MiR-9-5p调控瞬时受体电位M7对心肌缺血/再灌注大鼠的保护作用

doi:10.16098/j.issn.0529-1356.2022.02.016

摘要/Abstract

摘要：

目的探讨miR-9-5p调控瞬时受体电位M7（TRPM7）对心肌缺血/再灌注（MIR）大鼠的作用。方法将32只SD大鼠分为假手术组、模型组、miR-9-5p过表达组及空载体对照组，通过左冠状动脉结扎法建立MIR模型，假手术组不结扎，miR-9-5p过表达组和空载体对照组于模型建立前24 h分别尾静脉注入miR-9-5p agomir和agomir NC。通过HE染色观察各组大鼠心肌损伤情况；Real-time PCR检测各组大鼠心肌组织中miR-9-5p表达水平；酶联免疫吸附测定法检测各组大鼠血清中白细胞介素 (IL)-6、肿瘤坏死因子α(TNF-α)、IL-1β、肌酸激酶同工酶MB（CK-MB）、心肌肌钙蛋白Ⅰ（cTnI）、乳酸脱氢酶（LDH）水平以及心肌组织中丙二醛（MDA）、超氧化物歧化酶（SOD）含量；TUNEL法检测心肌细胞凋亡情况；双荧光素酶实验验证miR-9-5p与TRPM7的关系；免疫印迹法检测各组大鼠TRPM7、Bcl-2、Bax、磷酸化核因子κB 65（p-NF-κB p65）、toll样受体4(TLR4）蛋白表达。结果 MiR-9-5p在大鼠心肌组织中低表达（P<0.05）；miR-9-5p过表达能够降低CK-MB、cTnI、LDH表达水平，改善大鼠心肌损伤程度；与模型组相比，miR-9-5p过表达组心肌细胞凋亡率、Bax蛋白表达及MDA、IL-6、TNF-α及IL-1β含量均下降，而Bcl-2蛋白表达和SOD含量上升（P<0.05）；双荧光素酶实验结果显示，TRPM7为miR-9-5p的靶基因，且miR-9-5p过表达组TRPM7、p-NF-κB p65、TLR4蛋白表达均较模型组降低（P<0.05）。结论 MiR-9-5p通过调控TRPM7抑制心肌缺血再灌注导致的心肌细胞凋亡、氧化应激及炎症反应，并抑制TLR4/NF-κB通路。

关键词: 微小RNA-9-5p, 瞬时受体电位M7, 心肌缺血/再灌注, Toll样受体4/核因子κB信号通路, 实时定量聚合酶链反应, 酶联免疫吸附测定, 免疫印迹法, 大鼠

Abstract:

Objective To investigate the effect of microRNA-9-5p (miR-9-5p) regulating transient receptor potential melastatin 7 (TRPM7) on myocardial ischemia-reperfusion(MIR) in rats. Methods Thirty-two SD rats were divided into sham operation group, model group, miR-9-5p overexpression group and empty vector control group. The MIR model was established by ligation of left coronary artery. The sham operation group was not ligated. miR-9-5p agomir and agomir NC were injected into tail vein 24 hours before model establishment in miR-9-5p overexpression group and empty vector control group. The myocardial injury was observed by HE staining. The expression of miR-9-5p was detected by Real-time PCR. The serum levels of interleukin（IL）-6, tumor necrosis factor alpha（TNF-α）, IL-1β, creatine kinase isoenzyme MB (CK-MB), cardiac troponin Ⅰ (cTnI), lactate dehydrogenase (LDH) and the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were measured were measured by ELISA. Cardiomyocyte apoptosis was detected by TUNEL. Double luciferase assay verified the relationship between miR-9-5p and TRPM7. The protein expressions of TRPM7, Bcl-2, Bcl-2 associated X (Bax), phosphoryl ated nuclear factor kappa-B 65 (p-NF-κB p65) and toll like receptor 4 (TLR4) were detected by Western blotting. Results The expression of miR-9-5p was low in myocardial tissue of rats (P<0.05). Overexpression of miR-9-5p could reduce the expression levels of CK-MB, cTnI and LDH, and improve the degree of myocardial injury. Compared with the model group, the apoptosis rate, Bax protein expression, MDA, IL-6, TNF-α and IL-1β contents in myocardial cells of miR-9-5p overexpression group decreased, while Bcl-2 protein expression and SOD content increased (P<0.05). The result of dual luciferase assay showed that TRPM7 was the target gene of miR-9-5p, and the protein expressions of TRPM7, p-NF-κB p65 and TLR4 in miR-9-5p overexpression group were lower than those in model group (P<0.05). Conclusion MiR-9-5p can inhibit myocardial cell apoptosis, oxidative stress and inflammation induced by myocardial ischemia-reperfusion, and inhibit TLR4/NF-κB pathway by regulating TRPM7.

Key words: MicroRNA-9-5p, Transient receptor potential melastatin 7, Myocardial ischemia-reperfusion, Toll like receptor 4/nuclear factor kappa-B pathway, Real-time PCR, ELISA, Western blotting, Rat

中图分类号:

R542.2

刘向前周仪梦谢向荣倪进忠杨浩. MiR-9-5p调控瞬时受体电位M7对心肌缺血/再灌注大鼠的保护作用[J]. 解剖学报, 2022, 53(2): 246-253.

LIU Xiang-qian ZHOU Yi-meng XIE Xiang-rong NI Jin-zhong YANG Hao. Protective effect of miR-9-5p regulating transient receptor potential melastatin 7 on myocardial ischemia-reperfusion in rats[J]. Acta Anatomica Sinica, 2022, 53(2): 246-253.

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