过氧化物酶体增殖物激活受体γ在缺氧缺血性神经细胞死亡中的作用

Effect of peroxisome proliferation activated receptor -γ on neuronal cell death induced by hypoxia and ischemia in rats in vitro and in vivo

目的探讨过氧化物酶体增殖物激活受体γ(PPARγ)在缺氧缺血性神经细胞死亡中的作用。方法体外实验采用原代培养的大鼠胎鼠皮质神经细胞,给予缺氧再给氧处理;体内实验采用线栓法建立大鼠局灶性短暂性脑缺血再灌注损伤模型。用噻唑蓝比色法测定神经细胞生存率,用电泳迁移率变动分析法检测PPARγ的结合活性。结果缺氧处理后的神经细胞,其PPARγ的结合活性明显增加,以对照组活性为100,而缺氧3h组为160.3,缺氧3h再给氧的2、4、8h分别为157.5、136.6、103.3;缺氧3h及再给氧2h组与未处理组相比(P<0.01)。缺血处理后的神经细胞,其PPARγ的结合活性也明显增加,以假手术组为100,则手术组缺血侧、未缺血侧分别为144.8、102.6,缺血侧PPARγ的结合活性与未缺血侧及假手术组相比(P<0.01);PPARγ拮抗剂GW9662能明显增加缺氧再给氧后神经细胞的生存率,以对照组(CTL)生存率为100%计算,单纯缺氧组为58.6%,不同浓度的GW9662(0.5、1、2.5)预处理后分别为68%、73.6%和89.7%,GW9662预处理组与单纯缺氧再给氧组相比(P<0.05或P<0.01);GW9662能明显降低由于缺氧而增高的PPARγ结合活性,以未处理组为100,单纯缺氧组、GW9662预处理组分别为184、105,GW9662预处理组神经细胞PPARγ的结合活性与单纯缺氧再给氧组相比,P<0.01。

Objective To observe the effects of peroxisome proliferation activated receptor (PPAR)-γ on neuronal cell death induced by hypoxia/reoxygenation and ischemia/reperfusion.Methods Cortical neural cells of fetal SD rats were cultured for 12 days and exposed to hypoxia/ reoxygenation so as to establish a hypoxia/ reoxygenation model. Another primary fetal rat cortical neuronal cells were pre-treated with different concentrations of GW9662, antagonist of PPAR-γ, then underwent hypoxia for 3 hours, re-oxygenated for 21 hours. MTT was added one hour after to measure the cell viability. Eleven male SD rats underwent right middle cerebral artery occlusion (MCAO) using suture and reperfusion. Eleven rats underwent sham operation. after the rats were killed and their brains were taken out. Nucleoprotein was extracted from the cultured primary cortical cells and the cerebral cortexes of the rats and co-cultured withγ-~32-labeled PPAR-γ probe, EMSA to detect the PPAR-γ binding activity. Results The PPAR-γ activity of the cultured fetal rat cortical neurons that underwent hypoxia/reoxygenation significantly increased: It began to increase 1 hour after hypoxia and peaked in the 3rd hour of hypoxia, when the neurons underwent hypoxia for 3 hours and were reoxygenated for 2 hours, the binding activity still remained at a high level, and basically returned to the level of the untreated group 8 hours after reoxygenation. Data were quantified with control group as 100,3 h of hypoxia was 160.3, and 2,4,8 h after reoxygenation were 157.5,136.6,103.3 separately. One hour after reperfusion the PPAR-γ binding activity of the cortical cells at the ischemic side of the rats began to increase and peaked at the 4th hour, significantly higher than those of the cortical cells at the opposite side and of the sham operation group (both P<0.01) then remained at a high level for the following 24 hours. The survival rate of the cultured neurons that underwent hypoxia for 3 hours and reoxygenation for 21 hours was significantly lower than that of the untreated neurons. Data were quantified with sham surgery group as 100, the side of MCAO and the contra side in surgery group were 144.8 and 102.6 separately. The survival rate of the neurons that were pretreated with GW9662 and then underwent hypoxia/reoxygenation was significantly higher than that of those without pretreatment (P<0.01) with the peak protection effect of GW9662 at the concentration of 2.5-10 μmol/L. Data were quantified with control group as 100,hypoxia/ reoxygenation group was 184, GW9662 group was 105. The PPAR-γ binding activity of the primary cortical neurons pretreated with 5 μmol/L GW9662 for 30 minutes and than exposed to hypoxia for 3 hours and reoxygenation for 2 hours was significantly lower than that of the only hypoxia/reoxygenation group (P<0.01). Conclusion PPAR-γ is involved in the pathogenesis of neuron death induced by hypoxia/ ischemia and may become a new target of treatment of ischemic stroke.