cFLIPL Alleviates Myocardial Ischemia-Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress,Cardiovascular Drugs and Therapy

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cFLIPL Alleviates Myocardial Ischemia-Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress
Cardiovascular Drugs and Therapy ( IF 3.4 ) Pub Date : 2021-11-12 , DOI: 10.1007/s10557-021-07280-1
Yun Zhao Li _{1,

2,

3} , Hui Wu _{1,

2,

3} , Di Liu _{1,

2,

3} , Jun Yang _{1,

2,

3} , Jian Yang ₁ , Jia Wang Ding _{1,

2,

3} , Gang Zhou _{1,

2,

3} , Jing Zhang _{2,

3,

4} , Dong Zhang _{1,

2,

3}

Affiliation

Purpose

Endoplasmic reticulum stress (ERS) plays a crucial role in myocardial ischemia-reperfusion injury (MIRI). Cellular FLICE-inhibitory protein (cFLIP) is an essential regulator of apoptosis and plays a major role in regulating ERS. The present study aimed to investigate the effects of long isoform cFLIP (cFLIP_L) on endogenous apoptosis and the mechanism of ERS in MIRI.

Methods

The cFLIP_L recombinant adenovirus vector was used to infect H9c2 cells and Sprague–Dawley (SD) rats. After infection for 72 h, ischemia was induced for 30 min, and reperfusion was then performed for 2 h to establish the MIRI model in SD rats. H9c2 cells were hypoxic for 4 h and then reoxygenated for 12 h to simulate ischemia/reperfusion (I/R) injury. Model parameters were evaluated by assessing cardiomyocyte viability, cell death (apoptosis), and ERS-related protein expression. In addition, tunicamycin (TM), an ERS agonist, was also added to the medium for pretreatment. Coimmunoprecipitation (Co-IP) of cFLIP_L and p38 MAPK protein was performed.

Results

cFLIP_L expression was decreased in I/R injury and hypoxia/reoxygenation (H/R) injury, and cFLIP_L overexpression reduced myocardial infarction in vivo and increased the viability of H9c2 cells in vitro. I/R and H/R upregulated the protein expression of GRP78, IRE-1, and PERK to induce ERS and apoptosis. Interestingly, overexpression of cFLIP_L significantly inhibited ERS and subsequent apoptosis, which was reversed by an agonist of ERS. Moreover, Co-IP showed that cFLIP_L attenuated ERS and was associated with inhibiting the activation of p38 protein.

Conclusion

The expression of cFLIP_L is significantly downregulated in MIRI, and it is accompanied by excessive ERS and apoptosis. Upregulated cFLIP_L suppresses ERS to reduce myocardial apoptosis, which is associated with inhibiting the activity of p38 MAPK. Therefore, cFLIP_L may be a potential intervention target for MIRI.

中文翻译：

cFLIPL 通过抑制内质网应激减轻心肌缺血再灌注损伤

目的

内质网应激 (ERS) 在心肌缺血再灌注损伤 (MIRI) 中起着至关重要的作用。细胞 FLICE 抑制蛋白 (cFLIP) 是细胞凋亡的重要调节剂，在调节 ERS 中起主要作用。本研究旨在探讨长亚型 cFLIP (cFLIP _L ) 对内源性细胞凋亡的影响以及 MIRI 中 ERS 的机制。

方法

cFLIP _L重组腺病毒载体用于感染 H9c2 细胞和 Sprague-Dawley (SD) 大鼠。感染72 h后，诱导缺血30 min，再灌注2 h，建立SD大鼠MIRI模型。H9c2 细胞缺氧 4 小时，然后再充氧 12 小时以模拟缺血/再灌注 (I/R) 损伤。通过评估心肌细胞活力、细胞死亡（细胞凋亡）和 ERS 相关蛋白表达来评估模型参数。此外，还在培养基中加入 ERS 激动剂衣霉素 (TM) 进行预处理。对cFLIP _L和 p38 MAPK 蛋白进行免疫共沉淀 (Co-IP)。

结果

在 I/R 损伤和缺氧/复氧 (H/R) 损伤中cFLIP _L表达降低，cFLIP _L过表达减少体内心肌梗死并增加 H9c2 细胞的体外活力。I/R 和 H/R 上调 GRP78、IRE-1 和 PERK 的蛋白表达，从而诱导 ERS 和细胞凋亡。有趣的是，cFLIP _L的过表达显着抑制了 ERS 和随后的细胞凋亡，这可被 ERS 激动剂逆转。此外，Co-IP 表明 cFLIP _L减弱了 ERS，并且与抑制 p38 蛋白的激活有关。