Tat—NBD多肽抑制胰腺腺泡细胞AR42J细胞的核因子-κB相关性炎症反应

Tat-NBD pepfide inhibits inflammation effects related to NF-κB on AR42J cell

目的通过脂多糖在体外刺激大鼠胰腺腺泡细胞AR42J，诱导胰腺腺泡细胞发生炎性效应，探讨NF-KB必需调节蛋白结合域多肽对脂多糖诱导的炎性效应的干预作用与机制，为急性胰腺炎的治疗机制和研究提供新的途径。方法以脂多糖（0．1mg／L，1mg／L，10mg／L，100mg／L）刺激大鼠胰腺腺泡细胞AR42J构建急性胰腺炎的体外模型。不同浓度的免疫缺陷性病毒的Tat多肽的蛋白转导功能区和野生型NBD多肽融合成Tat-NBD多肽对AR42J细胞进行预处理，突变型Tat-NBD（MT）多肽，Tat，NBD作为对照。半定量逆转录-多聚酶链反应法观察TNF-α的mRNA表达的改变；定量酶联免疫吸附法检测培养液上清中TNF-α蛋白浓度的改变。链酶亲和素一生物素．过氧化物酶复合物免疫细胞化学法观察NF-κB-p65蛋白的合成和核转移的情况。结果Tat-NBD（WT）多肽可以抑制LPS诱导的AR42J炎症细胞因子TNFoamRNA和蛋白的表达，且抑制NF-κB—p65蛋白表达与移位，呈剂量依赖方式（P〈0．05）。对照的单纯NBD多肽、突变型Tat-NBD（MT）多肽、Tat均不能抑制TNF-RmRNA和蛋白的表达，且不能抑制NF-κB-p65蛋白的向核内转移。结论TAT-NBD（WT）多肽可以呈剂量依赖方式地抑制LPS诱导的AR42J促炎细胞因子TNF-α mRNA和TNF-α蛋白的表达，可能与阻止NF-κBp65蛋白表达及核移位有关。本研究的结果可为急性胰腺炎的治疗与研究提供新的途径。

Objective To investigate the effects of Tat-NBD（NEMO-binding domain, NBD） peptide on LPS-stimulated AR42J acinus cells inflammatory response. Method Lipopolysaecharide （LPS） was added to culture media at doses of 10 mg/kg for 24 hours to stimulate the AR42J cells. For pretreatment, cells were incubated with different peptides for 2 hours before LPS stimulation. The expression of TNF-α mRNA was conducted using a semi-quantitative RT-PCR method. TNF-α protein in culture medium were detected by enzyme linked immunoserbent assay（ELISA）. The expression and transloeation of the NF-κB-p65 protein of AR42J was determined by Strept Actividin-Biotin Complex （SABC） method. Results LPS （ 10 mg/L） resulted in an increase of TNF-α mRNA and TNF-α protein, whereas significant inhibitory effects were observed when cells were incubated with Tat-NBD（WT） just on dose of 0.1 mg/L （P 〈 0.05）. The Tat-NBD（WT） peptide decreased inflammatory cytokine expression by a dose-dependent mariner and its peak role was on dose of 100 mg/L. Consisting with TNF-α expression decrease, NF-κB-p65 expression significantly decreased in Tat-NBD（WT） pretreatment group, especially on the largest dose. NO significant changes in the control peptide group. Conclusions Tat-NBD（WT） peptide can inhibit the inflammation of acinus simulated by LPS.