能量限制激活Sirt1减轻心肌细胞缺血/再灌注损伤的作用

Effect and mechanism of calorie restriction-activated Sirt1 in protection of myocardial cells from ischemia/reperfusion injury

目的研究能量限制(calorie restriction,CR)激活沉默信息调节因子2相关酶Ⅰ(Sirt1)在缺血/再灌注损伤(ischemia reperfusion injury,I/RI)中对心肌细胞的保护作用及其机制。方法心肌细胞系H9c2以5×105/L接种于6孔细胞培养板中,随机分为4组:对照(Con)组、缺氧复氧(hypoxia/re-oxygenation,H/R)组、H/R+低糖预处理(H/R+CR)组和H/R+低糖预处理+Sirt1抑制剂EX527(H/R+CR+EX527)组,其中Con和H/R组使用葡萄糖浓度为4.5 g/L的培养基,而H/R+CR组和H/R+CR+EX527组则使用葡萄糖浓度为1.5 g/L的培养基,每组设置3个复孔,进行缺氧5 h复氧1 h,用DCFH-DA荧光探针检测细胞内活性氧(ROS)的水平,四甲基偶氮唑盐(MTT)比色法检测细胞的活性,末端标记法(TUNEL)染色法检测细胞的凋亡水平,蛋白免疫印迹法(Western blot)检测Sirt1和磷酸化腺苷酸活化蛋白激酶(P-AMPK)的表达。结果与H/R组(0.31±0.06)相比,H/R+CR组(0.11±0.02)细胞内ROS的水平明显减少(P<0.05);H/R+CR+EX527组(0.55±0.06)细胞内ROS的水平明显增多(P<0.05);与H/R组(0.23±0.01)相比,H/R+CR组(0.64±0.02)细胞活性明显上升(P<0.05);H/R+CR+EX527组(0.09±0.005)细胞活性明显下降(P<0.05);与H/R组(0.444±0.038)相比,H/R+CR组(0.222±0.556)细胞凋亡水平明显降低(P<0.05);H/R+CR+EX527组(0.578±0.0129)细胞凋亡水平明显提高(P<0.05);与H/R组(2.229±0.019)相比,H/R+CR组(3.712±0.010)Sirt1的表达水平明显增加(P<0.05);而H/R+CR+EX527组(1.603±0.134)Sirt1的表达水平明显降低(P<0.05);与H/R相(1.262±0.064)比,H/R+CR组(1.893±0.122)P-AMPK的表达水平明显增加(P<0.05);而H/R+CR+EX527组(0.734±0.069)P-AMPK的表达水平明显降低(P<0.05)。结论在I/RI中,CR可能通过AMPK途径激活Sirt1,进而发挥心肌保护作用。

AIM To investigate the protection and mechanism of calorie restriction-activated sirtuins 1 （Sirtl） on myocardial cells in ischemia/repeffusion injury （I/RI）. METHODS Myocardial cell line H9c2 seeded 5 x 105/L in a six-well cell culture plate was randomly divided into four groups ： control, hypoxia/re-oxygenation （H/R）, H/R ＋ CR and H/R ＋ CR ＋ EX527. In control group and hypoxia group, glucose concentration was 4. 5 g/L, whereas glucose concentration in low glucose preconditioning group and Sirtl inhibitor group was 1.5 g/L. DCFH-DA probe was used to detect the level of reactive oxygen species （ROS）, methyl thiazolyl tetrazolium （MTr） assay was used to detect cell activity, terminal labeling （TUNEL） staining was used to detect apoptotic levels of cells, and Western blot was used to detect Sirtl expression and phosphorylation amp activated protein kinase （P-AMPK） protein. RESULTS Compared with those in H/R group, intracellular levels of ROS in H/R ＋ CR group decreased significantly （0.31 ± 0. 06 vs. 0. 11±0. 02, P 〈0.05 ） and increased significantly in H/R ＋ CR ＋ EX527 group （0. 31 ±0. 06 vs. 0. 55 ± 0. 06, P 〈 0. 05 ）. Compared with those in H/R group, cell activity in H/R ＋ CR group increased significantly （0. 23 ±0. 01 vs. 0. 64 ±0.02, P 〈0. 05 ） and decreased significantly in H/R ＋ CR ＋ EX527 group （0.23 ± 0.01 vs. 0. 09 ± 0. 005, P 〈 0.05）. Compared with those in H/R group, apoptosis levels in H/R ＋ CR group significantly decreased （0. 444 ± 0. 038 vs. 0. 222 ± 0. 556, P 〈 0. 05） and significantly increased in H/R ＋ CR ＋ EX527 group （0. 444 ±0. 038 vs. 0. 578±0. 0129, P 〈 0.05 ）. Compared with those in H/R group, Sirtl expression levels significantly increased in H/R ＋ CR group （2. 229 ±0. 019 vs. 3.712 ±0. 010, P 〈 0.05 ） and significantly decreased in H/R ＋ CR ＋ EX527 group （2. 229 ± 0. 019 vs. 1. 603 ± 0. 134, P 〈 0. 05 ）. Compared with those in H/R group, P-AMPK expression levels in H/R ＋ CR group significantly increased （ 1. 262± 0. 064 vs. 1. 893 ± 0. 122, P 〈 0. 05 ） and significantly decreased in H/R ＋ CR ＋ EX527 group （ 1. 262 ± 0. 064 vs. 0. 734 ±0. 069, P 〈 0. 05 ）. CONCLUSION Calorie-restricted activated Sirtl may protect myocardial cells from ischemia/reperfusion injury through P-AMPK pathway.