复方银花解毒颗粒抗冠状病毒药效作用及初步机制研究

Antiviral effect of Fufang yinhua jiedu(FFYH)granules against coronavirus and its potential mechanism

为了探究复方银花解毒颗粒(FFYH)体外抗冠状病毒药效作用及机制,本研究首先采用细胞病变效应(cell pathogenic effect,CPE)初步评价了FFYH在Huh7、Huh7.5、H460和C3A细胞模型中的细胞毒性及其抗冠状病毒药效作用;然后采用实时荧光定量PCR法(quantitive reverse transcription PCR,qRT-PCR)探讨了FFYH对冠状病毒RNA复制及冠状病毒感染所致炎症因子mRNA的复制水平的影响;最后采用Western blot法和免疫荧光法对FFYH抑制冠状病毒蛋白表达及其潜在作用机制进行了探讨。结果显示,FFYH对Huh7、Huh7.5、H460和C3A细胞的半数毒性浓度(50%toxic concentration,TC_(50))分别为2035.21、5245.69、2935.28和520μg·mL^(-1);在Huh7和Huh7.5细胞上对冠状病毒HCoV-229E的半数抑制浓度(50%inhibitory concentration,IC_(50))分别为438.16和238.54μg·mL^(-1),治疗指数(safety index,SI)分别为4.64和21.99;在H460细胞上对冠状病毒HCoV-OC43的IC_(50)为165.13μg·mL^(-1),SI为17.78;FFYH在无毒浓度下不仅能够剂量依赖性地抑制冠状病毒HCoV-OC43和HCoV-229E的RNA复制与蛋白表达,而且能有效抑制冠状病毒感染所致炎症因子白介素-6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白介素-8(interleukin-8,IL-8)的表达,其机制可能与其抑制丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路及核转录因子(nuclear transcription factor-κB,NF-κB)的核转位有关。综上,本研究表明FFYH具有良好的体外抗冠状病毒的作用,这为其临床用于抗冠状病毒肺炎的治疗提供了理论依据。

To investigate the effect of Fufang yinhua jiedu(FFYH)granules against coronavirus and its potential mechanism,we used Huh7,Huh7.5,H460,and C3A cell lines as in vitro models to evaluate the cytotoxicity and antiviral activity of FFYH by observation of cell pathogenic effect(CPE);and then the inhibitory effect of FFYH on the transcription expression of coronavirus RNA and inflammatory factor mRNA were evaluated by quantitive reverse transcription PCR(qRT-PCR);finally,the inhibitory effect of FFYH on the expression of coronavirus protein and its underlying mechanism against coronavirus were investigated by Western blot and immunofluorescence.Our results indicated that 50%toxic concentration(TC_(50))FFYH on Huh7,Huh7.5,H460,and C3A cells were 2035.21,5245.69,2935.28 and 520µg·mL^(-1),respectively;50%inhibitory concentration(IC_(50))of FFYH on HCoV-229E in Huh7 and Huh7.5 cells were 438.16 and 238.54µg·mL^(-1)with safety index(SI)of 4.64 and 21.99,respectively;IC_(50) of FFYH on HCoV-OC43 in H460 cells was 165.13µg·mL^(-1) with SI of 17.78.Moreover,FFYH not only could inhibit the replication of coronaviruses(HCoV-OC43 and HCoV-229E)through inhibiting the transcription of viral RNA and the expression of viral protein,but also effectively suppress the expression of inflammatory factors interleukin-6(IL-6),tumor necrosis factorα(TNF-α)and interleukin-8(IL-8)at mRNA level caused by coronaviruses,which might be associated with the inhibitory effect of FFYH on mitogen-activated protein kinase(MAPK)pathway and the nuclear translocation of nuclear transcription factor-κB(NF-κB).In summary,our results demonstrated that FFYH exhibited a good in vitro anti-coronavirus effect,which provides a theoretical basis for its clinical use in the treatment of anti-coronavirus pneumonia.