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下调CopineⅠ抑制缺氧/复氧诱导的H9c2细胞凋亡(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2017年第3期
页码:
264-268
栏目:
基础研究
出版日期:
2017-01-25

文章信息/Info

Title:
Depletion of copine-I inhibits cell apoptosis induced by hypoxia/reoxygenation in H9c2 cells
作者:
高 群; 刘媛媛; 柏丹娜
解放军第323医院干一科，陕西 西安 710054
Author(s):
GAO Qun;  LIU Yuan-yuan;  BAI Dan-na
First Department of Cadres, PLA 323 Hospital, Xi’an 710054, Shaanxi, China
关键词:
H9c2; copineⅠ; 缺氧复氧; 细胞凋亡
Keywords:
H9c2;  copine-I;  hypoxia/reoxygenation;  apoptosis
分类号:
Q26
DOI:
-
文献标识码:
A
摘要:
目的 研究CopineⅠ（CPNE1）对缺氧/复氧（Hypoxia/Reoxygenation，H/R）诱导H9c2细胞凋亡的作用及可能的机制。方法 以H9c2心肌细胞为研究对象，建立H/R模型，细胞被随机分为对照组（CON）、H/R组、阴性对照(NC)+H/R和CPNE1 siRNA+H/R组，阻断实验用NF-κB的阻断剂PDTC（10 μmol/L）预处理细胞30 min。RT-PCR和Western blot方法用于检测CPNE1表达水平。细胞乳酸脱氢酶（LDH）活性采用ELISA方法检测。细胞经Annexin-V/PI染色后用流式细胞仪检测凋亡率，Western blot方法检测claved-caspase3（c-caspase3）、Bcl-2和Bax的蛋白表达水平。细胞中NF-κB活性采用ELISA方法检测。结果 与CON组相比，H/R组CPNE1表达水平上调、LDH活性升高、凋亡率上升、c-caspase3和Bax蛋白表达升高、Bcl-2蛋白表达水平下降。与NC+H/R组相比，CPNE1 siRNA+H/R组细胞的LDH活性下降、凋亡率降低、c-caspase3和Bax蛋白表达减少、Bcl-2表达增多。此外，沉默CPNE1细胞核中NF-κB活性增强且蛋白表达上升，PDTC可逆转CPNE1 siRNA对细胞凋亡的抑制作用。结论 下调CPNE1的表达能够抑制H/R诱导的H9c2细胞凋亡，其可能机制是通过增强NF-κB活性发挥心肌保护作用。
Abstract:
AIM To determine the effects and mechanisms of copine-I (CPNE1) on apoptosis induced by hypoxia/reoxygenation (H/R) in H9c2. METHODS H9c2 myocytes were subjected to H/R (24 h/6 h) and divided into control group (CON), H/R group, negative control (NC)+H/R and CPNE1 siRNA+H/R group. H9c2 myocytes were pretreated with NF-κB inhibitor PDTC (10 μM) for 30 min to determine the potential mechanisms of CPNE1 in cell apoptosis. CPNE1 expression was detected using RT-PCR and Western blot. Activity of LDH was assayed by ELISA and cell apoptosis was measured using flow cytometry after staining with annexin V/PI. Western blot was utilized to detect the expressions of cleaved-caspase 3 (c-caspase3), Bax, Bcl-2 and NF-κB and ELISA was used to determine the activity of NF-κB. RESULTS Compared with CON group, mRNA and protein levels of CPNE1 in the H/R group increased. Moreover, H/R resulted in a significant increase in apoptosis rate, activity of LDH, and protein expressions of c-caspase3 and Bax. In addition, the anti-apoptotic protein Bcl-2 was decreased. Depletion of CPNE1 decreased the activity of LDH, apoptosis rate, and expressions of c-caspase3 and Bax but augmented Bcl-2 expression. Silencing of CPNE1 enhanced the activity and expression of NF-κB in the nucleus. Regulation of CPNE1 in cell apoptosis was abolished partly by PDTC. CONCLUSION SCPNE1 silencing inhibits cell apoptosis induced by H/R in H9c2. The possible mechanism for a myocardial protective role of CPNE siRNA is enhancing the activity of NF-κB.

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备注/Memo

备注/Memo:
收稿日期：2016-04-17.通讯作者：刘媛媛，主任医师，主要从事心力衰竭基础及临床研究 Email：diana611@163.com 作者简介：高群，主治医师，硕士 Email：wenyinxian029@163.com
更新日期/Last Update: 2017-02-20