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

IntroductionIschemic cerebrovascular disease seriously endanger human life and health. We do not have effective treatment until now. In acute phase of cerebral ischemia, It is very important for healing, how to save the neurons in ischemic lesion sensitive areas effectively. ATP-sensitive potassium channel (KATP) openers could promote nerve cell survival and regeneration against hypoxia, which has become a target for anti-injury research.But its mechanism is not fully understood. In recent years, a new growth factor mediated signal transduction pathway-PI3K (phosphatidylinositol 3-kinase, phosphatidylinositol 3-kinase)/Akt(v-akt murine thymoma viral oncogene homolog)-gradually awareness. Research shows that it is involved in many cell's anti-apoptosis, proliferation, differentiation, it plays an especially significant facilitating role for the growth factor-mediated neuronal survival. PI3K/Akt can directly or indirectly phosphorylate cAMP response element binding protein (cAMP response element binding p-rotein, CREB), and play an important role in anti-apoptosis. In this study, through PC 12 cell culture methods, we observe the effect of KATP channel opener on hypoxia-ischemia PC 12 cells apoptosis and p-Akt, p-CREB protein and mRNA expression,to explore the KATP channel opener of cerebral protection mechanism.It will provide a theoretical basis for the KATP channel opener applied as early as possible in clinical treatment of ischemic cerebrovascular disease.ObjectiveTo observe the effect of KATP channel openers on hypoxic-ischemic apoptosis in PC 12 cells,and further explore the signal transduction mechanism of the KATP channel openers against PC 12 cells after hypoxia-ischemia apoptosis, to assess whether KATP channel openers regulate cell apoptosis through PI3K/AKT/CREB signal transduction pathways. for the KATP channel opener can be applied as soon as possible, it will provide a theoretical basis for the clinical treatment of ischemic cerebrovascular disease.Methods3 day after PC 12 cells passage,cells were divided into group A(normal control group), group B(ischemic control group), group C(pinacidil treatment group),group D(pinacidil and glipizide treatment group) 4 groups altogether. group C PC 12 cells at 20min before PC12 cells ischemia oxygen deficit add pinacidil(100μmol.L-1);D group add pinacidil(100μmol.L-1) and KATP channel blocking agent glipizide (500μmol.L-1). Neuronal apoptosis is detected by Annexin-v FITC/PI double-dyed flow cytometry, the expression of p-Akt and p-CREB proteins are detected by Immunofluorescent staining and Western-blotting methods. The expression of Akt and CREB mRNAs are detected by RT-PCR, to observe the protective effect of the KATP channel opener on PC 12 cells ischemia oxygen deficit.Result1,PC 12 cells apoptosis resultsThe apoptosis rate of B,C,D group increase along with time, peaked at 24 hours. With time after, coming down. Various time points B,C,D group apoptosis rate were higher than group A (P<0.05), the apoptosis rate for group C were lower than that of the B, D group (P<0.05),there are no differences between B,D groups (P>0.05).2,Western-blotting resultsThe expression of p-Akt and p-CREB protein in B,C,D groups increase along with the time,reach the peak at 12 hours. The expression of p-Akt and p-CREB protein in B,C,D groups are higher than that in A group (P<0.05).The expression of p-Akt and p-CREB protein in C group are higher than that in B,D groups(P<0.05). there is no differences between B,D groups (P>0.05).3,RT-PCR resultThe expression of p-Akt and p-CREB mRNAs in B,C,D groups increase along with time, reach the peak at 12 hours. The expression of p-Akt and p-CREB mRNAs in B,C,D groups are higher than that in A group (P<0.05).The expression of p-Akt and p-CREB mRNAs in C group are higher than that in B,D groups (P<0.05). there is no differences between B,D groups (P>0.05).ConclusionKATP channel opener, pinacidil, can decrease PC 12 cells apoptosis after ischemia and hypoxia,and increases p-Akt and p-CREB protein and mRNA expression, suggesting that KATP channel opener probably reduce PC 12 cell apoptosis after hypoxia-ischemia by activating PI3K/AKT/CREB pathway.