甲型流感病毒对人肺上皮细胞BEAS-2B的影响及疏风解毒胶囊含药血清的干预作用

Effect of Influenza A Virus on BEAS-2B in Human Lung Epithelial Cells and Intervention Effect of Shufeng Jiedu Capsule-containing Serum

摘要:

目的 观察疏风解毒胶囊（SFJD）含药血清对甲型流感病毒诱导的人肺上皮细胞的影响，探究药物对细胞的保护作用及潜在的抗病毒作用。方法 制备SFJD含药血清，体外培养人肺上皮细胞（BEAS-2B），通过细胞增殖与活性检测（CCK-8）试剂盒检测在不同浓度SFJD含药血清培养下的细胞存活率并筛选SFJD含药血清的最佳剂量用于后续实验。实验分为正常组、病毒感染组和SFJD含药血清组，采用CCK-8法检测病毒感染及给药后BEAS-2B细胞存活率。检测各组细胞中流感病毒核酸表达量，并采用荧光显微镜观察不同组细胞凋亡水平。实时荧光定量聚合酶链式反应（Real-time PCR）检测各组细胞中流感病毒核蛋白（NP）、Toll样受体4（TLR4）、髓样分化因子88（MyD88） mRNA水平及采用免疫荧光法检测肺上皮细胞TLR4、MyD88及磷酸化核转录因子-κB（p-NF-κB）的荧光强度。结果 与正常血清组比较，空白血清组、SFJD含药血清不同浓度组（5%、10%、20%）的细胞存活率分别为100.00%±0.00%、89.05%±4.80%、87.13%±5.90%、93.83%±6.03%和99.33%±3.39%（P<0.01），选择SFJD含药血清浓度为20%作为最佳剂量组进行后续实验；与正常组比较，病毒感染组的细胞存活率降低，细胞活力下降（P<0.01），与病毒感染组相比较，SFJD含药血清组的细胞存活率增加，细胞活力增加（P<0.01）；与病毒感染组比较，SFJD含药血清组能显著降低细胞中的病毒载量（P<0.01），减少细胞凋亡；与正常组比较，病毒感染组细胞中NP、TLR4和MyD88 mRNA表达水平显著增加（P<0.01），给予SFJD含药血清组治疗后细胞中的NP、TLR4和MyD88 mRNA表达水平明显降低（P<0.05，P<0.01）；与正常组比较，病毒感染后，细胞中TLR4、MyD88和p-NF-κB蛋白荧光强度明显增加（P<0.05，P<0.01），给予SFJD含药血清治疗后细胞中TLR4、MyD88和p-NF-κB蛋白荧光强度降低（P<0.05）。结论 SFJD含药血清在体外能有效抑制流感病毒，其通过提高BEAS-2B细胞存活率，减少细胞凋亡，下调BEAS-2B细胞中TLR4、MyD88和p-NF-κB的蛋白表达发挥抗流感病毒作用。

Abstract:

Objective To observe the effect of Shufeng Jiedu capsule （SFJD）-containing serum on human lung epithelial cells infected by influenza A virus， and investigate the protective effect of the drug on the cells and the potential antiviral effect.Method The SFJD-containing serum was prepared and used to treat human lung epithelial cells （BEAS-2B） cultured in vitro. The viability of cells treated with different concentrations of SFJD-containing serum was measured by the cell counting kit-8 （CCK-8）， and the optimal concentration of SFJD-containing serum was screened for subsequent experiments. BEAS-2B cells were classified into normal control， virus infection， and SFJD-containing serum groups， and the CCK-8 method was used to detect the survival rate of BEAS-2B cells after virus infection and drug administration. The expression of influenza virus nucleic acid in the cells of each group was determined， and the apoptosis of cells in different groups was observed by fluorescence microscopy. Real-time PCR was employed to determine the mRNA levels of influenza virus nucleoprotein （NP）， Toll-like receptor 4 （TLR4）， and myeloid differentiation primary response gene 88 （MyD88） in each group of cells. The immunofluorescence assay was used to detect the fluorescence intensities of TLR4， MyD88， and phosphorylated nuclear factor-κB （p-NF-κB） in lung epithelial cells.Result Compared with that in the control group （normal serum）， the cell survival rates in the blank serum and the SFJD-containing serum （5%， 10%， and 20%） groups were 100.00%±0.00%， 89.05%±4.80%， 87.13%±5.90%， 93.83%±6.03%， and 99.33%±3.39%， respectively （P<0.01）. The SFJD-containing serum of 20% was selected as the optimal treatment for subsequent experiments. Compared with the normal control group， the virus infection group showed reduced cell survival rate （P<0.01）， and the reduction was increased by the SFJD-containing serum （P<0.01）. Compared with the virus infection group， SFJD-containing serum reduced the virus load （P<0.01） to decrease apoptosis. Compared with the normal control group， the virus infection group showed up-regulated mRNA levels of NP， TLR4， and MyD88 （P<0.01）， and the up-regulation was down-regulated by the SFJD-containing serum （P<0.05， P<0.01）. The fluorescence intensities of TLR4， MyD88， and p-NF-κB proteins in the cells increased after virus infection compared with those in the normal control （P<0.05， P<0.01）， and they were decreased after administration with the SFJD-containing serum （P<0.05）.Conclusion The SFJD-containing serum can inhibit influenza virus in vitro by increasing the survival rate， reducing the apoptosis， and down-regulating the protein levels of TLR4， MyD88， and p-NF-κB in BEAS-2B cells.