摘要目的 探讨二硫代氨基甲酸吡咯烷(PDTC)对绿脓杆菌脂多糖(LPS)诱导的人角膜基质细胞核因子κB(NF-κB)活化与细胞因子释放的干预作用及其机制.方法 为实验研究.原代培养人眼角膜基质细胞(HCFs),选取生长良好的传代HCFs分为对照组、LPS刺激组及PDTC预处理组.LPS组给予单纯LPS刺激,PDTC预处理组则在LPS刺激前先加PDTC培养30 min,再给予相同浓度的LPS.在LPS刺激1、2、4及8 h时,分别收集各组细胞和上清液,Western blot检测HCFs NF-κB的活化表达;酶联免疫吸附实验(ELISA)检测HCFs培养上清液中白细胞介素6(IL-6)和IL-8的分泌情况;逆转录聚合酶链反应(RT-PCR)检测HCFs IL-6和IL-8 mRNA的表达.结果 与对照组相比较,LPS刺激后,HCFs胞核NF-κB p65的表达明显升高,HCFs中IL-6与IL-8蛋白的分泌和mRNA的表达均显著升高.PDTC预处理30 min后再给予LPS刺激,可部分抑制HCFs胞核中NF-κB p65的活化表达(tl h=9.3766,t2 h=15.9011,t4 h=12.5851,t8 h=10.8346;均P＜0.01);PDTC预处理可不同程度的下调LPS诱导的HCFs分泌IL-6、IL-8蛋白及IL-6、IL-8 mRNA的表达,二者均明显低于同时间点LPS组HCFs的表达,差异均有统计学意义(IL-6蛋白:t1 h=7.9154,t2 h=10.8630,t4 h=8.2451,t8 h=13.5063;IL-8蛋白:t1h=8.5663,t2 h=20.5169,t4 h=25.1580,t8 h=34.8699;IL-6 mRNA:t1 h=12.0235,t2 h=13.2894,t4 h=24.0799,t8 h=27.2261;IL-8 mRNA:t1 h=20.9424,t2 h=24.1314,t4 h=29.8580,t8h=47.9442;均P＜0.01).结论 绿脓杆菌LPS可引起HCFs NF-κB活化,促进细胞因子的表达,PDTC可部分抑制LPS对NF-κB的激活,下调相应细胞因子的表达.

abstractsObjeetive To investigate the effects of pyrrolidine dithiocarbamate(PDTC)on Lipopolysaccharide(LPS)-mediated activation of nuclear factor kappa B(NF-κB)and cytokine expression in cultured human corneal fibroblasts.Methods It was a experimental study.A completely random design was employed in this research.Human comeal fibroblasts(HCFs)were obtained from human specimen.HCFs were divided into three groups:control group(group 0 h),LPS alone group and PDTC treatment group.Cells were incubated with PDTC for 30 min in the PDTC pretreatment group before LPS challenged.At difierent time after LPS challenged,the activities of NF-κB were assessed by Western Blot analysis,the secretion of IL-6 and IL-8 from cultured corneal fibroblasts was measured with enzyme-linked immunosorbent assays (ELISA):the mRNAs expression of IL-6 and IL-8 was determined by reverse transcription polymerase chain reaction (RT-PCR).The effects of PDTC on activation of NF-κB and the expression of IL-6 and IL-8 were also assessed in HCFs challenged with LPS.Resuits Compared with control group.NF-κB level was significantly enhanced in the nucleus in the LPS alone group,indicating that LPS mediated the activation of NF-κB in HCFs.The activation of NF-κB by LPS was markedly inhibited by PDTC(t1 h=9.3766,t2 h=15.9011,t4 h=12.5851,t8 h=10.8346,P＜0.01).Compared with control group,LPS increased IL-6 and IL-8 expression both mRNA and protein in HCFs.At the same time,PDTC partly inhibited the expression of IL-6 and IL-8 in corneal fibroblasts induced by LPS.These inhibitory effects were significant at both the mRNA and protein levels(protein of IL-6:t1 h=7.9154,t2 h=10.863,t4 h=8.2451,t8 h=13.5063.protein of IL-8:t1 h=8.5663,t2 h=20.5169,t4 h=25.1580,t8 h=34.8699.mRNA of IL-6:t1 h=12.0235,t2 h=13.2894,t4 h=24.0799,t8 h=27.2261.mRNA of IL-8:t1 h=20.9424,t2 h=24.1314,t4 h=29.8580,t8 h=47.9442.P＜0.01).Condusion These results suggest that LPS can activation of NF-κB in cultured HCFs and PDTC partly inhibits NF-κB activation.