摘要
目的探讨脂多糖(LPS)诱导的气道上皮细胞基质金属蛋白酶9(MMP-9)表达的TNF-α/NF-κB信号转导机制及慢病毒介导的解整合素-金属蛋白酶17(ADAM17)RNA干扰(RNAi)对MMP-9表达的影响。方法构建ADAM17 siRNA慢病毒载体、包装重组慢病毒。以NF-κB抑制剂(pyrrolidine dithiocarbamate,PDTC)或TNF-α拮抗剂(etanercept)预处理HBE4-E6/E7细胞,以LPS刺激HBE4-E6/E7细胞24 h。以重组慢病毒感染HBE4-E6/E7细胞72 h后,以LPS或TNF-α刺激HBE4-E6/E7细胞24 h。以半定量RT-PCR检测MMP-9 mRNA表达;以酶联免疫吸附试验检测TNF-α蛋白含量;以Western blot检测MMP-9蛋白表达;以凝胶阻滞分析实验检测NF-κB活性。结果 LPS或TNF-α刺激均明显增加HBE4-E6/E7细胞MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05);etanercept和PDTC均明显抑制LPS诱导的MMP-9表达及NF-κB活性(P<0.05)。慢病毒介导的ADAM17 RNAi明显降低LPS诱导的HBE4-E6/E7细胞上清液中TNF-α蛋白含量(P<0.05),亦明显降低MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05),但不能降低TNF-α诱导的MMP-9mRNA和蛋白表达及NF-κB活性(P>0.05)。PDTC明显抑制TNF-α诱导的MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05)。结论 TNF-α/NF-κB信号通路参与调控LPS诱导的气道上皮细胞MMP-9的表达,ADAM17通过调节TNF-α释放在其信号通路上游起到重要作用。
Aim To study the role of TNF-α/NF-κB signaling in matrix metalloproteinase （ MMP）-9 expres-sion induced by lipopolysaccharide （ LPS ） in airway epithelial cells, and to investigate the effects of lenti-virus mediated RNAi targeting a disintegrin and metal-loproteinase 17 （ ADAM17 ） gene on MMP-9 expression induced by LPS. Methods The ADAM17 siRNA ex-pression vector was constructed, and packaged to re-combinant lentivirus in 293T cells. The HBE4-E6/E7 cells were pretreated for 30 min by NF-κB inhibitor （ PDTC） and a recombinant human TNFR p75-Fc fu-sion protein （ Etanercept） , or infected by the recombi-nant lentivirus for 72 h, and then stimulated for 24 h by LPS or TNF-α. The release of TNF-α was detected by ELISA. The mRNA and protein levels of MMP-9 were analyzed respectively by RT-PCR and Western blot. NF-κB activity was detected by electrophoretic mobility shift assay. Results LPS and TNF-α signifi-cantly increased MMP-9 mRNA and protein expressions and the activation of NF-κB in HBE4-E6/E7 cells （ P＆lt;0. 05 ） . Etanercept and PDTC significantly inhibited＆amp;nbsp;MMP-9 expression and the activation of NF-κB induced by LPS （ P＆lt;0. 05 ） . Lentivirus mediated RNAi targe-ting ADAM17 significantly decreased TNF-α produc-tion, inhibited MMP-9 mRNA and protein expressions and the activation of NF-κB induced by LPS in HBE4-E6/E7 cells （ P ＆lt;0. 05 ） . Lentivirus mediated RNAi targeting ADAM17 did not inhibit MMP-9 mRNA and protein expressions and the activation of NF-κB in-duced by TNF-α （ P＆gt;0. 05 ） . And PDTC significantly inhibited MMP-9 mRNA and protein expressions and the activation of NF-κB induced by TNF-α （ P ＆lt;0. 05 ） . Conclusions TNF-α/NF-κB signaling partic-ipates in the regulation of MMP-9 expression induced by LPS in airway epithelial cells, and lentivirus-media-ted RNAi targeting ADAM17 plays an important role in that signaling pathway upstream by regulating TNF-αrelease.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第4期559-565,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81000016)
浙江省医药卫生科学研究基金资助项目(No 2008B007)
