Li Hongyu, Qiu Jimiao, Liu Chang, Yu Guobing, Wu Danyu, Chu Yichun, Wang Kai
School of Nursing, NingBo College of Health Sciences, Ningbo, Zhejiang, China.
Department of Health Service, 906 Hospital of Joint Logistic Support Force of PLA, Ningbo, Zhejiang, China.
PLoS One. 2025 Jan 30;20(1):e0316887. doi: 10.1371/journal.pone.0316887. eCollection 2025.
To investigate the effects and mechanisms of miRNA 221 on myocardial ischemia/reperfusion injury (MIRI) in mice through the regulation of phospholamban (PLB) expression.
The MIRI mouse model was created and mice were divided into sham, MIRI, MIRI+ 221, and MIRI+ scr groups, with miRNA 221 overexpression induced in the myocardium of MIRI mice by targeted myocardial injection. Quantitative RT-PCR analysis was performed to observe the variation in miRNA 221, PLB, SERCA2, RYR2, NCX1, Cyt C and caspase 3 mRNA levels in myocardium, while Western blot assessed the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), SERCA2, RYR2, NCX1, Cyt C and caspase 3 proteins. Changes in the structural integrity of the mouse heart were identified with HE and MASSON staining, while TUNEL staining was used to evaluate the TUNEL-positive cells of cardiomyocytes. Changes in myocardium calcium concentration were detected with reagent kits and the targeting interaction between miRNA 221 and PLB was evaluated using a luciferase reporter assay.
In the myocardium of MIRI mice, miRNA 221 level was significantly reduced, while the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), and apoptosis-related genes caspase 3, and Cyt C were increased markedly, as well as calcium levels in myocardium. Following the overexpression of miRNA 221 in myocardium, there was a marked alleviation of myocardial injury and cardiomyocyte apoptosis and necrosis, significant enhancement of left ventricular systolic function, and marked decrease in the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), caspase 3 and Cyt C, as well as a significant decrease in total calcium levels in myocardium.
miRNA 221 can alleviate myocardial injury in mouse myocardial ischemia/reperfusion by suppressing the expression of PLB, thus reducing calcium overload in myocardium.
通过调控受磷蛋白（PLB）表达，研究微小RNA 221对小鼠心肌缺血/再灌注损伤（MIRI）的影响及机制。
建立MIRI小鼠模型，将小鼠分为假手术组、MIRI组、MIRI + 221组和MIRI + scr组，通过靶向心肌注射在MIRI小鼠心肌中诱导微小RNA 221过表达。采用定量逆转录聚合酶链反应分析观察心肌中微小RNA 221、PLB、肌浆网钙ATP酶2（SERCA2）、兰尼碱受体2（RYR2）、钠钙交换体1（NCX1）、细胞色素C（Cyt C）和半胱天冬酶3（caspase 3）mRNA水平的变化，同时采用蛋白质免疫印迹法评估PLB、磷酸化PLB（Ser16）、磷酸化PLB（Thr17）、SERCA2、RYR2、NCX1、Cyt C和caspase 3蛋白水平。用苏木精-伊红（HE）和马松（MASSON）染色鉴定小鼠心脏结构完整性的变化，采用TUNEL染色评估心肌细胞的TUNEL阳性细胞。用试剂盒检测心肌钙浓度变化，并用荧光素酶报告基因检测法评估微小RNA 221与PLB之间的靶向相互作用。
在MIRI小鼠心肌中，微小RNA 221水平显著降低，而PLB、磷酸化PLB（Ser16）、磷酸化PLB（Thr17）以及凋亡相关基因caspase 3和Cyt C的水平显著升高，同时心肌钙水平也升高。在心肌中过表达微小RNA 221后，心肌损伤、心肌细胞凋亡和坏死明显减轻，左心室收缩功能显著增强，PLB、磷酸化PLB（Ser16）、磷酸化PLB（Thr17）、caspase 3和Cyt C水平显著降低，心肌总钙水平也显著降低。
微小RNA 221可通过抑制PLB表达减轻小鼠心肌缺血/再灌注时的心肌损伤，从而减少心肌钙超载。
