香雪抗病毒口服液体外抗甲型流感病毒及炎症因子的作用机制研究

Mechanism of Xiangxue Kangbingdu Oral Liquid against influenza A virus and inflammatory factors in vitro

目的探讨香雪抗病毒口服液对甲型流感病毒的活性及其在炎症免疫应答中的潜在作用机制。方法采用噻唑蓝(MTT)染色法检测香雪抗病毒口服液对MDCK细胞和A549细胞的细胞毒性作用。采用细胞病变抑制法(CPE)及空斑减少实验对香雪抗病毒口服液进行抗病毒活性检测。采用免疫荧光法检测NP蛋白。实时荧光定量PCR测定炎症细胞因子的表达情况。采用蛋白印迹实验检测香雪抗病毒口服液对炎症相关信号通路的蛋白表达的影响。结果香雪抗病毒口服液对甲型流感病毒A/PR/8/34(H1N1)和A/Aichi/2/68(H3N2)均有一定的抗病毒抑制作用,半数细胞毒性浓度(TC50)分别为42.12、79.36 mg/mL,半数抑制浓度(IC_(50))分别为8.665、5.260 mg/mL,并可减少H1N1病毒空斑形成。香雪抗病毒口服液能呈剂量相关性地降低H1N1诱导的A549细胞炎症因子肿瘤坏死因子-α(TNF-α)、视黄酸(维甲酸)诱导基因蛋白I(RIGI)、趋化因子(CC基序)配体5(CCL5)、巨噬细胞炎症蛋白-1β(MIP-1β)、干扰素诱导蛋白-10(IP-10)、白细胞介素-10(IL-10)、λ干扰素(IFN-λ)、β干扰素(IFN-β)、白细胞介素-8(IL-8)m RNA的表达(P<0.05、0.01、0.001)。在流感病毒单复制周期中,香雪抗病毒口服液在早期阶段0~2 h干预,具有良好的抗流感病毒效果。香雪抗病毒口服液能显著抑制Toll样模式识别受体3(TLR3)、RIG-I、黑色素瘤分化相关基因5(MDA-5)受体的表达,下调核转录因子-κB亚基p65(NF-κB p65)、NF-κB抑制蛋白α(IκBα)、信号转导和转录激活因子1(STAT1)、STAT2、STAT3的磷酸化水平,且在20 mg/mL浓度下明显下调这些蛋白的表达。结论香雪抗病毒口服液不仅能有效抑制甲流感病毒在人宿主细胞中复制,降低流感病毒诱导的炎症因子表达,而且能显著抑制流感病毒诱导天然免疫信号通路的相关蛋白表达,表明香雪抗病毒口服液具有防治流感病毒的潜力。

Objective To investigate the activity of Xiangxue Kangbingdu Oral Liquid against influenza A virus,and its potential mechanism in inflammatory immune response.Methods Cytotoxic effects of Xiangxue Kangbingdu Oral Liquid on MDCK cells and A549 cells were detected by MTT staining.Antiviral activity of Xiangxue Kangbingdu Oral Liquid was detected by cytopathic inhibition(CPE)and plaque reduction test.NP protein was detected by immunofluorescence method.Real-time fluorescence quantitative PCR was used to determine the expression of inflammatory cytokines.Western blotting assay was used to detect the protein expression of antiviral and anti-inflammatory signaling pathway in Xiangxue Kangbingdu Oral Liquid.Results Xiangxue Kangbingdu Oral Liquid had certain antiviral inhibitory effect on influenza A virus A/PR/8/34(HIN1)and A/Aichi/2/68(H3N2),TC_(50) was 42.12 mg/mL and 79.36 mg/mL,respectively.ICso was 8.665 mg/mL and 5.260 mg/mL,respectively,and reduced plaque formation of HIN1 virus.The expression of TNF-a,RIG-I,CCL5,MIP-1β,IP-10,IL-10,IFN-2,IFN-β,IL-8 mRNA in A549 cells induced by H1N1 was decreased in a dose-dependent manner(P<0.05,0.01,0.001).In the single replication cycle of influenza virus,Xiangxue Kangbingdu Oral Liquid has a good anti-influenza virus effect after 0~2 h intervention in the early stage.Xiangxue Kangbingdu Oral Liquid can significantly inhibit the expression of TLR3,RIG-I,MDA-5 receptors.The phosphorylation levels of NF-kB p65,IkBa,STAT1,STAT2,and STAT3 were down-regulated,and the expression of these proteins was significantly down-regulated at 20 mg/mL.Conclusion Xiangxue Kangbingdu Oral Liquid could not only effectively inhibit the replication of influenza A virus in human host cells,reducing the expression of inflammatory factors induced by influenza virus,but also significantly inhibit the related proteins of the innate immune signaling pathway induced by influenza virus,which indicated that Xiangxue Kangbingdu Oral Liquid had the potential to prevent and treat influenza virus.