一氧化氮-核因子κB信号通路在支气管哮喘患者T细胞功能表达中的作用

Role of nitric oxide-nuclear factor κB signal pathway in functional expression of T lymphocytes from asthmatic patients

目的 探讨NO 核因子κB(NF κB)信号通路在哮喘患者T细胞细胞因子表达及细胞增殖、凋亡中的调控作用。方法 采用细胞原位杂交、电泳迁移率改变试验(EMSA)、免疫荧光、流式细胞术、双抗夹心ELISA、四甲基偶氮唑蓝微量比色法,观察不同浓度的硝普钠(SNP)及二硫代氨基甲酸吡咯烷(PDTC)对IL 4、IL 5、IFNγ表达及T细胞增殖、凋亡的影响。结果 (1)哮喘患者T细胞IL 4、IL 5mRNA和蛋白表达及细胞增殖均较对照组高,IFNγmRNA和蛋白表达及T细胞凋亡率均较对照组低(P<0 05); (2)低浓度的SNP能上调上述3种细胞因子表达,并使T细胞增殖反应增强、凋亡反应减弱;而中、高浓度的SNP却使上述3种细胞因子表达及细胞增殖率呈剂量依赖性地降低,并使T细胞凋亡率增加; ( 3 )PDTC可抑制10μmol/LSNP对IL 5和IFNγ表达的促进作用,增强1mmol/LSNP对IL 5和IFNγ表达的抑制作用,同时PDTC可减弱10μmol/LSNP对T细胞增殖反应的促进作用,增强1mmol/LSNP对T细胞增殖反应的抑制作用; (4)低浓度的SNP能明显增加NF κB活化细胞百分率及NF κB活性(P<0 05),而中、高浓度的SNP却显著减少NF κB活化细胞百分率及NF κB活性(P<0 05)。结论 哮喘患者T细胞细胞因子表达增多及细胞增殖反应的增强、凋亡反应的减弱与NF κB活化的异常增高有关。

Objective To investigate the regulatory role of nitric oxide (NO)-nuclear factor κB(NF-κB) signal pathway in cytokine expressions, cell proliferation and apoptosis of T lymphocytes from asthmatic patients Methods We observed the expressions and secretions of IL-4, IL-5 and IFNγ from T lymphocytes and measured the proliferation and apoptosis of these cells by using in situ hybridization, electrophoresis mobility shift assay(EMSA), immunofluorescence,flow cytometry, two antibodies sandwich enzyme linked immuno-sorbent assay and MTT, and by administering NO donor sodium nitroprusside (SNP) and NF-κB inhibitor pyrrolidine dithio-carbamic acid(PTDC) Results (1) The mRNA expression and protein secretion of IL-4 and IL-5 by, as well as the proliferation rate of T lymphocytes from asthmatic patients were augmented, while the mRNA expression and protein secretion of IFNγ by, as well as the apoptosis rate of T lymphocytes from asthmatic patients were decrease as compared with the values of the control groups( P <0 05). (2) Low dose of SNP (10 μmol/L) up-regulated the expression of these three cytokines, promoted the proliferation rate and suppressed the apoptosis rate of T lymphocytes However, middle or high dose of SNP (100 μmol/L, 1 mmol/L, 10 mmol/L) dose-dependently down-regulated cytokine expressions and cell proliferation rate and promoted apoptosis rate. (3)PDTC inhibited the higher expressions of IL-5 and IFNγ induced by 10 μmol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the expressions of IL-5 and IFNγ At the same time, PDTC weakened the promoting effect of 10 μmol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the proliferation of T lymphocytes (4) The percentage of NF-κB-activated cells and NF-κB activity were significantly increased in T lymphocytes treated with low dose of SNP ( P <0 05); whereas these parameters were decreased in cells treated with middle or high dose of SNP( P <0 05) Conclusions The hyper-expression of inflammatory cytokines and the increased proliferation and decreased apoptosis of T lymphocytes from asthmatic patients are related to the abnormal over-activation of NF-κB NO has bi-phasic effects on the expressions of IL-5 and IFNγ and on the proliferation and apoptosis of T lymphocytes from asthmatic patients via its bi-phasic regulations on the activity of NF-κB