摘要
研究P38对脂多糖(LPS)诱导的大鼠雪旺氏细胞(Schwann cell)肿瘤坏死因子-alpha(TNF-α)表达的作用.采用Western印迹检测细胞中P38激酶的活性;用P38的特异性抑制剂(SB202190)预处理细胞后,用ELISA检测细胞中TNF-α水平;RT-PCR检测细胞中TNF-αmRNA的表达;用免疫荧光双标法检测磷酸化P38的表达定位.LPS可显著激活雪旺氏细胞中的P38信号通路,其激活高峰在LPS刺激后15～30min,60min后,P38磷酸化水平开始逐渐下降,120min时又升高;用SB202190预处理细胞后,可显著抑制细胞TNF-αmRNA以及TNF-α的表达;LPS刺激细胞后通过与细胞膜表面Toll-like receptor 4(TLR4)结合后引起P38磷酸化,SB202190可抑制该过程.P38信号通路的激活可能是LPS诱导TNF-α表达的前提.通过阻断细胞内信号转导通路来减少TNF-α及其它细胞因子的产生,为抑制周围神经损伤后的炎症以及免疫反应发生提供了一条新的思路.
To explore the role of P38 in lipopolysaccharide （LPS） induced tumor necrosis factor-alpha （TNF- α） expression in Schwann cells. TNF-α mRNA levels were detected by RT-PCR. The protein level of phosphorylated P38 was determined by Western blot analysis. Schwann cells were pretreated with the inhibitor of P38 （SB202190） before treatment with vary doses of LPS. The intracellular concentrations of TNF-α were then detected by sandwich enzyme-linked immunosorbent assay （ELISA）. The location of phosphorylated P38 was observed by double immunofluorescent staining. LPS at dose of 10 μg/ml could significantly activate P38 signal pathway. The peak of phosphorylated P38 was detected at 15 - 30 min after stimulation with LIPS and the level of phosphorylated P38 began to fall-off after 60 min, then step up at 120 min again. Inhibition of P38 activation by using P38 inhibitor SB202190 markedly suppressed expression of TNF-α mRNA or TNF-α. LPS had the effect on promoting phosphorylation of P38 through binding to Toll-like receptor 4 on cytomembrane. The process was blocked distinctly by SB202190. Therefore,activation of P38 pathway might be a precondition for the induction of TNF-α expression. To reduce the biosynthesis of TNF-α and other cytokines by blocking the cell signal pathway may give us a new strategy against inflammatory and immunological response after injury of peripheral nerve.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第5期382-387,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.30300099
30500153)
江苏省自然科学基金(BK2003035)
江苏省高校自然科学研究项目(KJB180109
05KJB180100)~~
