紫菀酮对LPS诱导的气道细胞炎症反应及自噬的影响

Effect of Asterone on LPS-induced Inflammation and Autophagy in Airway Cells

目的通过构建LPS诱导的气道上皮细胞炎症模型,探讨紫菀酮对气道细胞自噬的影响及其机制。方法通过LPS诱导Beas-2B细胞(人支气管上皮细胞),构建炎症模型。通过CCK-8检测紫菀酮对Beas-2B细胞增殖活性的影响。通过ELISA检测细胞中IL-6和TNF-α的含量。通过RT-PCR检测各组细胞NF-κB和AP-1转录水平表达。Western blot检测NF-κB、p-NF-κB、AP-1、Beclin 1、LC3-Ⅱ、LC3-Ⅰ、mTOR、p-mTOR、p-S6K1和S6K1的蛋白表达。通过吖啶橙染色观察各组细胞自噬变化。结果与对照组比较,LPS组细胞中AP-1和p-NF-κB的相对表达显著升高(均P<0.05);细胞中出现橘、红色荧光,有很多点状红色斑点的酸性膜泡,自噬水平升高;细胞中Beclin 1和LC3-Ⅱ的相对表达显著增加(均P<0.05),LC3-Ⅰ、p-mTOR和p-S6K1的表达显著降低(均P<0.05)。与LPS组比较,LPS+紫菀酮组细胞中AP-1和p-NF-κB的相对表达显著降低(均P<0.05);细胞中橘色、红色荧光明显减少,自噬水平显著降低;细胞中Beclin 1和LC3-Ⅱ的相对表达显著降低(均P<0.05),LC3-Ⅰ、p-mTOR和p-S6K1的表达显著增加(均P<0.05)。结论紫菀酮能够抑制Beas-2B细胞自噬,其机制可能与mTOR信号通路的激活有关。

Objective To investigate the effect of asterone on airway epithelial cell autophagy and its mechanism through LPS-induced airway epithelial cell inflammation model.Methods Beas-2B cells(human bronchial epithelial cells)were induced by LPS to construct an inflammation model.The effect of asterone on the proliferative activity of Beas-2B cells was detected by CCK-8.The levels of IL-6 and TNF-αin the cells were detected by ELISA.The expression levels of NF-κB and AP-1 in each group of cells were detected by RT-PCR.The relative expression of NF-κB、p-NF-κB、AP-1、Beclin 1、LC3-Ⅱ、LC3-Ⅰ、mTOR、p-mTOR、p-S6K1 and S6K1 was detected by Western blotting.The changes of autophagy in each group of cells were observed by acridine orange staining.Results Compared with the control group,the relative expression of AP-1 and p-NF-κB was significantly higher than that of LPS group(P<0.05);orange and red fluorescence with many dotted red spots of acidic membrane vesicles as well as increased autophagy level were observed in cells;the relative expressions of Beclin 1 and LC3-Ⅱwere significantly increased in cells(P<0.05),and expressions of LC3-Ⅰ,p-mTOR and p-S6K1 were significantly reduced(P<0.05).Compared with the LPS group,the relative expressions of AP-1 and p-NF-κB in the LPS+asterone group were significantly decreased(P<0.05);less orange and red fluorescence was observed in cells,and the level of autophagy was decreased(P<0.05).The relative expressions of Beclin 1 and LC3-Ⅱwere reduced(P<0.05).The expressions of LC3-Ⅰ,p-mTOR and p-S6K1 were increased.Conclusion Asterone can inhibit the occurrence of autophagy in Beas-2B inflammatory cells,and the mechanism may be related to activation of mTOR signaling pathway.