MiR-146b在白藜芦醇保护缺氧/复氧心肌细胞损伤中的作用及其机制
| 作者: |
1李琼,
1王磊,
1高波
1 汉川市人民医院心血管II科，湖北 汉川 431600 |
| 通讯: |
高波
Email: 379759547@qq.com |
| DOI: | 10.3978/j.issn.2095-6959.2020.03.004 |
摘要
目的：探讨miR-146b在白藜芦醇保护缺氧/复氧(hypoxia/reoxygenation，H/R)心肌细胞损伤中的作用及其机制。方法：将体外培养的心肌细胞分为正常组、H/R组、白藜芦醇组、白藜芦醇+anti-miR-NC组和白藜芦醇+anti-miR-146b组。采用荧光定量PCR检测miR-146b的表达，ELISA法检测细胞上清液中IL-1β，TNF-α和IL-6含量，MTT法检测细胞存活率，比色法检测LDH漏出率，流式细胞仪检测细胞凋亡率，蛋白质印迹法检测Bcl-2，caspase-3，NF-κB和TRAF6蛋白的表达，双荧光素酶报告基因实验检测miR-146b和TRAF6的靶向关系。结果：与正常组相比，H/R组细胞中miR-146b和Bcl-2蛋白的表达水平、细胞存活率显著降低，而细胞上清液中IL-1β，TNF-α，IL-6含量和LDH漏出率、细胞凋亡率以及caspase-3，NF-κB蛋白的表达水平显著升高(P<0.05)；与H/R组相比，白藜芦醇组中miR-146b和Bcl-2蛋白的表达水平、细胞存活率显著升高，而IL-1β，TNF-α，IL-6含量和LDH漏出率、细胞凋亡率以及caspase-3，NF-κB蛋白的表达水平显著降低(P<0.05)；与白藜芦醇组相比，白藜芦醇+anti-miR-146b组中miR-146b和Bcl-2蛋白的表达水平、细胞存活率显著降低，而IL-1β，TNF-α，IL-6含量和LDH漏出率，细胞凋亡率以及caspase-3，NF-κB蛋白的表达水平显著升高(P<0.05)；而白藜芦醇+anti-miR-NC组与白藜芦醇组相比差异无统计学意义(P>0.05)。双荧光素酶报告基因和蛋白质印迹法证实TRAF6是miR-146b靶基因，且miR-146b可靶向调控其表达。结论：miR-146b抑制心肌细胞凋亡和炎症反应是白藜芦醇减轻H/R心肌细胞损伤的调控机制，其作用原理可能与靶向调控TRAF6有关。
关键词：
白藜芦醇；心肌细胞；缺氧/复氧；miR-146b；TRAF6
Role of miR-146b in resveratrol protecting hypoxic/reoxygenated cardiomyocytes from injury
CorrespondingAuthor: GAO Bo Email: 379759547@qq.com
DOI: 10.3978/j.issn.2095-6959.2020.03.004
Abstract
Objective: To investigate the role and mechanism of miR-146b in resveratrol protecting hypoxic/reoxygenated cardiomyocytes from injury. Methods: The cultured cardiomyocytes were divided into a normal group, a H/R group, a resveratrol group, a resveratrol + anti-miR-NC group, and a resveratrol + anti-miR-146b group. The expression of miR-146 b was detected by fluorescence quantitative PCR, the contents of IL-1β, TNF-α and IL-6 in cell supernatant were measured by ELISA, the cell survival rate was checked by MTT, the leakage rate of LDH was tested by colorimetry, the apoptotic rate was examined by flow cytometry, and the expression of Bcl-2, caspase-3, NF-κB and TRAF6 proteins were detected by Western blot. Double luciferase reporter gene assay was used to detect the targeting relationship between miR-146b and TRAF6. Results: Compared with the normal group, the expression levels of miR-146b and Bcl-2 protein, cell survival rate in the H/R group were significantly decreased, while the contents of IL-1β, TNF-α, IL-6 in cell supernatant and LDH leakage rate, apoptosis rate and the expression levels of caspase-3 and NF-κB proteins were significantly increased (P<0.05); compared with the H/R group, the expression levels of miR-146b and Bcl-2 protein, cell survival in resveratrol group were significantly increased, while the contents of IL-1β, TNF-α, IL-6 in cell supernatant and LDH leakage rate, apoptosis rate and the expression level of caspase-3 and NF-κB proteins decreased significantly (P<0.05). Compared with the resveratrol group, the expression level of miR-146b and Bcl-2 protein, cell survival rate in the resveratrol + anti-miR-146b group decreased significantly, while the contents of IL-1β, TNF-α, IL-6 in cell supernatant and LDH leakage rate, apoptosis rate and the expression levels of caspase-3 and NF-κB proteins were significantly increased (P<0.05); however, there was no significant difference between the resveratrol+anti-miR-NC group and the resveratrol group (P>0.05). Double luciferase reporter gene and Western blotting experiments confirmed that TRAF6 was a target gene of miR-146b, and miR-146b can target regulating its expression. Conclusion: MiR-146b inhibits apoptosis and inflammation in cardiomyocytes, which is the regulatory mechanism of resveratrol to alleviate H/R cardiomyocyte injury. Its mechanism may be related to the targeting regulation of TRAF6.
Keywords：
resveratrol; cardiac myocytes; hypoxia/reoxygenation; miR-146b; TRAF6