LY294002对热预处理抗低钾诱导的大鼠小脑颗粒神经元凋亡的影响

Effects of LY294002 on Antagonistic Action of Thermal Preconditioning against Low Potassium-induced Apoptosis in Cultured Rat Cerebellar Granule Neurons

【目的】观察磷酸肌醇3位羟基激酶(PI3-K)的特异性阻断剂LY294002对热预处理抗低钾诱导的大鼠小脑颗粒神经元(CGN)凋亡的影响。探讨磷酸肌醇3位羟基激酶/AKT(PI3-K/AKT)信号转导通路是否参与了热预处理的保护作用及其机制。【方法】以低钾诱导的大鼠小脑颗粒神经元凋亡为模型。用相差显微镜观察形态学,DNA琼脂糖凝胶电泳和Hoechst33258核染色分析神经元凋亡,二乙酸荧光素(FDA)染色法检测细胞的存活率。蛋白质印迹(Westernblot)法检测蛋白的表达。【结果】热预处理(44℃,1h)可以保护低钾诱导的CGN的凋亡,使神经元存活率由(33.2±4.1)%上升至(76.1±5.6)%(P <0.01),核染色出现的核固缩和凋亡小体减少,DNA凝胶电泳DNA梯状条带明显减弱。LY294002(20μmol/L)可以明显抑制热预处理的保护作用,使神经元的存活率降至(37.4±3.5)%(P<0.01),核染色出现的核固缩和凋亡小体,DNA凝胶电泳出现梯状条带。热预处理可使HSP70、磷酸化AKT增高。LY294002可以抑制磷酸化AKT(p-AKT)的增加,而对HSP70则无明显影响。【结论】在低钾诱导CGN凋亡的模型中,LY294002可以抑制热预处理的抗凋亡作用,并可抑制p鄄AKT的增加,显示PI3鄄K/AKT信号转导通路参与了热预处理的保护作用,但该抑制作用可能不是通过抑制热休克蛋白(HSP)

To observe the effects of a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, on antagonistic action of thermal preconditioning against low potassium-induced apoptosis in cultured rat cerebellar granule neurons (CGN). To explore weather phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) signal pathway is involved in anti-apoptotic action produced by thermal preconditioning and its mechanism. Apoptosis was induced by low potassium in cultured rat CGN. Morphology of neurons was observed by phase-contrast microscopy and Hoechst 33258 nucleus staining. The neuronal viability was measured by fluorescein diacetate (FDA) staining. DNA fragmentation was analyzed by agarose gel electrophoresis. Expressions of HSP70, phospho-AKT (p-AKT), AKT were detected by Western blot analysis. Thermal preconditioning (44 ℃, 1 h) protected CGN from apoptosis induced by low potassium. The neuronal viability was elevated from (33.2±4.1)% to (76.1±5.6)% (P < 0.01), nuclear condensation/aggregation was reduced and a typical apoptotic DNA ladder almost disappeared. LY294002 (20 μmol/L) reversed the protection of thermal preconditioning, making neuronal viability decreased from (76.1±5.6)% to (37.4±3.5)% (P <0.01), nuclear condensation/aggregation was increased and a typical apoptotic DNA ladder appeared again. Thermal preconditioning elevated the expression of heat shock protein 70 (HSP70) and p-AKT, LY294002 inhibited the expression of p-AKT, HSP70 was not induced by thermal preconditioning. [Conclusion] LY294002 can reverse antagonistic action of thermal preconditioning against low potassium-induced apoptosis in cultured rat CGN, suggesting PI3-K/AKT pathway is involved in anti-apoptotic action produced by thermal preconditioning, but this action may not be produced by inhibiting the expression of HSP70.