吡格列酮对谷氨酸诱导的皮质神经元损伤的保护作用

Protective effect of pioglitazone against glutamate induced neurotoxicity in cultured cortical neurons

目的观察吡格列酮对谷氨酸所致培养皮质神经元损伤的保护作用及其机制。方法乳大鼠大脑皮质神经元,培养7d后用于实验。实验分为对照组,加入0.1%二甲亚砜;谷氨酸损伤组,加入谷氨酸100μmol·L-1作用2h或24h;吡格列酮组,先分别加入吡格列酮0.01,0.1和1μmol·L-1作用1h,然后加入谷氨酸;谷氨酸+吡格列酮+GW9662组,先加入GW966210μmol·L-1作用30min,然后加入吡格列酮1μmol·L-1,作用1h后加入谷氨酸。MTT法测定细胞存活率;Hoechst33258核染色观察细胞凋亡的形态学改变;Western印迹法检测Bcl-2蛋白、钙蛋白酶Ⅰ蛋白和磷酸化c-Jun氨基端激酶1(JNK1)表达水平;免疫荧光染色法检测钙蛋白酶Ⅰ及磷酸化活化转录因子2(ATF2)表达。结果谷氨酸作用24h可使体外培养神经元细胞存活率明显下降,从对照组的(100.0±15.4)%降低至(71.5±6.1)%;细胞凋亡百分率明显增加,从对照组的(8.7±1.3)%增加至(35.4±6.9)%;磷酸化JNK1、钙蛋白酶Ⅰ蛋白和磷酸化ATF2表达增加,Bcl-2蛋白表达水平降低。吡格列酮0.1及1μmol·L-1明显对抗谷氨酸引起的神经元损伤,神经元细胞存活率分别为(91.1±4.7)%和(96.6±3.4)%;细胞凋亡百分率分别为(15.5±3.8)%和(9.2±0.9)%。过氧化物酶体增殖物激活受体γ(PPARγ)的特异性阻断剂GW9662不能拮抗吡格列酮对神经元的保护作用,谷氨酸+吡格列酮+GW9662组细胞存活率为(91.3±6.7)%,细胞凋亡百分率为(10.2±1.8)%。单独应用GW966210μmol·L-1对细胞存活率和细胞凋亡百分率没有影响。吡格列酮也可抑制谷氨酸引起的磷酸化JNK1、磷酸化ATF2、钙蛋白酶Ⅰ表达增多及Bcl-2蛋白表达减少。结论吡格列酮对谷氨酸引起的培养皮质神经元损伤具有明显的保护作用,可能与吡格列酮抑制磷酸化JNK1和钙蛋白酶Ⅰ表达,以及增强Bcl-2蛋白表达有关,与PPARγ激活无关。

OBJECTIVE To investigate whether pioglitazone has protective effect against glutamate induced neurotoxicity in cultured cortical neurons and the mechanism. METHODS The cultured cortical neurons were randomly divided into control group, in which neurons were incubated with 0.1% DMSO; glutamate group, in which neurons were incubated with glutamate 100 μmol·L-1 for 2 h to detect phospho-JNK1(p-JNK1) or 24 h; glutamate + pioglitazone group, in which neurons were incubated with pioglitazone (0.01, 0.1 and 1 μmol·L-1, respectively) for 1 h and then coincubated with glutamate for 2 h or 24 h, and glutamate+pioglitazone+GW9662 group, in which neurons were first incubated with GW9662 10 μmol·L-1 for 30 min and with pioglitazone 1 μmol·L-1 for 1 h and finally with glutamate for 24 h. MTT and Hoechst 33258 staining were performed to investigate cell viability and apoptosis of neurons. Western blotting was performed to investigate the protein expression of Bcl-2, μ-calpain and p-JNK1. Immunostaining was used to investigate the expression of μ-calpain and phospho-activating transcription factor-2(p-ATF2) in neuronal cells. RESULTS Glutamate 100 μmol·L-1 showed apparent damage to cultured cortical neurons, decreasing cell viability from (100.0±15.4)% to (71.5±6.1)% and increasing the apoptotic rate from (8.7±1.3)% to (35.4±6.9)%. p-JNK1, μ-calpain and p-ATF2 protein expression was increased and Bcl-2 expression decreased in the model group. Pioglitazone 0.1 and 1 μmol·L-1 markedly reduced the damage caused by glutamate. Cell viability increased to (91.1±4.7)% and (96.6±3.4)% and the apoptotic rate decreased to (15.5±3.8)% and (9.2±0.9)%. GW9662, an antagonist of peroxisome proliferators-activited receptor γ (PPARγ), could not reverse the protective effect of pioglitazone. In glutamate+pioglitazone (1 μmol·L-1)+GW9662 (10 μmol·L-1) group, cell viability was (91.3±6.7)% and the apoptotic rate was (10.2±1.8)%. GW9662(10 μmol·L-1) had no effect on cell viability and the apoptotic rate. Pioglitazone inhibited glutamate induced down-regulation of the Bcl-2 protein level and up-regulation of the level of μ-calpain, p-ATF2 and p-JNK1 protein. CONCLUSION Pioglitazone can protect cultured cortical neurons from glutamate induced damage. Up-regulation of Bcl-2 and down-regulation of μ-calpain and p-JNK1 protein may account for its protective effect that is independent of PPARγ.