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-αand IL-6 Expression in Bronchial Epithelial Cells

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100μmol/L)作用BEAS-2B细胞24h,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50μmol/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、50μmol/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。

Objective To investigate the effects of dihydroartemis(DHA) on influenza A virus(IAV A/PR/8/34(H1 N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase(ERK)signaling pathway expression in bronchial epithelial cells. Methods The BEAS-2 B 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-2 B cells were measure by CCK8 method. The BEAS-2 B 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-2 B 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-2 B cells. Results The BEAS-2 B 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-2 B cells. Conclusion These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2 B cells through ERK signaling pathway.