Study Of Cerebral Ischemic Preconditioning Attenuates Ischemic Brain Injury And Neuron Apoptosis Possibly Through PI3K/Akt/GSK3β Signaling Pathways

Stroke is the second most common cause of death and the leading cause of long-term disability in worldwide adults. Stroke can be subdivided into two categories, ischemic and hemorrhagic stroke. Ischemic brain injury develops from a complex series of pathophysiological events, which are involved in excitotoxicity, oxygen free radical injury, inflammatory reaction and apoptosis. PI3K/Akt pathway has been shown to play critical role in regulating apoptosis,cell proliferation and survival in dieffrent systems. Several studies have reported that the PI3K/Akt pathway in heart ischemie injury. However, it remains to clarify the PI3K/Akt pathway that is involved in cerebral ischemic injury. We explored whether the antiapoptotic Bcl-2 family member MCL-1 plays a role in CIPC-mediated apoptosis. The aim of our study was to examine the effects of PI3-kinase inhibitor LY294002 and GSK3βinhibitor LiCL on the CIPC. Such as p-Akt、p-PDK1、p-GSK3β、PP2A and MCL-1, to investigated the role of PI3K/Akt pathway involved in CIPC.PartΙA model of cerebral ischemic preconditioning and the effect of CIPC on rat hippocampus neuronal apoptosisObjective:To introduce a model in vivo of ischemic tolerance induced by a cerebral ischemic preconditioning (CIPC) in SD rats, to explore the CIPC of endogen- ous neuroprotection and the effect on rat hippocampus neuronal apoptosis through PI3K/Akt/GSK3βsignaling pathways. To valuated the effectiveness of this CIPC model.Methods: Focal-focal CIPC model was established by improved suture method of Zea Longa and 3×10min middle cerebral artery occlusion (MCAO) were used for CIPC and followed by 1d reperfusion n before 24h MCAO. Adult male Sprague-Daw- ley rats were randomly assigned into five groups: Sham group, ischemia group (IS),ischemia+cerebral ischemia preconditioning group (CIPC),chemian+cerebral ischemia preconditioning +a selective PI3K inhibitor LY294002 group (LY294002), ischemia+ cerebral ischemia preconditioning+ a selective GSK3βinhibitor LiCL group (LiCL).Neurological deficit scores, infarct volume and pathological injury were evaluated by Bederson method, TTC staining . The apoptosis of the hippocampus neurons were detected in each group with TUNEL apoptosis detection kit.Result:Compared with ischemia group,CIPC decreased the neurological deficit scores,the volume of the cerebral infarction and reduced the apoptosis of neurons(P<0.05).LY294002 blocked the neuroprotection of CIPC. Compared with ischemia and CIPC groups, the selective GSK3βinhibitor LiCL decreased the neurological deficit scores, the volume of the cerebral infarction and reduced the apoptosis of neurons(P<0.05).Conclusion:Focal CIPC induced endogenous neuroprotection through PI3K/Akt/ GSK3βsignaling pathways.a selective PI3K inhibitor LY294002 blocked the neuroprotection of CIPC.The selective GSK3βinhibitor LiCL increased this effect.Part II: Study of the role and mechanism of PI3K/Akt/GSK3βsingnaling pathway in cerebral ischemia preconditioning neuroprotection against ischemic brain injury.Objective: To investigate the effects of PI3K/Akt/GSK3βcell signal pathway on cerebral ischemia preconditioning and its possible relationship with activation of PI3K/Akt/GSK3βsignal transduction pathway.Methods: Adult male Sprague-Dawley rats were randomly assigned into five groups: Sham group, ischemia group (IS),ischemia+cerebral ischemia preconditioning group (CIPC),ischemian+cerebral ischemia preconditioning+a selective PI3K inhibitor LY294002 group(LY294002),Ischemia+cerebral ischemia preconditioning+ a selective GSK3βinhibitor LiCL group (LiCL).Then cerebral ischemia preconditioning underwent sublethal CIPC 3×10 min and lethal pMCAO .After cerebral ischemia 24h, expression of p-PDK1、Akt、p-Akt、GSK-3β、p-GSK-3β(ser9) and MCL-1 were determined by Western blot, and the expression of GSK-3βand PP2A at the mRNA level by RT-PCR, respectively.Result: Compared with ischemia group, CIPC increased the expressions of p-PDK1、p-Akt、MCL-1 p-GSK-3β(ser9) in ischemic hippocampus (P<0.05).This effect was also abolished by the selective PI3K inhibitor LY294002.Compared with ischemia and CIPC groups ,the selective GSK3βinhibitor LiCL increased the expressions of p-PDK1,p-Akt,MCL-1, p-GSK-3β(ser9)(P<0.05).Compared with sham group, ischemia group and LY294002 increased the expressions of PP2A mRNA, CIPC decreased the expressions of PP2A mRNA. But no distinguished difference was found among CIPC and LiCL group (P>0.05). Compared with ischemia group, increased the expressions of GSK3βmRNA (P<0.05).Conclusion: The CIPC induced neuroprotective effect could be exerted via the activation of the PI3K/Akt/GSK3βpathway.PartⅢ: CIPC provides neuroprotection against ischemic brain injury by inhibiting mitochondrial apoptosis through PI3K/Akt/GSK3βsignaling pathways.Objective: To explore the mitochondrial permeability transition pore opening and activation of caspase-3,9 in ischemic rat hippocampus after pMCAO, and further investigate whether ischemic preconditioning inhibited hippocampus neuronal mitochondrial apoptosis pathway through PI3K/Akt /GSK3βsignaling pathways.Methods: Adult male Sprague-Dawley rats were randomly assigned into five groups: Sham group, ischemia group (IS),ischemia+cerebral ischemia preconditioning group (CIPC),Ischemian+cerebral ischemia preconditioning+a selective PI3K inhibitor LY294002 group (LY294002), Ischemia + cerebral ischemia preconditioning+ a selective GSK3βinhibitor LiCL group (LiCL).Then cerebral ischemia preconditioning underwent sublethal CIPC 3×10 min and lethal pMCAO . After cerebral ischemia 24h, expression of caspase-3,9 were determined by Western blot, the mitochondrial permeability transition pore opening were determined by spectrophotometric method.Result: Compared with sham group, ischemia increased the expressions of caspase-3,9 and MPTP opening in ischemic hippocampus (P<0.05). Compared with ischemia group, cerebral ischemia preconditioning decreased the expressions of caspase-3,9 and MPTP opening in ischemic hippocampus (P<0.05). This effect was also abolished by the selective PI3K inhibitor LY294002. The selective GSK3βinhibitor LiCL decreased the expressions of caspase-3,9 and MPTP opening (P<0.05).Conclusion:Cerebral ischemia induced the mitochondrial permeability transition pore opening and activation of caspase-3,9 in ischemic rat hippocampus.Cerebral ischemia preconditioning could inhibit inactivating the mitochondrial pathway of apoptosis and provide neuroprotection against ischemic brain injury through PI3K/Akt/GSK3βsignaling pathways.