皮质抑素拮抗环孢素诱导的大鼠心肌细胞凋亡及其对心肌细胞凋亡基因表达的影响。

Antagonism of cortistatin against cyclosporine-induced apoptosis in rat myocardial cells and its effect on myocardial apoptosis gene expression.

机构信息

Department of International Medical, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China.

出版信息

Eur Rev Med Pharmacol Sci. 2018 May;22(10):3207-3213. doi: 10.26355/eurrev_201805_15082.

DOI:10.26355/eurrev_201805_15082

PMID:29863267

Abstract

OBJECTIVE

To investigate the role of cortistatin (CST) on cyclosporine A (CsA)-induced myocardial apoptosis in rats and determine its effect on the expressions of myocardial apoptosis genes.

MATERIALS AND METHODS

H9C2 cells were treated with different concentrations of CsA solution (0.04, 0.2, 1 and 5 μM) for 24, 48 and 72 h, respectively. The cell viability was detected via methyl thiazolyl tetrazolium (MTT) assay, and the appropriate dose and time were compared and determined. At the same time, CST in different concentrations (0.08, 0.04, 0.2, 1, 5 and 25 μM) was added into cell culture, and the appropriate dose was identified using MTT assay. The cellular morphology in each group was observed, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed for the detection of cell apoptosis. Moreover, in molecular mechanism research, the apoptosis-associated factors, factor associated suicide (Fas), Fas ligand (FasL) and B-cell lymphoma-2-associated X protein (Bax), were detected via quantitive Real-time polymerase chain reaction (qPCR). Finally, the levels of a protein related to myocardial apoptosis in rats were investigated via Western blotting.

RESULTS

The treatment with 1 μM CsA for 48 h caused significant apoptosis. The results of TUNEL staining showed the inhibitory role of CST on the myocardial apoptosis in rats induced by CsA. The detection of apoptosis factors via Real-time PCR revealed that after the induction of CsA, the expressions of Fas, FasL and Bax mRNA in cells were significantly higher than those in control group, but were significantly decreased after administration of CST. Western blotting showed that the protein expressions of Caspase 3 and Caspase 9 were remarkably elevated in cells after the use of CsA, but were significantly reduced after administration of CST (p < 0.01).

CONCLUSIONS

CST contributes to antagonistic function against the CsA-induced apoptosis of rat myocardial cells, and its effect is related to the down-regulation of expressions of apoptotic factors, Fas, FasL, Bax, Caspase 3, and Caspase 9.

摘要

目的

探讨脑啡肽（CST）在环孢素 A（CsA）诱导大鼠心肌细胞凋亡中的作用及其对心肌细胞凋亡相关基因表达的影响。

材料和方法

采用不同浓度 CsA 溶液（0.04、0.2、1 和 5 μM）处理 H9C2 细胞 24、48 和 72 h，通过噻唑蓝（MTT）比色法检测细胞活力，比较并确定合适的剂量和时间。同时，加入不同浓度（0.08、0.04、0.2、1、5 和 25 μM）的 CST，通过 MTT 比色法确定合适的剂量。观察各组细胞形态，末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色检测细胞凋亡。此外，在分子机制研究中，通过定量实时聚合酶链反应（qPCR）检测凋亡相关因子、自杀相关因子（Fas）、Fas 配体（FasL）和 B 细胞淋巴瘤-2 相关 X 蛋白（Bax）。最后，通过 Western blot 法检测大鼠心肌细胞凋亡相关蛋白水平。

结果

1 μM CsA 处理 48 h 可引起明显的细胞凋亡。TUNEL 染色结果显示 CST 对 CsA 诱导的大鼠心肌细胞凋亡具有抑制作用。qPCR 检测凋亡因子结果显示，CsA 诱导后细胞 Fas、FasL 和 Bax mRNA 表达明显高于对照组，CST 给药后表达明显降低。Western blot 结果显示，CsA 作用后细胞 Caspase 3 和 Caspase 9 蛋白表达明显升高，CST 给药后表达明显降低（p<0.01）。