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《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:

2017年第4期

页码:

382-388

栏目:

基础研究

出版日期:

2017-02-25

文章信息/Info

Title:

Protective role of Parkin-mediated mitophagy in cardiomyocyte injury induced by high glucose and high fat

作者:

高蓓蕾¹; 张国勇¹; 余文军²; 黎 翔¹; 林 晨¹; 王婷婷¹; 张英梅¹

1.第四军医大学西京医院心血管内科，陕西 西安 710032；2.解放军第306医院心内科，北京 100101

Author(s):

GAO Bei-lei¹;  ZHANG Guo-yong¹;  YU Wen-jun²;  LI Xiang¹;  LIN Chen¹;  WANG Ting-ting¹;  ZHANG Ying-mei¹

1.Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi, China; 2.Department of Cardiology, 306th Hospital of PLA, Beijing 100101, China

关键词:

E3泛素化连接酶; 线粒体自噬; 糖尿病心肌病

Keywords:

Parkin;  mitophagy;  diabetic cardiomyopathy

分类号:

R392.3

DOI:

-

文献标识码:

A

摘要:

目的 明确Parkin（一种E3泛素化连接酶）介导的线粒体自噬对高糖高脂导致的原代心肌细胞损伤的保护作用。方法 以LV-lacZ(LacZ空病毒)或LV-Parkin（Parkin过表达慢病毒）转染SD大鼠原代心肌细胞48 h，再用含葡萄糖(5.5 mmol/L NG)的培养基或含棕榈酸盐(500 μmol/L HF)和葡萄糖(25 mmol/L HG)的高糖高脂培养基培养心肌细胞24 h。 实验分组：①阴性对照组(NG-LacZ)，②正常Parkin过表达组(NG-Parkin)，③高糖高脂阴性对照组（HG-HF-LacZ），④高糖高脂Parkin过表达组(HG-HF-Parkin)。用Western blot法检测PTEN介导的假定激酶蛋白1（PINK1）、Parkin、P62（一种自噬相关蛋白）、微管相关蛋白1轻链3（LC3）蛋白表达水平。采用JC-1染色法检测活细胞内线粒体膜电位水平。免疫荧光法检测自噬体数量，TUNEL法检测细胞凋亡率。结果 与对照组相比，高糖高脂处理的原代心肌细胞自噬相关蛋白LC3-II，P62表达水平上调（P<0.05），PINK1表达未发生统计学差异，Parkin表达水平下调（P<0.05），自噬体数量增多，线粒体膜电位下降功能损伤，心肌细胞凋亡率升高（P<0.05）。而用高糖高脂处理LV-Parkin转染的心肌细胞，LC3-II蛋白表达水平进一步升高（P<0.05），而P62表达水平显著下降（P<0.05），自噬体数量进一步增多，细胞内线粒体膜电位水平上升，心肌细胞凋亡率下降（P<0.05）。结论 高糖高脂可引起SD大鼠原代心肌细胞自噬流量降低，自噬小体增多，线粒体自噬发生障碍。Parkin过表达慢病毒通过激活心肌细胞内线粒体自噬途径，提高自噬流量，改善线粒体功能，降低心肌细胞凋亡率。

Abstract:

AIM To determine the role of Parkin-mediated mitophagy in cardiomyocytes exposed to high glucose and saturated fatty acid stimulation. METHODS Neonatal mouse ventricular myocytes were separated and cultured in DMEM with normal (5.5 mmol/L) dose of glucose. LV-LacZ or LV-Parkin was transfected into cardiomyocytes. After transfected for 48 hrs, cardiomyocytes were cultured in DMEM with normal (5.5 mmol/L) or high dose of glucose (25 mmol/L HG) and palmitate (16:0; 500 μmol/L HF) for 24hrs. So we divided the experiment into four groups: ①NG-LacZ; ②NG-Parkin; ③HG-HF-LacZ; ④HG-HF-Parkin. The expression of proteins was analyzed by Western blot and the number of autophagosomes was counted by immunofluorescence. The mitochondrial membrane potential was measured by JC-1 staining and the apoptotic index was evaluated by TUNEL.RESULTS Compared with that in control group, mitophagy associated protein Parkin was significantly reduced by high glucose and high palmitate( P<0.05), suggesting that mitophagy might be reduced in this condition. The expression of LC3II was significantly increased (P<0.05), indicating the accumulation of autophagosomes. Up-regulated P62 (P<0.05) suggested that HG and HF resulted in impaired clearance of autophagosomes. Compared with those in HG-HF group,,LV-Parkin up-regulated the expression of Parkin and LC3II (P<0.05) and decreased the level of P62 significantly (P<0.05) following HG-HF treatment, indicating that Parkin over-expression enhanced mitophagy and autophagy flux. Parkin reduced the apoptosis (P<0.05) caused by high glucose and high plamitate in cardiomyocytes. CONCLUSION Parkin-mediated mitophagy plays a protective role in cardiomyocyte injury induced by high glucose and high palmitate.

参考文献/References

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备注/Memo

备注/Memo:

收稿日期：2016-09-03.基金项目：国家自然科学基金项目资助（81370195） 通讯作者：张英梅，副教授，主要从事代谢异常心肌损伤的分子机制研究 Email:zhangym197951@126.com 作者简介：高蓓蕾，硕士生 Email:975254951@qq.com

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