吡格列酮对Aβ_(25-35)引起的皮层神经元损伤保护作用部分机制的研究

The mechanisms of neuroprotective effect of pioglitazone on amyloid beta-protein fragment 25-35-induced neurotoxicity in the cultured cortical neurons

目的研究吡格列酮对抗淀粉样β蛋白片段25-35(Amyloid-β,Aβ25-35)所致培养皮层神经元损伤作用的机制。方法取培养7d大鼠乳鼠大脑皮层神经元,Aβ组加入Aβ25-35(20μmol.L-1)作用24h;吡格列酮组和各种阻断剂组,先加入吡格列酮(0.1、1、10μmol.L-1)或各种阻断剂作用1h,然后加入Aβ25-35(20μmol.L-1)作用24h;正常对照组加入等量培养基。MTT法测定细胞存活率;免疫荧光染色法测定活性的caspase-3细胞内定位;Westernblot检测活性的caspase-3表达水平;Griess法测定培养细胞上清液中一氧化氮(NO)含量。结果神经元经NSE和NF200免疫荧光鉴定,其阳性率可达90%以上。Aβ25-35(20μmol.L-1)可使神经元细胞存活率下降、caspase-3表达明显增加,同时神经元培养液中的NO含量也明显增加。吡格列酮可明显抑制Aβ25-35诱导的神经元细胞存活率下降、抑制caspase-3表达的增加,吡格列酮还可明显抑制Aβ25-35诱导的神经元培养液中NO含量增加,且呈浓度依赖性。GW9662(10μmol.L-1)能明显对抗吡格列酮对Aβ25-35诱导的神经元细胞存活率下降、活性的caspase-3表达增加、NO增加的抑制作用。SP600125(5μmol.L-1)、SB203580(20μmol.L-1)和SMT(1mmol.L-1)可明显对抗Aβ25-35诱导的神经元细胞存活率下降及培养液中NO含量增加。结论吡格列酮能够明显的抑制Aβ25-35引起的皮层神经元损伤作用,这种作用可能与激活PPARγ受体、抑制JNK信号传导通路和p38MAPK信号传导通路有关。

Aim To investigate the possible mechanisms responsible for neuroprotective effect of pioglitazone against 25-35（Aβ25-35）-induced neurotoxicity in cultured cortical neurons.Methods After 7 d cultures,cultured cortical neurons were incubated with Aβ25-35 20 μmol·L^-1for 24 h with or without other drugs.In co-incubation experiments,other drugs were added to the neurons 30 min or 1 h prior to incubation with Aβ25-35.Tne cell viability was assessed by MTT assay.The protein expression of active caspase 3 by Western blot and immunocytochemical stain with active caspase-3 antibody.Nitric oxide（NO）generation was measured by Griess method.Results The percentage of NSE and NF200 positive cells was about 90%.The decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed incubated with Aβ25-35 for 24 h compared with the normal controls.Aβ25-35 induced the increases of active caspase-3 expression and NO generation.Pioglitazone inhibited Aβ25-35-induced decrease in cell viability and increase of active caspase-3 expression and NO generation in cultured neurons.The PPARγ receptor antagonist GW9662（10 μmol·L^-1did reverse the effects of pioglitazone.JNK inhibitor SP600125（5 μmol·L^-1,p38MAPK inhibitor SB203580（20 μmol·L^-1and iNOS inhibitor SMT（1 mmol·L^-1significantly inhibited Aβ25-35-induced increases of active caspase-3 expression and NO generation.Conclusion Pioglitazone protects cortical neurons against Aβ25-35 insult through PPARγ receptor activation and inhibiting JNK and p38MAPK pathway.