and TRAF6 at 24, 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.
为研究麻黄汤抗甲型流感病毒的作用及潜在机制,本研究以流感病毒感染的犬肾传代细胞(MDCK)为载体,采用细胞病变效应(CPE)法检测甲型流感病毒株(A/PR8/34)对MDCK细胞的半数组织培养感染剂量(TCID₅₀)。以阻断流感病毒侵入宿主细胞和抗流感病毒生物合成作为两种不同给药方式,然后采用甲基噻唑基四氮唑蓝(MTT)法测定麻黄汤的抗病毒有效率(ER)、半数有效浓度(EC₅₀)和治疗指数(TI)。采用定量实时聚合酶链反应(RT-PCR)检测处理后24、48 h MDCK细胞中的病毒载量以及TLR4、TLR7、MyD88和TRAF6的mRNA表达水平。实验结果表明,A/PR8/34对MDCK细胞的TCID₅₀为1×10⁻⁴.³²/mL。两种不同处理方式的EC₅₀值分别为4.92、1.59 g·L⁻¹,TI值分别为12.53、38.78,当麻黄汤浓度为5.00 g·L⁻¹时,ER值分别为50.21%、98.41%,表明麻黄汤可阻断流感病毒进入宿主细胞并显著抑制其生物合成。同时,与病毒组相比,麻黄汤各给药组病毒载量均显著降低,且麻黄汤高、中剂量组在处理后24、48 h对降低TLR4、TLR7、MyD88和TRAF6的mRNA表达有显著作用。由此证明,麻黄汤抗甲型流感病毒的机制与抑制流感病毒复制以及TLR4和TLR7信号通路相关基因的mRNA表达有关。