PTD-NBD多肽对大鼠胰腺腺泡细胞炎性反应的抑制作用

Inhibitory effects of cell-permeable PTD-NBD peptide on lipopolysaccharide-induced inflammation of pancreatic acinar cell line AR42J

目的:探讨透细胞性PTD-NBD多肽对脂多糖诱导的炎性效应的干预与作用机制.方法:以脂多糖刺激大鼠胰腺腺泡细胞AR42J,构建急性胰腺炎的体外模型.不同浓度免疫缺陷性病毒PTD多肽蛋白转导功能区与野生型NBD多肽融合成PTD-NBD多肽,对AR42J细胞进行预处理,突变型PTD-NBD(MT)多肽、PTD、NBD为对照.RT-PCR法观察ICAM-1及IL-1βmRNA的表达;定量酶联免疫吸附法检测培养液上清中IL-1β蛋白浓度的改变.结果:透细胞性PTD-NBD多肽可以抑制脂多糖诱导的AR42J炎症细胞因子ICAM-1和IL-1βmRNA及蛋白的表达,且呈剂量依赖方式.PTD-NBD(WT)多肽与单纯NBD多肽、突变型PTD-NBD(MT)多肽及PTD组比较(相对灰度值:0.449±0.088vs1.132±0.069,1.158±0.095,1.206±0.078),能够抑制ICAM-1mRNA表达.上述4组的IL-1βmRNA相对灰度值分别为0.526±0.077,0.993±0.065,1.143±0.086和1.128±0.117,IL-1β蛋白表达分别为278.82±61.80ng/L、898.08±74.65ng/L、945.25±42.49ng/L和947.86±38.66ng/L,结果显示后3组不能抑制IL-1βmRNA及其蛋白的表达.结论:PTD-NBD(WT)多肽可以呈剂量依赖方式抑制LPS诱导的AR42J促炎细胞因子IL-1β和ICAM-1的表达,其可直接影响胰腺腺泡细胞炎性反应.

AIM： To explore the effects of cell permeable PTD-NBD peptide on rat pancreatic acinus AR42J cell induced by lipopolysaccharide. METHODS： AR42J cell lines were stimulated by lipopolysaccharide with a dose of 10 mg/L for 24 h. The wild type PTD-NBD peptide was incubated with AR42J cells with different concentrations （10L102 mg/L） before the inflammation induced by lipopolysaccharide. At the same time, the mutant type PTD-NBD peptide, PTD and NBD peptide were used as control peptide fragments. The expressions of ICAM-1 and IL-1β mRNA were detected using reverse transcription-polymerase chain reaction （RT-PCR）, and IL-1β protein was measured by enzyme-linked irnmunosorbent assay （ELISA）. RESULTS： The PTD-NBD （WT） peptide inhibited the expression of ICAM-1 and IL-1β mRNA （0.449 ± 0.088, 0.526 ± 0.077）, but also decreased the IL-1β protein level （278.82 ± 61.80 ng/L） in a dose-dependent manner. NBD （1.132 ± 0.069）, PTD- NBD （MT） （1.158 ± 0.095） and PTD （1.206 ± 0.078） did not inhibit the expression of ICAM-1 mRNA. NBD （0.993 ± 0.065）, PTD- NBD （MT） （1.143 ± 0.086） and PTD （1.128 ± 0.117） did not inhibit the expression of IL-1β mRNA. NBD （898.08±74.65 ng/L）, PTD-NBD （MT） （945.25 ± 42.49 ng/L） and PTD （947.86 ± 38.66 ng/L） failed to inhibit the expression of IL-1β protein. CONCLUSION： The wild type PTD-NBD peptide is able to inhibit the expression of ICAM- 1and IL-1β induced by LPS on AR42J cell lines in a dose-dependent manner. We confirmed PTD-NBD peptide can take effect on acinus cell directly. The result showed the early event and new therapeutic pathway of acute pancreatitis.