天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (11): 1262-1266.doi: 10.11958/j.issn.0253-9896.2015.11.011

• 细胞与分子生物学 • 上一篇    下一篇

脑源性神经营养因子可减轻 H9c2 心肌细胞缺氧/复氧损伤

王诗才， 陈太军， 黄美松， 朱少铭△#br#   

  1. 湖北省十堰市中西医结合医院内科 （邮编442000）
  • 收稿日期:2015-05-18 修回日期:2015-07-06 出版日期:2015-11-15 发布日期:2015-11-15
  • 通讯作者: 朱少铭 E-mail： zhusaoming@163.com E-mail:zhusaoming@163.com
  • 作者简介: 王诗才 （1971）， 男， 副主任医师， 主要从事心血管疾病病理基础与临床研究

BDNF reduces the hypoxia/reoxygenation injury of H9c2 myocardial cells

WANG Shicai, CHEN Taijun, HUANG Meisong, ZHU Shaoming△   

  1. Department of Internal Medicine, Shiyan Hospital of Integrated Traditional and Western Medicine, Shiyan, Hubei 442000, China
  • Received:2015-05-18 Revised:2015-07-06 Published:2015-11-15 Online:2015-11-15
  • Contact: ZHU Shaoming E-mail： zhusaoming@163.com E-mail:zhusaoming@163.com

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摘要： 目的 探讨脑源性神经营养因子（BDNF）预处理对 H9c2 心肌细胞缺氧/复氧（H/R）损伤的影响及其作用机制。方法 体外培养 H9c2 心肌细胞并以缺氧 （95%N2+5%CO2） 培养 4 h 后复氧 （95%O2+5%CO2） 培养 12 h 建立 H/R 模型， 并分为 Control 组、 H/R 组、 不同浓度 （1、 10、 100 μg/L） BDNF 预处理后 H/R 组、 酪氨酸蛋白激酶受体 B （TrkB）inhibitor 组 （同时加入 100 μg/L BDNF 和 1∶1 000 的 TrkB inhibitor 预处理后 H/R 组）。利用 MTT 方法检测各组心肌细胞的细胞存活率； 并测定各组心肌细胞 H/R 损伤后乳酸脱氢酶（LDH）、 肌酸激酶（CK）、 丙二醛（MDA）、 超氧化物歧化酶（SOD）含量及活性； 流式细胞术测定各组心肌细胞的凋亡率； Western-blot 检测 TrkB、 Bcl-2、 Bax 蛋白表达。结果 与 Control 组相比， H/R 组细胞存活率明显下降， LDH、 CK 和 MDA 含量升高， SOD 活性降低， 细胞凋亡率升高， 抗凋亡 Bcl-2 蛋白表达下降， 促凋亡 Bax 蛋白表达升高（均 P < 0.05）。与 H/R 组相比， 不同浓度 BDNF 预处理H9c2 心肌细胞后 H/R， 各组细胞存活率均明显上升， CK、 LDH、 MDA 含量逐渐下降， SOD 活性升高， 细胞凋亡率下降， TrkB 和 Bcl-2 蛋白表达升高， 而 Bax 蛋白表达降低， 但 BDNF 的作用均受到 TrkB inhibitor 的抑制。结论 BDNF预处理能够提高 H9c2 心肌细胞 H/R 损伤的细胞存活率， 通过降低细胞凋亡率和提升细胞抗氧化能力而发挥保护作用， 并与 BDNF-TrkB 信号通路有关。

关键词: 脑源性神经营养因子, 肌细胞, 心脏,  , 细胞低氧, 受体, trkB, 氧化性应激, 细胞凋亡, 缺氧/复氧损伤

Abstract: Objective To investigate the effects of brain-derived neurotrophic factor (BDNF) pretreatment on H9c2 myocardial hypoxia/reoxygenation (H/R) injury, and explore its mechanism. Methods The H9c2 myocardial cells were cultured in vitro and (95%O2+5%CO2) oxygen cultured 12 h after (95%N2+5%CO2) hypoxia cultured 4 h to establish the H/R model. The cells were divided into normal control group, H/R group, different concentrations (1, 10, 100 μg/L) BDNF pretreatment in H/R groups and TrkB-inhibitor group (with 100 μg/L BDNF and 1∶1 000 TrkB inhibitor pre-treatment in H/R group). The cell survival rate was measured by MTT method in different groups. The lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and superoxide dismutase (SOD) content and activity were detected after H/R injury. The apoptotic rate of H9c2 myocardial cells were detected by flow cytometry, and the expressions of TrkB, Bcl-2 and Bax protein were detected by Western blot assay. Results Compared with the normal control group, the survival rate of H9c2 myocardial cells was decreased significantly in H/R model group (P < 0.05), LDH, CK and MDA contents were increased and SOD activity was decreased (P < 0.05). The cell apoptosis rate was increased significantly (P < 0.05). The antiapoptosis Bcl-2 protein expression was decreased, pro-apoptosis Bax protein expression was increased in H/R model group (P < 0.05). Compared with the H/R model group, the cell survival rates of H9c2 myocardial cells were increased after pre-treatment with different concentrations of BDNF (P < 0.05); LDH, CK and MDA contents were decreased and SOD activity were increased respectively (P < 0.05). The cell apoptotic rates were decreased (P < 0.05). The expressions of TrkB receptor and Bcl-2 protein gradually increased, while the expression of Bax protein was gradually decreased (P < 0.05). The role of BDNF was inhibited by TrkB inhibitor. Conclusion BDNF pre-treatment can promote the cell survival rate of H9c2 myocardial cells after H/R injury, which plays a protective role by inhibiting the cell apoptotic rate and maintaining antioxidant capacity, and associates with BDNF-TrkB signaling pathways.

Key words: brain-derived neurotrophic factor, myocytes, cardiac, cell hypoxia, receptor, trkB, oxidative stress, apoptosis, hypoxia/reoxygenation injury