Effects Of ATP Sensitive Potassium Channel Opener On The Apoptosis And The PI3K/Akt/Bcl-2 Signal Passway Of PC12 Cells Induced By Ischemia And Hypoxia

IntroductionIschemic cerebrovascular disease are currently seriously endanger human health, and death and disability rates are higher.The key to the treatment of cerebral ischemia is to reduce the cerebral ischemic penumbra neuronal apoptosis.Clinical doctors always look for really effective drugs as a brain protective agent research focus of the work content.The ATP-sensitive potassium channel(K_ATP) opener in recent years is a new brain protective agent.But the specific mechanism of the protective effect remains unclear.PI3K(phosphatidylinositol 3-kinase,phosphatidylinositol 3-kinase) / AKT (v-akt murine thymoma viral oncogene homolog) signal transduction pathways are important in intracellular signal transduction pathway in apoptosis,survival, proliferation events.PI3K/AKT signaling pathway is a cell survival signaling pathway and its neuroprotective effect has been confirmed.PI3K/Akt signaling pathway can directly induce anti-apoptotic protein Bcl-2 increase.In this study,we through PC12 cell culture methods,observe the effects of K_ATP channel openers on ischemic hypoxia-inducible PC12 cell apoptosis and p-Akt and Bcl-2 mRNA and protein expression,to explore the K_ATP channel opener of the cerebral protection mechanism and for the K_ATP channel opener can be applied as soon as possible for the clinical treatment of ischemic cerebrovascular disease provides a theoretical basis.ObjectiveTo observe the effect of K_ATP channel openers on hypoxic-ischemic apoptosis in PC12 cells,and further explore the signal transduction mechanism of the K_ATP channel opener against PC12 cells after hypoxia-ischemia apoptosis,to clear K_ATP channel opener to inhibit PC12 cells apoptosis through PI3K / Akt/Bcl-2 signal transduction pathway,for the KATP channel opener can be applied as soon as possible the clinical treatment of ischemic cerebrovascular disease provides a theoretical basis.Methods3d after PC12 cells passsge,divided into A group(normal control group),B group (ischemic control group),C group(pinacidil treatment group),D group(pinacidil +Glipizide treatment group) the altogether 4 groups.C group PC12 cells at 20min before PC12 cells ischemia oxygen deficit add pinacidil(100μmol.L-1);D group add pinacidil(100μmol.L-1) and K_ATP,channel blocking agent glipizide(500μmol. L-1).Neuronal apoptosis is detected by Annexin-v FITC/PI double-dyed flow cytometry, the expression of Bcl-2 and p-Akt proteins are detected by Immunofluorescent staining and Western-blotting methods,the expression of Bcl-2 and Akt mRNAs are detected by RT-PCR,to observe the protective effect of the K_ATP openiner on PC12 cells ischemia oxygen deficit.Results1.PC12 apoptosis resultsB,C,D group with the apoptosis rate increased with increasing time points, peaked at 24 hours.With time after,coming down.Various time points B,C,D Group apoptosis rate were higher than group A(P＜0.01),C group the apoptosis rate significantly lower than that of the B,D group,(P＜0.01),B and D group has no significant difference(P＞0.05).2.Immunofluorescent staining resultsB,C,D group at different time points were seen P-Akt,Bcl-2 expression,and the P-Akt,Bcl-2 expression is mainly in cytoplasm,the expression of an occasional at nucleus.K_ATP channel opener group expresses the most obviously.Fluorescence intensity of PC12 cells in B,C,D group were significantly higher than that of in A group(P＜0.01或P＜0.05).However,C group was significantly higher than the B and D group(P＜0.01).B and D group has no significant difference(P＞0.05).3.Western-blotting resultsThe expression of p-Akt and Bcl-2 proteins in B,C,D groups increase along with the time spot increases,12 hours reach the peak.The expression of p-Akt and Bcl-2 proteins in B,C,D groups are higher than that in A group(P＜0.05或P＜0.01).The expression of p-Akt and Bcl-2 proteins in C group are significant higher than that in B,D groups(P＜0.01).there are no differences between B,D groups(P＞0.05)4.RT-PCR resultsThe expression of p-Akt and Bcl-2 mRNAs in B,C,D groups increase along with the time spot increases,12 hours reach the peak..The expression of p-Akt and Bcl-2 mRNAs in B,C,D groups are higher than that in A group(P＜0.05 or P＜0.01).The expression of p-Akt and Bcl-2 mRNAs in C group are significant higher than that in B,D groups(P＜0.01).there are no differences between B,D groups(P＞0.05)ConclusionK_ATP channel opener pinacidil significantly decreases PC12 cell apoptosis after ischemia and hypoxia,and increases P-akt,Bcl-2 protein and mRNA expression, suggesting that K_ATP channel openers probably reduce PC12 cell apoptosis after hypoxia-ischemia by activating PI3K/Akt/Bcl-2pathway.