双氢青蒿素对甲型流感病毒H1N1诱导人支气管上皮细胞TNF-α和IL-6表达的影响及机制研究

欧利; 秦克; 杨子宵; 别明江

doi:10.12182/20200360604

双氢青蒿素对甲型流感病毒H1N1诱导人支气管上皮细胞TNF-α和IL-6表达的影响及机制研究

The Effects and Mechanisms of Dihydroartemisinin on Influenza A Virus H1N1 Induces TNF-α and IL-6 Expression in Bronchial Epithelial Cells

摘要

摘要:
目的探讨双氢青蒿素（dihydroartemisinin，DHA）对甲型流感病毒（influenza A virus，IAV）A/PR/8/34（H1N1）诱导人支气管上皮细胞（BEAS-2B）促炎症因子和细胞外信号调节蛋白激酶（extracellular signal regulated kinase，ERK）信号通路蛋白表达的影响。
方法采用不同浓度的DHA（即0、12.5、25、50和100 μmol/L）作用BEAS-2B细胞24 h ，CCK8法检测DHA对BEAS-2B细胞活性的影响。IAV吸附BEAS-2B细胞1 h ，分别采用不同浓度的DHA（低、中、高浓度DHA ，即12.5、25和50 μmol/L）作用24 h，同时设置正常对照组和IAV组。采用实时荧光定量PCR法（real time quantitative PCR, RT-qPCR）和酶联免疫吸附法（enzyme linked immunosorbent assay，ELISA）分别检测肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和白细胞介素-6（interleukin 6，IL-6）的mRNA和蛋白表达水平，蛋白质印迹法（Western blot）检测磷酸化ERK（phospho-ERK, p-ERK）蛋白表达水平。采用特异性ERK激动剂（ERK agonist，20 ng/mL）作用BEAS-2B细胞（分组为正常对照、IAV、DHA、DHA+IAV、ERK agonist、DHA+IAV+ERK agonist组）24 h，观察对DHA抑制IAV诱导BEAS-2B细胞TNF-α、IL-6和p-ERK表达的影响。
结果 DHA浓度在12.5、25、50 μmol/L时，BEAS-2B细胞存活率与正常对照组比较差异无统计学意义；与正常对照组比较，IAV组细胞TNF-α、IL-6 mRNA和蛋白及p-ERK蛋白表达水平增高 (P<0.05)，与IAV组比较，IAV+ DHA低、中、高浓度组细胞TNF-α、IL-6 mRNA和TNF-α、IL-6、p-ERK蛋白表达水平呈剂量依赖性的下降(P<0.05)；而ERK激动剂减弱了DHA抑制IAV诱导BEAS-2B细胞ERK信号通路蛋白p-ERK表达和促炎症因子TNF-α、IL-6的分泌。
结论 DHA可通过ERK信号通路抑制IAV诱导BEAS-2B细胞TNF-α和IL-6的表达。

Abstract:
Objective To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells.
Methods The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0, 12.5, 25, 50 and 100 μmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 μmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells.
Results The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 μmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group (P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group (P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells.
Conclusion These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.

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