尼氟灭酸对气道平滑肌细胞增殖的抑制作用

Inhibitory effect of niflumic acid on the proliferation of airway smooth muscle cells

目的探讨尼氟灭酸(NFA)对气道平滑肌细胞(ASMCs)增殖的影响及机制。方法采用含胎牛血清的DMEM原代培养BALB/c小鼠ASMCs,[3H]-TdR掺入法检测不同剂量NFA(10和50μmol/L)对ASMCs增殖活性的影响。激光共聚焦显微镜下观察NFA及硝苯地平对组胺致ASMCs中[Ca2+]i变化的影响。间接免疫荧光法观察NFA对ASMCs表达MAPK蛋白的影响。结果10μmol/L和50mol/LNFA组细胞的活性明显低于对照组,并且高浓度50μmol/LNFA组比低浓度10mol/LNFA组更显著(P<001),表现出剂量依赖性。共聚焦显微镜下,对照组ASMCs胞浆和胞核呈现亮绿色荧光,胞膜周围较淡。加入激动剂组胺后,荧光亮度逐渐增强。相反,当同时存在NFA或硝苯地平干预时,镜下观察荧光强度变化不够明显。间接免疫荧光染色结果表明,培养24h后,对照组中ASMCs细胞沿梭形胞浆出现大斑片状亮绿色荧光,而NFA干预组细胞表现为弱绿色荧光,胞浆内分布区域局限。结论NFA能有效抑制培养ASMCs的增殖活性,这种作用可能是通过阻断钙激活性Cl-通道对[Ca2+]i的正反馈效应,以及降低MAPK的表达来实现的。

AIM: Niflumic acid (NFA) is known as a kind of inhibitor of calcium-activated chloride channel. The inhibition and mechanism of NFA on the proliferation of airway smooth muscle cells (ASMCs) were investigated. METHODS: Using [ 3H]-TdR incorporation method, we examined the effect of NFA (at concentration of 10 and 50 μmol/L) on the proliferation of primarily ASMCs from BALB/c mouse. With confocal laser scanning microscope the [Ca 2+ ]i in ASMCs exposed to histamine was observed, and the opposed effects of NFA and nifedipine on histamine were also checked. Finally the effect of NFA on expression of MAPK in ASMCs was examined by indirect immunofluorescent assay. RESULTS: Compared with control group, the proliferation of NFA group was reduced markedly with dependent concentration. Histamine significantly improved the [Ca 2+ ]i in ASMCs, but NFA and nifedipine showed the inhibition on the effect of histamine. NFA reduced the level of MAPK expression in ASMCs. CONCLUSION: It is demonstrated that NFA inhibits the proliferation of ASMCs by reducing [Ca 2+ ]i and the expression level of MAPK. [