Department of Anesthesiology, Qingdao Women and Children's Hospital, Shandong University, Qingdao, Shandong 266034, China.
Department of Anesthesiology, Weifang Medical University, Weifang, Shandong 261053, China.
QJM. 2020 Dec 1;113(12):859-869. doi: 10.1093/qjmed/hcaa088.
Our previous study has demonstrated that morphine post-conditioning (MpostC) protects cardiomyocytes from ischemia/reperfusion (I/R) injury partly through activating protein kinase-epsilon (PKCε) signaling pathway and subsequently inhibiting mitochondrial permeability transition pore (mPTP) opening.
In this study, we aim to investigate the relationship between long non-coding RNA TINCR and PKCε in cardiomyocytes under MpostC-treated I/R injury.
The myocardial I/R rat model was established by the ligation of lower anterior descending coronary artery for 45 min followed by the reperfusion for 1 h, and MpostC was performed before the reperfusion.
H/R and MpostC were performed in the rat cardiomyocyte cell line (H9C2), and the Cytochrome-c release in cytosol and mPTP opening were determined. Cell viability was detected by using Cell Counting Kit-8, and cell apoptosis was determined by using flow cytometry or TUNEL assay.
The results indicated that MpostC restored the expression of TINCR in I/R rat myocardial tissues. In cardiomyocytes, the therapeutic effect of MpostC, including reduced mPTP opening, reduced Cytochrome-c expression, increased cell viability and reduced cell apoptosis, was dramatically negated by interfering TINCR. The expression of fibroblast growth factor 1 (FGF1), a protein that activates PKCε signaling pathway, was positively correlated with TINCR. The RNA immunoprecipitation and RNA pull-down assay further confirmed the binding between FGF1 and TINCR. Furthermore, TINCR was demonstrated to inhibit the degradation and ubiquitination of FGF1 in cardiomyocytes using the cycloheximide experiment and the ubiquitination assay. The TINCR/FGF1/PKCε axis was revealed to mediate the protective effect of MpostC against hypoxia/reoxygenation injury both in vitro and in vivo.
In conclusion, our findings demonstrated that MpostC-induced up-regulation of TINCR protects cardiomyocytes from I/R injury via inhibiting degradation and ubiquitination of FGF1, and subsequently activating PKCε signaling pathway, which provides a novel insight in the mechanism of TINCR and PKCε during MpostC treatment of I/R injury.
我们之前的研究表明，吗啡后处理（MpostC）通过激活蛋白激酶-epsilon（PKCε）信号通路，随后抑制线粒体通透性转换孔（mPTP）开放，从而部分保护心肌细胞免受缺血/再灌注（I/R）损伤。
本研究旨在探讨吗啡后处理（MpostC）治疗 I/R 损伤时心肌细胞中长链非编码 RNA TINCR 与 PKCε 之间的关系。
结扎大鼠冠状动脉前降支 45min 后再灌注 1h 建立心肌 I/R 大鼠模型，并在再灌注前进行 MpostC。
在大鼠心肌细胞系（H9C2）中进行 H/R 和 MpostC，测定细胞质中细胞色素 c 的释放和 mPTP 的开放。采用细胞计数试剂盒-8 检测细胞活力，采用流式细胞术或 TUNEL 检测细胞凋亡。
结果表明，MpostC 恢复了 I/R 大鼠心肌组织中 TINCR 的表达。在心肌细胞中，MpostC 的治疗作用，包括减少 mPTP 开放、减少细胞色素 c 表达、增加细胞活力和减少细胞凋亡，被干扰 TINCR 显著否定。成纤维细胞生长因子 1（FGF1）的表达，一种激活 PKCε 信号通路的蛋白质，与 TINCR 呈正相关。RNA 免疫沉淀和 RNA 下拉实验进一步证实了 FGF1 与 TINCR 的结合。此外，通过环己酰亚胺实验和泛素化实验，证明 TINCR 抑制了心肌细胞中 FGF1 的降解和泛素化。TINCR/FGF1/PKCε 轴被证明在体外和体内介导了 MpostC 对缺氧/复氧损伤的保护作用。
总之，我们的研究结果表明，MpostC 诱导的 TINCR 上调通过抑制 FGF1 的降解和泛素化，从而激活 PKCε 信号通路，保护心肌细胞免受 I/R 损伤，为 MpostC 治疗 I/R 损伤时 TINCR 和 PKCε 的作用机制提供了新的见解。
