FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究

doi:10.11958/20220348

天津医药 ›› 2022, Vol. 50 ›› Issue (8): 791-795.doi: 10.11958/20220348

FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究

郑君毅(), 张莹莹, 刘园园, 陈孟英, 郭绪昆()

天津市胸科医院心内科,天津市心血管病研究所（邮编300222）

收稿日期:2022-03-05 修回日期:2022-06-16 出版日期:2022-08-15 发布日期:2022-08-12
通讯作者: 郭绪昆 E-mail:zhjunyi0305@163.com;guoxukun206@163.com
作者简介:郑君毅（1979）,男,副主任医师,主要从事心血管疾病的临床和基础方面研究。E-mail： zhjunyi0305@163.com

Mechanism of FUNDC1 affacting apoptosis of H9c2 cardiomyocytes with high glucose injury by regulating mitochondrial fission

ZHENG Junyi(), ZHANG Yingying, LIU Yuanyuan, CHEN Mengying, GUO Xukun()

Department of Cardiology, Tianjin Chest Hospital, Tianjin Institute of Cardiovascular Disease, Tianjin 300222, China

Received:2022-03-05 Revised:2022-06-16 Published:2022-08-15 Online:2022-08-12
Contact: GUO Xukun E-mail:zhjunyi0305@163.com;guoxukun206@163.com

郑君毅, 张莹莹, 刘园园, 陈孟英, 郭绪昆. FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究[J]. 天津医药, 2022, 50(8): 791-795.

ZHENG Junyi, ZHANG Yingying, LIU Yuanyuan, CHEN Mengying, GUO Xukun. Mechanism of FUNDC1 affacting apoptosis of H9c2 cardiomyocytes with high glucose injury by regulating mitochondrial fission[J]. Tianjin Medical Journal, 2022, 50(8): 791-795.

摘要/Abstract

摘要：

目的探讨含FUN14域蛋白1（FUNDC1）在高糖损伤的H9c2心肌细胞中通过调控线粒体分裂影响心肌细胞凋亡的作用机制。方法体外培养H9c2心肌细胞并建立高糖损伤模型,分别进行细胞转染,使用腺苷酸活化蛋白激酶（AMPK）激活剂5-氨基-4-咪唑羧基酰胺核苷（AICAR）后,分为正常对照组（CTRL组）、高糖组（HG组）、HG+shRNA-NC组（转染shRNA-NC的HG组细胞）、HG+shRNA-FUNDC1组（转染shRNA-FUNDC1的HG组细胞）和HG+AICAR组（使用AICAR的HG组细胞）。MTT法检测细胞存活率;酶标仪检测细胞乳酸脱氢酶（LDH）释放量;激光共聚焦显微镜观察线粒体结构;Western blot检测线粒体动力相关蛋白1（DRP1）、分裂蛋白1（FIS1）、Bcl-2相关X蛋白（Bax）、B淋巴细胞瘤-2（Bcl-2）、活化的胱天蛋白酶3（Cleaved Caspase-3）、FUNDC1和GAPDH的蛋白表达水平。结果与CTRL组比较,HG组细胞存活率降低,LDH释放量升高,线粒体DRP1（DRP1-mito）、FIS1、Bax和Cleaved Caspase-3蛋白表达水平升高,胞浆DRP1（DRP1-cyto）和Bcl-2蛋白表达水平降低（P<0.05）。与HG组比较,HG+shRNA-FUNDC1组细胞存活率升高,LDH释放量明显降低,DRP1-mito、FIS1、Bax和Cleaved Caspase-3蛋白表达水平降低,DRP1-cyto和Bcl-2蛋白表达水平升高（P<0.05）。HG+shRNA-NC组和HG组比较,各指标差异均无统计学意义（P>0.05）。CTRL组线粒体主要呈线状结构,HG组和HG+shRNA-NC组线粒体均主要呈点状结构;与HG组比较,HG+shRNA-FUNDC1组线粒体线状结构增多,点状结构减少。与CTRL组比较,HG组FUNDC1蛋白表达水平升高（P<0.05）。与HG组比较,HG+AICAR组FUNDC1蛋白表达水平降低（P<0.05）。结论 FUNDC1通过调控线粒体分裂影响高糖引起的H9c2心肌细胞凋亡,AMPK信号通路参与该过程。

关键词: 肌细胞,心脏, 线粒体, 细胞凋亡, FUNDC1, 信号通路

Abstract:

Objective To investigate the mechanism of FUNDC1 affecting high glucose injured H9c2 cardiomyocyte apoptosis by regulating mitochondrial fission. Methods H9c2 cardiomyocytes were cultured in vitro, and the high glucose-induced injury model of cells was established. After cell transfection or AMPK activation by AICAR, the cells were divided into the control (CTRL) group, the high glucose injury (HG) group, the HG with transfected shRNA-NC (HG+shRNA-NC) group, the HG with transfected FUNDC1 shRNA (HG+shRNA-FUNDC1) group and the HG with AICAR (HG+AICAR) group. MTT method was used to detect the cell survival rate. The level of released LDH was measured with microplate reader. The structure of mitochondria was observed by laser confocal microscope. The proteins expression levels of mitochondrial dynamin-related protein1 (DRP1), fission protein 1 (FIS1), Bcl-2 associated X protein (Bax), B-lymphocytoma-2 (Bcl-2), cleaved cysteine-containing aspartate-spicific protease 3 (Cleaved Caspase-3), FUNDC1 and GAPDH were detected by Western blot assay. Results Compared with the CTRL group, the cell survival rate decreased, and the protein levels of DRP1-cyto and Bcl-2 were also decreased, the release of LDH and the protein expression levels of DRP1-mito, FIS1, Bax and Cleaved Caspase-3 increased in the HG group (P<0.05). Compared with the HG group, knocking down FUNDC1 increased the cell survival rate and the protein levels of DRP1-cyto and Bcl-2, decreased the release of LDH and the protein expression levels of DRP1-mito, FIS1, Bax and Cleaved Caspase-3 (P<0.05). There was no difference in each index between the HG group and the HG+shRNA-NC group (P>0.05). The mitochondria were mainly linear structure in the CTRL group, and the mitochondria were mainly pucta structure in the HG group and the HG+shRNA-NC group. Compared with the HG group, the mitochondrial linear structure increased and the puncta structure decreased in the HG+shRNA-FUNDC1 group. Compared with the CTRL group, the protein expression level of FUNDC1 increased in the HG group (P<0.05). Compared with the HG group, the protein level of FUNDC1 significantly decreased in the HG+AICAR group (P<0.05). Conclusion FUNDC1 affects H9c2 cardiomyocyte apoptosis induced by high glucose through regulating mitochondrial fission, and AMPK signaling pathway is involved in this process.

Key words: myocytes, cardiac, mitochondria, apoptosis, FUNDC1, signaling pathway

中图分类号:

R541

图/表 5

表1 各组细胞存活率和LDH释放量对比（ $\bar{x}±s$）

Tab.1 Comparison of cell viability and LDH release between the four groups

组别	n	细胞存活率（%）	LDH释放量（A₄₉₀）
CTRL组	6	100.00±18.24	0.26±0.03
HG组	6	54.41±8.69^a	0.61±0.06^a
HG+shRNA-NC组	6	55.51±12.99	0.60±0.04
HG+shRNA-FUNDC1组	6	83.63±9.86^b	0.40±0.06^b
F		17.708^＊	71.294^＊

图1 激光共聚焦显微镜检测各组线粒体结构（免疫荧光染色） A：CTRL组;B：HG组;C：HG+shRNA-NC组;D：HG+shRNA-FUNDC1组。

Fig.1 Laser confocal microscope assay of mitochondrial structure (Immunofluorescence staining)

图2 Western blot检测线粒体分裂和凋亡相关蛋白 A：CTRL组;B：HG组;C：HG+shRNA-NC组;D：HG+shRNA-FUNDC1组。

Fig.2 Western blot assay of mitochondrial fission and apoptosis proteins

表2 各组细胞线粒体分裂和凋亡相关蛋白表达水平比较 (n=3, $\bar{x}±s$)

Tab.2 Comparison of fission and apoptosis proteins between the four groups

组别		DRP1-mito	DRP1-cyto	FIS1
CTRL组		0.62±0.09	0.86±0.09	0.48±0.05
HG组		1.03±0.15^a	0.42±0.05^a	0.71±0.08^a
HG+shRNA-NC组		1.06±0.14	0.31±0.04	0.63±0.11
HG+shRNA-FUNDC1组		0.62±0.09^b	0.61±0.07^b	0.46±0.07^b
F		12.704^＊	38.953^＊	8.006^＊
组别	Bax	Bcl-2	Cleaved Caspase-3
CTRL组	0.38±0.05	0.84±0.05	0.72±0.09
HG组	1.10±0.14^a	0.60±0.03^a	1.00±0.09^a
HG+shRNA-NC组	1.18±0.15	0.59±0.06	0.94±0.11
HG+shRNA-FUNDC1组	0.48±0.06^b	0.79±0.08^b	0.75±0.09^b
F	42.624^＊	14.560^＊	5.891^＊

图3 Western blot检测AICAR对FUNDC1蛋白表达的影响 A：CTRL组;B：HG组;C：HG+AICAR组。

Fig.3 Western blot assay of AICAR on FUNDC1 protein

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FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究

Mechanism of FUNDC1 affacting apoptosis of H9c2 cardiomyocytes with high glucose injury by regulating mitochondrial fission

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