Effects of LY294002 on Antagonistic Action of Thermal Preconditioning against Low Potassium-induced Apoptosis in Cultured Rat Cerebellar Granule Neurons
CAO Lin 1, JIANG Wei-jian 2, SU Xing-wen 2, CAO De-xiong 1, QIU Peng-xin 2, YAN Guang-mei 2(1. Department of Anesthesiology of The Second Affiliated Hospital, 2. Department of Pharmacology, Preclinical Medical School, SUN Yat-sen University, Guangzhou 510120, China)
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.
【Fund】： 国家杰出青年基金资助项目(39625022);; 国家自然科学基金资助项目(39900181);; 广东省科技计划基金资助项目(2KM028091)
【CateGory Index】： R96
【CateGory Index】： R96