目的 探討香雪抗病毒口服液對(duì)甲型流感病毒的活性及其在炎癥免疫應(yīng)答中的潛在作用機(jī)制。方法 采用噻唑藍(lán)（MTT）染色法檢測(cè)香雪抗病毒口服液對(duì)MDCK細(xì)胞和A549細(xì)胞的細(xì)胞毒性作用。采用細(xì)胞病變抑制法（CPE）及空斑減少實(shí)驗(yàn)對(duì)香雪抗病毒口服液進(jìn)行抗病毒活性檢測(cè)。采用免疫熒光法檢測(cè)NP蛋白。實(shí)時(shí)熒光定量PCR測(cè)定炎癥細(xì)胞因子的表達(dá)情況。采用蛋白印跡實(shí)驗(yàn)檢測(cè)香雪抗病毒口服液對(duì)炎癥相關(guān)信號(hào)通路的蛋白表達(dá)的影響。結(jié)果 香雪抗病毒口服液對(duì)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，半數(shù)細(xì)胞毒性濃度（TC₅₀）分別為42.12、79.36 mg/mL，半數(shù)抑制濃度（IC₅₀）分別為8.665、5.260 mg/mL，并可減少H1N1病毒空斑形成。香雪抗病毒口服液能呈劑量相關(guān)性地降低H1N1誘導(dǎo)的A549細(xì)胞炎癥因子腫瘤壞死因子-α（TNF-α）、視黃酸（維甲酸）誘導(dǎo)基因蛋白I （RIG-I）、趨化因子（CC基序）配體5（CCL5）、巨噬細(xì)胞炎癥蛋白-1β（MIP-1β）、干擾素誘導(dǎo)蛋白-10（IP-10）、白細(xì)胞介素-10（IL-10）、λ干擾素（IFN-λ）、β干擾素（IFN-β）、白細(xì)胞介素-8（IL-8） mRNA的表達(dá)（P<0.05、0.01、0.001）。在流感病毒單復(fù)制周期中，香雪抗病毒口服液在早期階段0~2 h干預(yù)，具有良好的抗流感病毒效果。香雪抗病毒口服液能顯著抑制Toll樣模式識(shí)別受體3（TLR3）、RIG-I、黑色素瘤分化相關(guān)基因5（MDA-5）受體的表達(dá)，下調(diào)核轉(zhuǎn)錄因子-κB亞基p65（NF-κB p65）、NF-κB抑制蛋白α（IκBα）、信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子1（STAT1）、STAT2、STAT3的磷酸化水平，且在20 mg/mL濃度下明顯下調(diào)這些蛋白的表達(dá)。結(jié)論 香雪抗病毒口服液不僅能有效抑制甲流感病毒在人宿主細(xì)胞中復(fù)制，降低流感病毒誘導(dǎo)的炎癥因子表達(dá)，而且能顯著抑制流感病毒誘導(dǎo)天然免疫信號(hào)通路的相關(guān)蛋白表達(dá)，表明香雪抗病毒口服液具有防治流感病毒的潛力。

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 (H1N1) and A/Aichi/2/68 (H3N2), TC₅₀ was 42.12 mg/mL and 79.36 mg/mL, respectively. IC₅₀ was 8.665 mg/mL and 5.260 mg/mL, respectively, and reduced plaque formation of H1N1 virus. The expression of TNF-α, RIG-I, CCL5, MIP-1β, IP-10, IL-10, IFN-λ, 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-κB p65, IκBα, 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.

R978.7

廣東省基礎(chǔ)與應(yīng)用基礎(chǔ)研究基金資助項(xiàng)目（2020B1515120045）