中文摘要：

目的 在原代培养皮质神经元中,观察过氧化氢（H2 O2）损伤是否诱导了过氧化物酶体增殖物激活受体γ （PPARγ）的磷酸化修饰及活性改变,从调节PPARγ的角度探讨H2 O2的损伤机制.方法 体外原代培养SD大鼠皮质神经元,完全随机分为正常对照组、H2O2损伤组（分别给予250、500、750 μmol/L H2O2作用2h）、U0126（ERK1/2激活抑制剂）组（10 μmol/L U0126预处理30min后给予500 μmol/L H2O2作用2h）,采用细胞形态学观察、MTT法测定细胞存活率、锥虫蓝染色测定细胞死亡率、Western印迹法检测PPARγ、p-PPARγ（磷酸化的PPARγ）蛋白表达水平及PPARγ的核移位（即PPARγ活性）的改变.结果 （1）与正常对照组相比,经不同浓度（250、500、750 μmol/L） H2O2损伤2h后,神经元相对存活率降低（74.8％±5.2％、53.6％±6.7％和26.5％±5.8％,均P＜0.05）,死亡率增加（正常对照组6.6％±1.0％,H2 O2损伤组23.1％±2.8％、48.2％±4.1％、75.9％±4.4％,均P＜0.05）.（2）与正常对照组相比,H2O2 500μmol/L损伤组神经元PPARγ蛋白表达无明显变化（正常对照组1.25±0.07,H2 O2损伤组1.16±0.08,t=1.44,P＞0.05）,而p-PPARγ蛋白表达增加（正常对照组0.90 ±0.04,H2O2损伤组1.26±0.09,t=-6.48,P＜0.05）.同时PPARγ胞质蛋白表达增加（正常对照组0.49±0.04,H2O2损伤组0.77±0.03,t=-10.35,P＜0.05）,胞核蛋白表达下降（正常对照组0.76±0.03,H2 O2损伤组0.20±0.06,t=14.82,P＜0.05）（即核移位下降）.（3）与H2 O2损伤组相比,抑制ERK1/2激活降低p-PPARγ蛋白表达水平（H2O2损伤组0.85±0.05,U0126组0.42±0.14,t =4.91,P＜0.05）、增加PPARγ核移位（胞质损伤组1.03±0.16,U0126组0.60±0.04,t=4.58,P＜0.05;胞核损伤组0.16±0.04,U0126组0.87±0.11,t=-10.41,P＜0.05）,同时增加神经元存活率（70.8％±1.3％,P＜0.05）,降低神经元死亡率（29.8％±3.4％,P＜0.05）.结论 原代皮质神经元中,PPARγ的磷酸化参与了H2 O2的细胞?

英文摘要：

Objective To investigate whether H2O2 treatment negatively regulates PPARγ in primary cortical neurons by increasing PPARγphosphorylation.Methods Primary cultured cortical neurons were treated with H2O2（250,500,and 750 μmol/L） for2 h.30 min before the H2O2（500 μmol/L）,the specific inhibitor of ERK1/2 activation,U0126,was added to the culture.Morphological observation,MTT assay and the trypan blue exclusion method were used to detect cell damage.Western blot was carried out to evaluate the expressions of p-PPARγ （phospho-PPARγ） and total PPARγ,as well as to investigate the nuclear translocation of PPARγ （PPARγ activity）.Results （1） Compared with the control group,cell survival rates were decreased by H2O2 at concentrations of 250,500,and 750 μmol/L （74.8% ± 5.2%,53.6% ±6.7% and 26.5% ±5.8%,respectively,P 〈 0.05）,while cell death rate were increased （ctrl group 6.6% ± 1.0%,H2O2-injured groups：23.1% ±2.8%,48.2% ±4.1% and 75.9% ±4.4% respectively,P 〈 0.05）.（2） Compared with the control group,the expression of total PPARγ failed to show significant change in H2O2-injured group,whereas the expression of p-PPARγ increased.Neurons injured by H2O2（500 μmol/L） also showed a reduction of PPARγ nuclear translocation （an increase in cytosol PPARγand a simultaneous decrease in nuclear PPARγ）.（3） Compared with H2 O2-injured group,inhibition of ERK1/2 activation decreased p-PPARγ expression,and increased PPARγ nuclear translocation,as well as improved cell survival rate （53.6% ±6.7% vs 70.8% ± 1.3%,P 〈0.05） and decreased cell death rate （48.2% ± 4.1% vs 29.8% ± 3.4%,P 〈 0.05）.Conclusion Phosphorylation of PPARγ may be involved in cell death induced by hydrogen peroxide in primary cultured cortical neurons.