冬凌草甲素对脂多糖诱导小鼠肺泡上皮细胞炎症因子的抑制作用及其机制研究

Mechanism underlying the inhibitory effect of oridonin on lipopolysaccharide-induced inflammatory factors in mouse alveolar epithelial cells

目的探讨冬凌草甲素对脂多糖诱导小鼠肺泡上皮细胞(MLE-12)炎症因子的影响及其潜在机制。方法采用CCK-8法检测不同浓度(0、0.3125、0.625和1.25μg/ml)冬凌草甲素对MLE-12的毒性影响;利用实时荧光定量聚合酶链反应(PCR)和酶联免疫吸附试验分别在mRNA和蛋白质水平检测不同浓度冬凌草甲素对脂多糖诱导MLE-12炎症因子[肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β]表达的抑制作用;通过蛋白免疫印迹法检测不同浓度冬凌草甲素对脂多糖刺激的MLE-12中核因子-κB(NF-κB)/p65的核移位及磷酸化水平。结果CCK-8结果显示不同浓度的冬凌草甲素对MLE-12均无毒性作用;实时荧光定量PCR和酶联免疫吸附试验结果表明冬凌草甲素可以剂量依赖性地抑制脂多糖诱导的MLE-12、TNF-α、IL-6和IL-1βmRNA和蛋白质的表达;蛋白印迹免疫结果显示冬凌草甲素剂量依赖性地抑制NF-κB/p65的核移位和磷酸化水平。结论冬凌草甲素能有效抑制脂多糖诱导的MLE-12炎症因子的产生,其机制可能与NF-κB/p65信号通路的抑制有关。

Objective To investigate the effect of oridonin on lipopolysaccharide(LPS)induced inflammatory factors in mouse alveolar epithelial cells(MLE-12)and its underlying mechanism.Methods CCK-8 assay was used to detect the toxicity of oridonin at different concentrations(0,0.3125,0.625,and 1.25μg/ml)on MLE-12 cells.Fluorescence-based quantitative real-time PCR(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to examine the inhibitory effect of different doses of oridonin on LPS-induced expression of inflammatory factors(TNF-α,IL-6 and IL-1β)in MLE-12 cells at the mRNA and protein levels.Western blotting was used to measure the nuclear translocation and phosphorylation of NF-κB/p65 in LPS-stimulated MLE-12 cells.Results CCK-8 assay showed that oridonin at any of the tested concentrations caused no toxicity on the MLE-12 cells.qRT-PCR and ELISA showed that oridonin could dose-dependently inhibit the LPS-induced expression of TNF-α,IL-6 and IL-1βmRNA and protein in MLE-12 cells.Western blotting showed that oridonin inhibited nuclear translocation and phosphorylation of NF-κB/p65in a dose-dependent manner.Conclusion Oridonin can effectively inhibit the LPS-induced production of inflammatory factors in mouse alveolar epithelial cells,and its mechanism may be related to the inhibition of NF-κB/p65 signaling pathway.