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目的探讨AKT通路在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大中的作用,及硫氧环蛋白过氧化物酶3(Peroxiredoxin-3,Prdx-3)对此作用的影响。方法体外培养心肌细胞(H9C2)随机分为对照组、AngⅡ组(10-7 mol/L)、AngⅡ+转染对照组和AngⅡ+Prdx-3转染组。脂质体转染法将Prdx-3表达质粒转染心肌细胞,western blot法检测Prdx-3及磷酸化AKT(p-AKT)蛋白表达,Realtime PCR法检测脑钠素(BNP)mRNA表达,二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平。结果 Prdx-3表达质粒转染心肌细胞后,Prdx-3蛋白表达值为0.94±0.10,高于转染对照组(0.35±0.04),差异有统计学意义(P<0.01)。与对照组比较,AngⅡ组BNP mRNA[(1.0±0.0)比(1.63±0.24)]、ROS水平[(3 631±317)比(4 678±270)]及p-AKT蛋白表达[(0.13±0.05)比(0.44±0.09)]均明显增加,差异有统计学意义(P<0.01)。AngⅡ+转染对照组和AngⅡ组的BNP mRNA[(1.77±0.22)比(1.63±0.24)]、ROS[(4 401±308)比(4 678±270)]及p-AKT蛋白水平[(0.53±0.08)比(0.44±0.09)]差异无统计学意义(P>0.05)。与AngⅡ组比较,AngⅡ+Prdx-3转染组BNP mRNA[(1.63±0.24)比(1.28±0.18)]、ROS[(4 678±270)比(3 933±237)]及p-AKT蛋白水平[(0.44±0.09)比(0.20±0.05)]明显下降,差异有统计学意义(P<0.05)。结论AngⅡ通过线粒体来源的ROS激活AKT通路,诱导心肌细胞肥大,而高表达Prdx-3可通过降低ROS水平来抑制AngⅡ的作用。
Objective To investigate the role of AKT pathway in cardiomyocyte hypertrophy induced by angiotensin Ⅱ (AngⅡ) and the effect of peroxiredoxin-3 (Prdx-3) on this effect. Methods H9C2 cells were randomly divided into control group, Ang Ⅱ group (10-7 mol / L), Ang Ⅱ + transfected control group and Ang Ⅱ + Prdx-3 transfected group. The expression of Prdx-3 and phosphorylated AKT (p-AKT) protein was detected by western blot and BNP mRNA expression by Realtime PCR. Fluorescein diacetate (DCFH-DA) was used to detect reactive oxygen species (ROS) levels. Results Prdx-3 protein expression was 0.94 ± 0.10 in transfected cardiomyocytes, which was significantly higher than that of the transfected control group (0.35 ± 0.04) (P <0.01). Compared with the control group, the levels of BNP mRNA [(1.0 ± 0.0) vs (1.63 ± 0.24), ROS (3631 ± 317) vs (4678 ± 270) 0.05) than that of (0.44 ± 0.09)], the difference was statistically significant (P <0.01). The levels of BNP mRNA [(1.77 ± 0.22) (1.63 ± 0.24)], ROS [(4 401 ± 308) vs 4678 ± 270) and the levels of p-AKT protein in AngⅡ + 0.53 ± 0.08) (0.44 ± 0.09), there was no significant difference (P> 0.05). Compared with AngⅡgroup, BNP mRNA (1.63 ± 0.24) (1.28 ± 0.18), ROS [(4678 ± 270) vs 3933 ± 237) and p-AKT protein (0.44 ± 0.09 vs 0.20 ± 0.05)], the difference was statistically significant (P <0.05). Conclusion AngⅡ can activate cardiac myocyte hypertrophy through activation of mitochondria-derived ROS, and high expression of Prdx-3 can inhibit the effect of AngⅡ by reducing ROS level.