The Effects And Mechanism Of Ca²⁺/CaN/Cbl-b/Akt In The Oxygen-glucose Deprivation And Pharmacological Preconditioning

Objective：Stroke is the major cause of disability and death among adults, and it is the heavyload of patients’ family and society, but no effective therapeutic strategy has beenproduced to improve the prognosis of ischemic cerebrovascular disease.Transientsublethal ischemia known as ischemic preconditioning can protect cells and tissues tosurvive subsequent prolonged lethal ischemic injury. This phenomenon was termed as“ischemic tolerance”.The phosphatidylinositol3-kinase （PI3K）/Akt pathway is related to survivalactivity in many kinds of neuronal cell types. Cbl-b belongs to the Casitas B-lineagelymphoma （Cbl） family and can regulate the signal transduction. In T cell, Cbl-bnegatively regulate PI3K/Akt signaling pathway by ubiquitinating the P85subunit ofPI3K, and suppress the activation of T cell. This negative regulation of Cbl-b to PI3Kwas reported in many kinds of cells such as T lymphocytes, osteoblasts, B cell, mastcell. Our group previously reported that Cbl-b was involved in the apoptosis regulationin animal brain ischemia model.In T cell elevated calcium combine with the calmodulin （CAM） and activatecalcineurin （CaN）. The activated CaN makes transcription factor nuclear factor ofactivated T cells （NFAT） dephosphorylated and transfer into nucleus. NFAT, as atranscription factor, regulates the expression of Cbl-b gene.In the present study, we investigated the relationship between Ca²⁺/CaN/Cbl-b andPI3K/Akt and their regulation to ischemic preconditioning in PC12cell oxygen-glucosedeprivation （OGD） model. We also explored the effects and mechanism ofpharmacological preconditioning of calcium ion antagonist （nimodipine and MK801）.Methods： 1. The model of ischemia and preconditioning was established throughoxygen-glucose deprivation.2. MTT assay was used to determinate the cell viability.3. Morphologic change and staining nuclear by Hoechst33258was employed toinvestigate the apoptosis of cells.4. Real-time PCR method was employed to determinate the mRNA level ofCbl-b.5. Western blot method was used to determinate the expression of CaN, Cbl-b,p-Akt, p-GSK3β, Caspase-3and Bcl-2.6. Statistical analysis: all data are tested independently six to eight times, shownas mean±standard deviation. One-way analysis of variance was performed bySPSS13.0statistical software.Results：1. Cell viability was significantly changed by OGD and OGD preconditioning:OGD greatly decreased cell viability compared with control group, and preconditioningcould rescue this damage demonstrated by the increase of cell viability.2. Real-time PCR: The transcription of Cbl-b was significantly higher in theischemia group than the control group （P<0.05）. The transcription of Cbl-b was greatlylower in the preconditioning group （P<0.05）. LY294002partly inhibited the decrease ofCbl-b transcription.3. Western blot: the expressions of CaN and Cbl-b was significantly increasedafter OGD treatment. However, the activation of Akt and GSK3β was greatly inhibited.Preconditioning could inhibit the increase of CaN and Cbl-b caused by OGD, theactivation of Akt and GSK3β was increased. LY294002, a specific inhibitor of PI3K,could effectively inhibit the increase of Akt and GSK3β after preconditioning treatment.And it partly inhibited the decrease of CaN and Cbl-b expressions after preconditioningtreatment.4. CaN, Cbl-b and Akt were differently activated in OGD preconditioning: theexpression of Cbl-b was increased at beginning, and arrived expression peak at6h. Theexpression of Cbl-b decreased gradually with time prolonged. Contrary to Cbl-b, theAkt were continuously activated. The expression of CaN decreased gradually from0h.5. OGD preconditioning protected cell from damage by inhibiting cell apoptosis:the expression of Bcl-2was significantly increased and the Caspase-3was decreased inthe preconditioning group, resulting in the inhibition of cell apoptosis. LY294002could greatly block the increase of Bcl-2and decrease of Caspase-3, and finally block theprotective effect of OGD preconditioning.6. Ca²⁺was involved in OGD preconditioning process: expressions of CaN andCbl-b were inhibited by OGD preconditioning. Pre-incubation of A23187increasedexpressions of CaN and Cbl-b reduced by OGD preconditioning, and theneuro-protective effect of OGD preconditioning was blocked.7. Nimodipine or MK801protected PC12cells from OGD damage: pre-incubationof nimodipine or MK801protected cells from OGD damage demonstrated by theincrease of cell viability, and this effect could be inhibited by LY294002.8. CaN, Cbl-b and PI3K/AKT pathway were involved in neuro-protective effectof Nimodipine or MK801: Nimodipine or MK801significantly inhibited the increase ofCaN and Cbl-b, activated Akt and GSK3β; LY294002had no effect on expressions ofCaN and Cbl-b when cells were pre-treated with Nimodipine or MK801, but couldeffectively inhibited the activation of Akt and GSK3β.9. Nimodipine or MK801protected cell from damage by inhibiting cell apoptosis:the expression of Bcl-2was significantly increased and Caspase-3was decreased,resulting in the inhibition of cell apoptosis when cells were pre-incubated withNimodipine or MK801before OGD. LY294002could specific block thisneuro-protective effect of Nimodipine or MK801.Conclusion1. Expressions of CaN and Cbl-b increased and p-Akt/p-GSK decreased in OGDgroup, OGD reduced the expression of anti-apoptotic protein Bcl-2and up-regulatedcaspase-3, resulting in cell injury. But OGD preconditioning reversed this process andprotect PC12cell from OGD injury. CaN de-phosphorylate transcription factor NFAT,which regulates the expression of Cbl-b gene, and then transfer into nucleus. Cbl-bnegatively regulates the PI3K/Akt pathway by ubiquitinating the P85subunit of PI3K,and protects PC12cells from OGD damage.2. The regulation of calcium ionophore A23187and calcium mobilization blockercyclosporine A in OGD and preconditioning process is very important. Which indicatesthe decrease of Ca²⁺might be the initiation factor of OGD preconditioning protection. Itimplies that Ca²⁺antagonist could be used as preconditioning drug.3. Two different kinds of Ca²⁺antagonist nimodipine and MK801both decreasedthe expression of CaN and Cbl-b, and then acted on PI3K/Akt survival pathway whichregulates the balance of pro-apoptotic and anti-apoptotic protein, they might works through the ways similar to ischemic preconditioning. Compared with ischemicpreconditioning the pharmacological preconditioning is easy to realize and littledangerous in clinical practice.