高糖通过诱导自噬障碍促进心肌细胞H9c2凋亡  

High glucose induced apoptosis in H9c2 cells by promoting autophagy dysfunction

作　　者：方媛[1] 郭统帅[1] 吴岳[1] 刘洋[1] 曹瑜梦 雷新军[1] Fang Yuan;Guo Tongshuai;Wu Yue;Liu Yang;Cao Yumeng;Lei Xinjun(Department of Cardiology,First Affiliated Hospital of Xi＇an Jiaotong University,Xi＇an 710061,China)

机构地区：[1]西安交通大学第一附属医院心内科,710061

出　　处：《中华细胞与干细胞杂志（电子版）》2018年第3期134-139,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金资助项目(30900616)

摘　　要：目的探讨自噬对高糖(HG)诱导的心肌细胞H9c2凋亡的影响。方法 MTT法检测H9c2细胞活力;hoechst33258染色法检测凋亡细胞;Western Blot检测H9c2细胞促凋亡蛋白Bax和自噬相关蛋白(Beclin-1和P62)的表达。各组的OD值和蛋白条带灰度值均采用析因设计的方差分析,各组间差异用单因素ANOVA分析。结果 HG能诱导H9c2细胞活力降低:12、24、48 mmol/L的HG细胞活力分别为Control组(100﹪)的[(79.5±2.23)﹪](t=3.143,P=0.043)、[(54.6±3.08)﹪](t=12.425,P=0.000)和[(37.2±2.59)﹪](t=13.761, P=0.000);与Control组(100﹪)比较,甘露醇等渗对照组的细胞活力值为[(101.0±1.27)﹪](t=0.012,P=0.094)。HG诱导H9c2细胞hoechst33258阳性细胞增加,且能诱导促凋亡蛋白Bax表达增加:与Control组比较,12、24、48 mmol/L的HG处理组凋亡蛋白Bax/β-actin灰度值分别为(1.29±0.25,t=2.32,P=0.045)、(1.42±0.23,t=10.247,P=0.000)和(1.81±0.29,t=16.324,P=0.000)。HG诱导自噬障碍:与Control组比较,自噬相关蛋白Beclin-1/β-actin灰度值分别为(0.82±0.16,t=4.243,P=0.032)、(0.78±0.19,t=11.341,P=0.000)和(0.62±0.11,t=13.455,P=0.000),P62蛋白/β-actin蛋白灰度值分别为(1.29±0.25,t=4.442,P=0.014)、(1.42±0.23,t=13.341,P=0.000)和(1.81±0.29,t=15.851,P=0.000)。自噬诱导剂雷帕霉素可逆转HG诱导的hoechst33258阳性细胞增加,且逆转HG诱导的Bax表达升高:与Control组比较,HG组、HG和雷帕霉素共处理组、雷帕霉素组的Bax/β-actin灰度值分别为(1.51±0.31,t=14.342,P=0.000)、(1.42±0.23,t=9.621,P=0.004)和(1.81±0.12,t=0.172,P=0.124)。结论 HG可促进心肌细胞H9c2凋亡,且能诱导自噬障碍,自噬诱导剂的运用逆转了HG对H9c2细胞的凋亡作用,表明自噬障碍是HG诱导H9c2细胞凋亡的重要机制。Objective To investigate the effect of autophagy on high glucose （HG）-induced cardiomyocytes H9c2 cells apoptosis. Methods The viability of H9c2 cells was detected by MTT assay. The apoptotic cells were observed by hoechst33258 staining. The expression of pro-apoptotic proteins Bax and autophagy-related proteins （Beclin-1 and P62） in H9c2 cells were detected by Western Blot. The factorial design analysis of variance was used for the OD value and the protein band gray value of each group, and the difference between the groups was analyzed by single factor ANOVA. Results High glucose induced the decrease in viability of H9c2 cells： The viability of high glucose cells at 12, 24, and 48 mmol/L was [（79.5±2.23）﹪] （t = 3.143, P = 0.043）, [（54.6±3.08）﹪] （t = 12.425, P = 0.000） and [（37.2±2.59）﹪] （t = 13.761, P = 0.000） of the control group （100﹪） . Compared with the control group （100﹪）, the cell viability of the mannitol isotonic Control group was [（101.0±1.27）﹪] （t = 0.012, P = 0.094）. High glucose induced the increase of hoechst 33258 positive cells in H9c2 cells and increased the expression of pro-apoptotic protein Bax： Compared with the control group, the gray levels of apoptotic protein Bax/β-actin in the high glucose treatment groups of 12, 24, and 48 mmol/L were （1.29±0.25, t = 2.32, P = 0.045）, （1.42±0.23, t = 10.247, P = 0.000） and （1.81±0.29, t = 16.324, P = 0.000）. High glucose induced autophagy dysfunction： Compared with the control group, the gray values of autophagy-related protein Beclin-1/β-actin were （0.82±0.16, t = 4.243, P = 0.032）, （0.78±0.19, t = 11.341, P = 0.000）, and （0.62±0.11, t = 13.455, P = 0.000）, and the gray levels of P62 protein/β-actin protein were （1.29±0.25, t = 4.442, P = 0.014）, （1.42±0.23, t = 13.341, P = 0.000）, and （1.81±0.29, t = 15.851, P = 0.000）. The autophagy inducer rapamycin reversed high glucose-induced increase in hoechst33258-positive cells and the hi

关 键 词：自噬 高糖 细胞凋亡 心肌细胞 

分 类 号：R587.2[医药卫生—内分泌] R542.2[医药卫生—内科学]