Toll样受体4及核因子-κB在脂多糖诱导的气道上皮细胞基质金属蛋白酶9表达中的作用

Effect of Toll-like receptor-4 and NF-κB on expression of matrix metalloprotease 9 induced by lipopolysaccharide in human airway epithelial cells

目的探讨Toll样受体4(TLR4)和核因子(NF-)κB在脂多糖(LPS)诱导的气道上皮细胞基质金属蛋白酶9(MMP-9)表达中的作用,观察TLR4抑制剂TAK-242对MMP-9表达的影响。方法以终浓度为0、0.1、1、10、100μg/L的LPS刺激HBE4-E6/E7细胞24h,或以LPS(终浓度10μg/L)刺激细胞0、8、12、16、24h,检测LPS刺激与MMP-9表达的量效和时效性关系。以TAK-242(100μg/L)或PDTC(50mg/L)预处理HBE4-E6/E7细胞后以100μg/L LPS刺激24h,采用RT-PCR检测MMP-9mRNA表达,用Western blotting检测MMP-9蛋白表达,以明胶酶谱法检测MMP-9酶活性,采用凝胶阻滞实验检测活化NF-κB的含量。结果 MMP-9mRNA表达随着LPS刺激剂量的增加而增加(P<0.05),且随着LPS刺激时间延长,MMP-9mRNA表达量亦显著增加(P<0.05)。TAK-242和PDTC均能显著抑制LPS诱导的MMP-9mRNA和蛋白的表达,降低其酶活性(P<0.05)。TAK-242和PDTC均能显著降低LPS诱导的活化NF-κB含量(P<0.05)。结论 TLR4/NF-κB信号通路在LPS诱导的气道上皮细胞MMP-9表达中发挥着重要的调控作用;TAK-242可抑制LPS诱导的MMP-9的表达。

Objective To study the effects of toll-like receptor-4（TLR-4） and nuclear factor-κB（NF-κB） on the expression of matrix metalloprotease 9（MMP-9） induced by lipopolysaccharide（LPS） in human airway epithelial cells,and explore the influence of TLR-4 inhibitor TAK-242 on the expression of MMP-9.Methods In order to study the effects of LPS on MMP-9 expression,HBE4-E6/E7 cells were challenged for 24 hours by different concentrations of LPS（0,0.1,1,10,100 μg/L）,or challenged for 0,8,12,16 and 24 hours by LPS at a concentration of 10 μg/L.HBE4-E6/E7 cells were pretreated for 30min with TAK-242（100μg/L） or PDTC（50mg/L）,and challenged for 24h by LPS at 100 μg/L thereafter.The expression of MMP-9 mRNA was assessed by RT-PCR and of MMP-9 protein by Western blotting.MMP-9 activity was determined by gel zymography.The active NF-κB production was determined by electrophoretic mobility shift assay（EMSA）.Results The expression of MMP-9 mRNA in HBE4-E6/E7 cells was induced by LPS in both dose-and time-dependent manner（P0.05）.Both TAK-242 and PDTC significantly inhibited the expression of MMP-9 mRNA and protein（P0.05）,down-regulated MMP-9 activity（P0.05）,and significantly inhibited active NF-κB production induced by LPS（P0.05）.Conclusion MMP-9 expression is induced by LPS in human airway epithelial cells via TLR-4/NF-κB signal pathway,which can be inhibited by TAK-242.