吡格列酮对脂多糖引起大鼠大脑皮质神经元损伤的抑制作用

Protective effects of pioglitazone against lipopolysaccharides-induced neurotoxicity in cultured cortical neurons in rats

目的探讨吡格列酮对脂多糖(LPS)引起的神经元损伤的抑制作用,并探讨其信号传导机制。方法取培养7d的大鼠大脑皮质神经元细胞,除正常对照组外,其余各组先给予相应的处理30min～1h后,再加LPS10mg.L-1处理4～24h。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;免疫荧光染色法测定磷酸化c-Jun氨基端激酶1(JNK1)细胞内定位;Western蛋白印迹法检测活性胱天蛋白酶3(caspase3)蛋白表达和磷酸化JNK1水平;Griess法测定细胞培养上清液中一氧化氮(NO)含量。结果与对照组相比,LPS可使体外培养神经元细胞存活率明显下降,(100.0±10.9)%vs(72.3±2.1)%;凋亡细胞百分率明显增加,(11.5±4.2)%vs(39.5±8.2)%;活性caspase3蛋白表达和磷酸化JNK1水平增加;细胞培养上清液中NO含量明显增加。吡格列酮0.01,0.1和1μmo.lL-1可明显对抗LPS引起的培养神经元细胞存活率下降,并呈一定浓度依赖性。吡格列酮0.1和1μmol.L-1也可明显对抗LPS引起的培养神经元凋亡细胞百分率、活性caspase3蛋白表达、磷酸化JNK1水平和NO含量增加。LPS+吡格列酮(1μmo.lL-1)组,细胞存活率为(97.8±9.7)%,凋亡细胞百分率为(20.6±5.0)%,NO含量为(6.8±1.3)μmol.L-1。过氧化物酶体增殖物激活受体γ(PPARγ)的特异性阻断剂GW9662不能去除吡格列酮对LPS引起的神经元细胞损伤的抑制作用,在LPS+吡格列酮(1μmol.L-1)+GW9662(10μmo.lL-1)组,细胞存活率为(90.7±6.9)%,凋亡细胞百分率为(23.4±4.1)%,NO含量为(5.8±0.7)μmol.L-1。GW966210μmol.L-1对LPS引起的细胞存活率降低没有影响。与LPS组相比,JNK特异性阻断剂SP6001255μmol.L-1可明显对抗LPS引起的神经元损伤,细胞存活率明显增加,(72.3±2.1)%vs(109.8±11.8)%;凋亡细胞百分率降低,(39.5±8.2)%vs(19.1±4.8)%;NO含量降低,(21.1±5.0)μmol.L-1vs(4.0±1.3)μmol.L-1。结论吡格列酮能明显抑制LPS引起的皮质神经元损伤,这种作用可能与抑制JNK信号传导通路有关,与PPARγ激活无关。

AIM To investigate whether pioglitazone can protect cortical neurons from lipopolysaccharides（LPS）-induced neurotoxicity and the mechanisms responsible for this protective effect.METHODS After 7 d cultures,cultured cortical neurons were incubated with LPS 10 mg·L-1 for 4-24 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 LPS.The cell viability was assessed by MTT assay.The neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining.The cultured cells were then fixed on the 7th day and immunocytochemically stained with phosphorylated JNK1 antibody.The protein expressions of active caspase 3 and phosphorylated JNK1 were measured by Western blot.Nitric oxide（NO） generation was measured by Griess method.RESULTS The decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed incubated with LPS for 24 h compared with the normal controls.The cell viability of cortical neurons was decreased from（100.0±10.9）% in the normal control group to（72.3±2.1）% in the LPS-treated group and the apoptotic cell percentages were increased from（11.5±4.2）% in the normal control group to（39.5±8.2）% in the LPS group.LPS induced the increases in phospho-JNK1,active caspase 3 expression,and NO generation.Pioglitazone 0.01,0.1 and 1 μmol·L-1,respectively inhibited LPS-induced decrease in cell viability and increase of apoptotic morphology,active caspase 3 expression in cultured neurons.In LPS＋pioglitazone 1 μmol·L-1 group,cell viability was（97.8±9.7）%,the apoptotic cells percentage was（20.6±5.0）%,NO generation（6.8±1.3）μmol·L-1.Furthermore,pioglitazone also inhibited LPS-induced the increase in JNK1 phosphorylation and NO generation.JNK inhibitor SP600125 5 μmol·L-1 significantly inhibited LPS-induced neurotoxicity,cell viability was increased from（72.3±2.1）% to（109.8±11.8）%,the apoptotic cells percentage from（39.5±8.2）% decreased to（19.1±4.8）%,NO generation from（21.1±5.0）μmol·L-1 decreased to（4.0±1.3）μmol·L-1.The PPARγ antagonist GW9662 10 μmol·L-1 did not reverse the effects of pioglitazone.In LPS＋pioglitazone 1 μmol·L-1＋GW9662 10 μmol·L-1 group,cell viability was（90.7±6.9）%,the apoptotic cells percentage was（23.4±4.1）%,and NO concentration was（5.8±0.7）μmol·L-1.CONCLUSION Pioglitazone protects cortical neurons against LPS insult at least via inhibiting JNK activity and NO generation,but not PPARγ activation.