生长分化因子15过表达对心肌缺血再灌注的保护作用及与其自噬的关系

Protective effect and its related with autophagy of growth differentiation factor 15 over expression on myocardial ischemia/reperfusion

目的通过构建生长分化因子15(GDF15)稳定过表达的心肌细胞株,探讨其对H9c2心肌细胞缺血/再灌注(I/R)损伤凋亡和自噬的影响。方法建立H9c2 I/R损伤模型,用靶向GDF15过表达慢病毒和阴性空载体病毒转染H9c2细胞,筛选出GDF15稳定过表达细胞株。分为3组:对照组、空载体组和GDF15过表达组。用MTT法检测I/R后细胞存活率,以LDH试剂盒检测细胞培养上清液中乳酸脱氢酶活力,以免疫印迹法检测凋亡相关蛋白Caspase-3、Bcl-2、Bax和自噬相关蛋白Beclin-1、LC3-B的表达。结果 I/R 2 h,对照组、空载体组和过表达组的细胞存活率分别为(60.47±5.03)%,(63.21±17.13)%,(74.93±10.03)%,过表达组细胞活性较对照组与空载体组均增高,差异有统计学意义(P<0.01)。对照组和过表达组LDH活力值分别为108.72±7.20,50.29±10.05,与对照组比较差异有统计学意义(P<0.01)。在I/R干预下,凋亡相关蛋白Caspase-3在对照组和过表达组的灰度值分别为99.73±0.61,67.68±0.47,过表达组低于对照组,差异有统计学意义(P<0.01)。Bcl-2/Bax在对照组、空载体组和过表达组的灰度值分别为72.52±2.05,39.55±0.46,82.52±1.39,过表达组明显高于对照组和空载体组,差异有统计学意义(P<0.01)。在I/R后,自噬相关蛋白Beclin-1在对照组、空载体组和GDF15过表达组的灰度值分别为58.93±0.64,36.71±0.35,88.39±0.72,过表达组明显高于对照组和空载体组,差异有统计学意义(P<0.01)。LC3-Ⅱ/LC3-Ⅰ在空载体组和GDF15过表达组的灰度值分别为2.11±0.02,3.32±0.04,过表达组明显高于空载体组,差异有统计学意义(P<0.01)。结论上调H9c2细胞的GDF15水平可保护H9c2大鼠心肌细胞,其作用机制可能通过促进细胞的自噬作用得以实现。

Objective To investigate the effect of growth differentiation factor 15（ GDF15 ） on H9c2 cardiomyocytes against ischemia/reperfusion injury（I/R） by constructing GDF15 stable over expression of myocardial cell lines. Methods To establish the model of hypoxia/reoxygenation, transfect targeted GDF15 over expression lentivirus and negative empty vector lentivirus into H9c2 cells, and select the H9c2 cells of GDF15 gene over expression stably. Randomly assigned to control group , trans- fected with empty vector（H9c2/Sham） and GDF15 over expression group （H9c2/GDF15）. Cell viability was detected by MTT. And lactate dehydrogenase （LDH） in supernatant of cell culture was measured by LDH kit. The expression levels of apoptosis - related protein Caspase - 3, Bcl - 2, Bax and autophagy - related protein Beclin- 1, LC3 -B protein were observed with Western- blotting. Results Compared with control group （60. 47 ± 5.03） % and H9c2/Sham group （63.21 ± 17. 13 ） % , the H9c2/GDF15 group （74.93 ± 10.03 ） % in- creased significantly, the difference was statistically significant after I/R 2 h （P〈 0. 01 ） . Compared with control group 108.72 ±7.20, the activity of LDH in H9c2/GDF15 group 50.29 ± 10.05, the difference was statistically significant （P〈0.01）. To apoptosis- related protein Caspase- 3, the expression in H9c2/GDF15 group （gray value 67.68 ± 0. 47 ） was lower than control group （gray value 99.73 ± 0. 61 ）, the difference was statistically significant after IZR2 h（P 〈0.01）. The Bcl - 2/Bax in H9c2/GDFI5 group 82. 52 ± 1.39 was higher than control group 72. 52 ±2.05 and H9c2/Sham group 39.55 ± 0.46, the difference was statistically significant after I/R 2 h （P〈0.01）. To autophagy- related protein Beclin- 1, the expression in H9c2/GDF15 group （gray value 88.39 ±0.72） was higher than control group （gray value 58.93 ± 0.64） and H9c2/Sham group （gray value 36.71 ±0. 35）, the difference was statistically significant（P 〈0.01 ）. The LC3 -Ⅱ/LC3 -Ⅰ in H9c2/GDF15 group （3.32 ± 0.04） was higher than control group （2. 11 ± 0. 02 ）, the difference was statistically significant （P 〈 0.01 ）. Conclusion Up - regulation of GDF15 in H9c2 cells protected myocardial cells H9c2 and its protective mechanism may be achieved by promoting the autophagy of cells.