CAJ | 학술논문

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的细胞损伤机制.
목적 재원대배양피질신경원중,관찰과양화경(H2 O2)손상시부유도료과양화물매체증식물격활수체γ (PPARγ)적린산화수식급활성개변,종조절PPARγ적각도탐토H2 O2적손상궤제.방법 체외원대배양SD대서피질신경원,완전수궤분위정상대조조、H2O2손상조(분별급여250、500、750 μmol/L H2O2작용2h)、U0126(ERK1/2격활억제제)조(10 μmol/L U0126예처리30min후급여500 μmol/L H2O2작용2h),채용세포형태학관찰、MTT법측정세포존활솔、추충람염색측정세포사망솔、Western인적법검측PPARγ、p-PPARγ(린산화적PPARγ)단백표체수평급PPARγ적핵이위(즉PPARγ활성)적개변.결과 (1)여정상대조조상비,경불동농도(250、500、750 μmol/L) H2O2손상2h후,신경원상대존활솔강저(74.8％±5.2％、53.6％±6.7％화26.5％±5.8％,균P＜0.05),사망솔증가(정상대조조6.6％±1.0％,H2 O2손상조23.1％±2.8％、48.2％±4.1％、75.9％±4.4％,균P＜0.05).(2)여정상대조조상비,H2O2 500μmol/L손상조신경원PPARγ단백표체무명현변화(정상대조조1.25±0.07,H2 O2손상조1.16±0.08,t=1.44,P＞0.05),이p-PPARγ단백표체증가(정상대조조0.90 ±0.04,H2O2손상조1.26±0.09,t=-6.48,P＜0.05).동시PPARγ포질단백표체증가(정상대조조0.49±0.04,H2O2손상조0.77±0.03,t=-10.35,P＜0.05),포핵단백표체하강(정상대조조0.76±0.03,H2 O2손상조0.20±0.06,t=14.82,P＜0.05)(즉핵이위하강).(3)여H2 O2손상조상비,억제ERK1/2격활강저p-PPARγ단백표체수평(H2O2손상조0.85±0.05,U0126조0.42±0.14,t =4.91,P＜0.05)、증가PPARγ핵이위(포질손상조1.03±0.16,U0126조0.60±0.04,t=4.58,P＜0.05;포핵손상조0.16±0.04,U0126조0.87±0.11,t=-10.41,P＜0.05),동시증가신경원존활솔(70.8％±1.3％,P＜0.05),강저신경원사망솔(29.8％±3.4％,P＜0.05).결론 원대피질신경원중,PPARγ적린산화삼여료H2 O2적세포손상궤제.
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.