Compound C抑制腺苷酸活化蛋白激酶的磷酸化后对缺氧预处理的影响及其机制

Effect and Mechanism of Phosphorylated AMPK on Hypoxia Preconditioning with Its Special Inhibitor-Compound C

【目的】探讨Compound C抑制腺苷酸活化蛋白激酶(AMPK)的磷酸化后对缺氧预处理的影响。【方法】筛选合适的Compound C浓度。将细胞分为Compound C组和non-Compound C组,以上两组再各自分为3个亚组:对照组(control);缺氧预处理+缺氧组(Hyp+OGD);单纯缺氧组(OGD);噻唑蓝(MTT)比色法测定细胞活性。ATP荧光检测试剂盒测定胞内的ATP水平。Western Blot法测定各组细胞内AMPKα,磷酸化的AMPKα(P-AMPKα),过氧化物酶体增生激活受体-γ共刺激因子-1α(PGC-1α)的蛋白表达水平。【结果】OGD后Compound C组(0.418±0.021)和non-Compound C组(0.640±0.028)相比,细胞活性下降更明显(P<0.01),经预处理后细胞活性均增加(P均<0.05),加入Compound C后,control组细胞活性下降3.5%(P=0.473),OGD组细胞活性下降34.6%(P<0.01),而缺氧预处理后细胞活性下降21.1%(P<0.05)。OGD后Compound C组(0.042±0.001)和non-Compound C组(0.051±0.001)相比,ATP下降更明显(P<0.05),经预处理后ATP水平分别增加了21.5%及28.0%(P均<0.05)。Compound C 3组AMPKα蛋白的表达与non-Compound C对应的3组相比差异无统计学意义(P均>0.05)。OGD后,Compound C组与non-Compound C组细胞P-AMPKα蛋白的表达均明显增加(与各自的对照组比较,P均<0.05)。经预处理后,non-Compound C组P-AMPKα蛋白的表达较单纯OGD组增加(P<0.05),但Compound C组P-AMPKα蛋白的表达与单纯OGD组间的差异无统计学意义(P=0.935)。Compound C组细胞PGC-1α蛋白的表达与non-Compound C组对应的3组比较明显下降(P均<0.05),其中control组下降了68.1%,Hyp+OGD组下降了24.7%,OGD组下降了39.6%,Hyp+OGD及OGD相对于各自control组均上调其表达(P均<0.05),且预处理组的上调更明显(与OGD组比较,P均<0.05)。【结论】Compound C抑制AMPK的激活后,PGC-1α的表达下调,AMPK为缺氧预处理或缺氧处理上调PGC-1α的其中一条途径,抑制其活体P-AMPKα后其他的途径仍然在缺氧预处理中发挥重要的细胞保护作用。

[ Objective ] To investigate the effect of AMP-activated protein kinase （AMPK） on hypoxia preconditioning （Hyp） with its special inhibitor-Compound C. [ Methods ] MTF assay an Western blot were used to select the suitable Compound C concentration. Cells with Compound C or without Compound C were divided into control, hypoxia preconditioning ＋Oxygen-glucose deprivation （Hyp ＋OGD） and OGD respectively. MTT assay was used to test the cell viability. The ATP level was evaluated using ATP Fluorometric Assay Kit. The expression of AMPK, Peroxisome proliferators-activated receptor Y coactivator-lα （PGC-lα and phosphorylated-AMPK （P-AMPK） were assessed at the protein level by Western blot. [Results] After OGD, the cell activity decreased more in Compound C compared with non-Compound C （0.418-＋0.002 vs 0.640＋0.028, P 〈 0.01）, but were increased in Hyp＋OGD respectively （P 〈 0.05）. After adding Compound C, the cell activity decreased 3.5% in control （P=0.473）, 34.6% in OGD （P 〈 0.01） and 21.1% in Hyp＋OGD （P 〈 0.05）. After OGD, the ATP levels decreased more in Compound C compared with non-Compound C （0.042＋0.001 vs 0.051＋0.001 ,P 〈 0.05）, but were increased 21.5% and 28.0% in Hyp＋OGD respectively （P 〈 0.05）. The expression of AMPK had no statistical difference in the three groups of Compound C compared with non-Compound C respectively （P 〉 0.05）.After OGD, the expression of P-AMPKα were increased significantly in Compound C and non-Compound C compared with its control （P 〈 0.05）. After preconditioning, the expression of P-AMPKct were increased in non-Compound C compared with OGD （P 〈 0.05）, while had no statistical difference in Compound C compared with OGD （P = 0.935 〉 0.005）. The expression of PGC-lα were declined significantly in the three groups of Compound C compared with non-Compound C respectively （P 〈 0.01）, it decreased 68.1% in control, 24.7% in Hyp＋OGD and 39.6% in OGD, but were both increased in Hyp＋OGD and OGD compared with control （P 〈 0.05）, and were increased more in Hyp＋OGD compared with OGD （P 〈 0.05）. [ Conclusion ] The expression of PGC-lα were decreased with Compound C （a special inhibitor of AMPK） and AMPK was one of the ways that hypoxia preconditioning or hypoxia up regulated the expression of PGC-lα.