Activation of PI3-K/Akt pathway for thermal preconditioning to protect cultured cerebellar granule neurons against low potassium-induced apoptosis
Abstract
Aim: This study was designed to investigate whether the activation of the
phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning
to protect rat cerebellar granule neurons (CGN) against apoptosis
induced by low potassium, and to explore the possibility of a link between the
upregulated heat shock protein (HSP)70 expression and Akt activation in the
acquisition of neuroprotection induced by thermal preconditioning. Methods:
CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal
preconditioning (TP; 43.5 °C for 90 min, then 37 °C for 1 h). To study the role of the
PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 μmol/L) was added into the
cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide
(MTT) assay and fluorescein diacetate staining were used to determine cell
viability. Hoechst 33258 staining and agar gel electrophoresis were used to test
the morphological and biological characters of CGN. Western blot analysis was
employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase
3β (GSK3β) Akt, GSK3β, and HSP70. Results: TP protected CGN against
apoptosis induced by low potassium. LY294002 inhibited the neuroprotective
effect on CGN induced by TP. TP induced a robust activation of Akt and the
inactivation of GSK3β via PI3-K. Furthermore, the activation of the PI3-K/Akt
pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 μmol/L) failed
to inhibit the upregulated HSP70 expression induced by TP. Conclusion: The
activation of the PI3-K/Akt pathway is required for TP to protect CGN against
apoptosis induced by low potassium, but the neuroprotective effect by Akt activation
is not mediated through the downstream induction of HSP70 expression.
Keywords:
phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning
to protect rat cerebellar granule neurons (CGN) against apoptosis
induced by low potassium, and to explore the possibility of a link between the
upregulated heat shock protein (HSP)70 expression and Akt activation in the
acquisition of neuroprotection induced by thermal preconditioning. Methods:
CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal
preconditioning (TP; 43.5 °C for 90 min, then 37 °C for 1 h). To study the role of the
PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 μmol/L) was added into the
cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide
(MTT) assay and fluorescein diacetate staining were used to determine cell
viability. Hoechst 33258 staining and agar gel electrophoresis were used to test
the morphological and biological characters of CGN. Western blot analysis was
employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase
3β (GSK3β) Akt, GSK3β, and HSP70. Results: TP protected CGN against
apoptosis induced by low potassium. LY294002 inhibited the neuroprotective
effect on CGN induced by TP. TP induced a robust activation of Akt and the
inactivation of GSK3β via PI3-K. Furthermore, the activation of the PI3-K/Akt
pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 μmol/L) failed
to inhibit the upregulated HSP70 expression induced by TP. Conclusion: The
activation of the PI3-K/Akt pathway is required for TP to protect CGN against
apoptosis induced by low potassium, but the neuroprotective effect by Akt activation
is not mediated through the downstream induction of HSP70 expression.