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中华细胞与干细胞杂志(电子版) ›› 2018, Vol. 08 ›› Issue (03) : 134 -139. doi: 10.3877/cma.j.issn.2095-1221.2018.03.002

所属专题: 文献

论著

高糖通过诱导自噬障碍促进心肌细胞H9c2凋亡
方媛1, 郭统帅1, 吴岳1, 刘洋1, 曹瑜梦1, 雷新军1,()   
  1. 1. 710061 西安交通大学第一附属医院心内科
  • 收稿日期:2017-11-24 出版日期:2018-06-01
  • 通信作者: 雷新军
  • 基金资助:
    国家自然科学基金资助项目(30900616)

High glucose induced apoptosis in H9c2 cells by promoting autophagy dysfunction

Yuan Fang1, Tongshuai Guo1, Yue Wu1, Yang Liu1, Yumeng Cao1, Xinjun Lei1,()   

  1. 1. Department of Cardiology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
  • Received:2017-11-24 Published:2018-06-01
  • Corresponding author: Xinjun Lei
  • About author:
    Corresponding author:Lei Xinjun, Email:
引用本文:

方媛, 郭统帅, 吴岳, 刘洋, 曹瑜梦, 雷新军. 高糖通过诱导自噬障碍促进心肌细胞H9c2凋亡[J/OL]. 中华细胞与干细胞杂志(电子版), 2018, 08(03): 134-139.

Yuan Fang, Tongshuai Guo, Yue Wu, Yang Liu, Yumeng Cao, Xinjun Lei. High glucose induced apoptosis in H9c2 cells by promoting autophagy dysfunction[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2018, 08(03): 134-139.

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 high-glucose-induced increase in Bax expression: Compared with the control group, the Bax /β-actin gray value of the high glucose group, high glucose and rapamycin co-treatment group, and rapamycin group rapamycin group were (1.51±0.31, t = 14.342, P = 0.000), (1.42±0.23, t = 9.621, P = 0.004), and (1.81±0.12), t = 0.172, P = 0.124).

Conclusion

High glucose may promote apoptosis and induces autophagy dysfunction in cardiomyocytes H9c2 cells. The application of autophagy inducer may reverse the effect of high glucose on apoptosis of H9c2 cells, indicating that autophagy is an important mechanism of high glucose-induced apoptosis in H9c2 cells.

表1 高糖对H9c2细胞损伤的影响(±s
图1 荧光显微镜下观察高糖对H9c2细胞凋亡形态(Hoechst 33258染色,×200)
表2 高糖对H9c2细胞自噬相关蛋白表达的影响(灰度值比较,±s
图2 荧光显微镜下观察雷帕霉素对高糖诱导的H9c2细胞凋亡的形态(Hoechst 33258染色,×200)
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