脑源性神经营养因子可减轻H9c2心肌细胞缺氧/复氧损伤

BDNF reduces the hypoxia/reoxygenation injury of H9c2 myocardial cells

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、100μg/L)BDNF预处理后H/R组、酪氨酸蛋白激酶受体B(Trk B)inhibitor组(同时加入100μg/L BDNF和1∶1 000的Trk B 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Trk B、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,Trk B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Trk B 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-Trk B信号通路有关。

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 cul？tured 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, 100μg/L） BDNF pre？treatment in H/R groups and TrkB-inhibitor group （with 100μg/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 anti-apoptosis 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 in？creased 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.