腺苷酸活化蛋白激酶在缺氧预处理中的保护作用及机制

Role of AMP-activated protein kinase in the neuroprotection against hypoxia injury in hypoxic preconditioning

目的探讨腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)参与缺氧预处理的保护作用及机制。方法将大鼠肾上腺嗜铬细胞瘤PC12细胞分为空白对照组(Control)、单纯缺氧预处理组(Hyp)、缺氧预处理+缺氧组(Hyp+OGD)、单纯缺氧组(OGD)。通过噻唑蓝(MTT)细胞活力测定及DAPI核染色法判定细胞的损伤程度;Western Blot测定细胞内腺苷酸活化蛋白激酶α亚基(AMPKα)、磷酸化的AMPKα(P-AMPKα)及过氧化物酶体增生激活受体的共刺激因子-1α(PGC-1α)的蛋白表达水平。结果 4组间细胞活性有统计学差异(F=127,P<0.01),OGD组细胞活性减少至48%(与Control组相比,P<0.01),而Hyp+OGD组细胞的活力回升至62.5%(与OGD组相比,P<0.01)。4组细胞间三磷酸腺苷(ATP)含量有统计学差异(F=584.833,P<0.01),Hyp+OGD组ATP含量为0.114507±0.001837,较OGD组增加0.048266(P<0.01)。Hyp+OGD组和OGD组AMPKα的蛋白表达增加(与Control组相比,P<0.01)。4组间P-AMPKα、PGC-1α的表达均有统计学差异(F分别为17.496、13.421,P均<0.01),缺氧预处理及缺氧刺激均可上调2种蛋白的表达(与Control组相比,P<0.05),Hyp+OGD组较OGD组蛋白表达的增加更明显(P<0.05)。结论缺氧预处理可产生细胞保护作用,缺氧预处理后AMPK可能通过PGC-1α促进ATP的生成,进而发挥重要的细胞保护作用。

Objective To investigate the mechanisms that AMP-activated protein kinase（AMPK）contributes to protect PC12 cells from Oxygen-Glucose Deprivation（OGD）damage in hypoxic preconditioning.Methods PC12 cells were randomly divided into control,hypoxic preconditioning,OGD with and without hypoxic preconditioning groups.MTT assay and DAPI staining were used for testing the cells viability.ATP Colormetric/Fluorometric assay kit was used to measure ATP levels.The expression of AMPKα,P-AMPKα and PGC-1α were assessed at the protein level by Western blot.Results Cell viability among the four groups were in statistics differences（F=127,P〈0.01）,MTT values following 6h OGD were around 48% in the OGD group but around 62.5% in the Hyp＋OGD group compared with the control group（P〈0.01）.ATP among the four groups were in statistics different（F=584.833,P〈0.01）.ATP was 0.114507±0.001837 in Hyp＋OGD and increased 0.048266 compared with OGD group（P〈0.01）.Compared with the control group,the expression of AMPKα protein in Hyp＋OGD and OGD groups were increased（P〈0.01）.The expression of P-AMPKα and PGC-1α proteins among the four groups were in statistics different respectively（F=17.496,13.421,P〈0.01）,and increased in Hyp＋OGD group compared with that in OGD group（P〈0.05）.Conclusion Hypoxic preconditioning can protect PC12 cells from hypoxic injury and one of the possible mechanisms in hypoxic preconditioning was that AMPK led ATP production through PGC-1α passway.