丹红注射液通过改善线粒体功能障碍减轻缺氧/复氧诱导的心肌细胞损伤

Danhong injection alleviates hypoxia/reoxygenation induced cardiomyocyte injury by improving mitochondrial dysfunction

关键词:  丹红注射液  线粒体能量代谢  线粒体膜电位  缺氧复氧  Akt  ERK1/2

Key words:  Danhong injection  mitochondrial energy metabolism  mitochondrial membrane potential  hypoxia/reoxygenation  Akt  ERK1/2

摘要:

[目的] 研究丹红注射液（DHI）对缺氧/复氧（H/R）损伤心肌细胞线粒体功能的调节作用机制。[方法] 建立原代心肌细胞H/R损伤模型。四甲基噻唑蓝（MTT）法检测磷脂酰肌醇-3-羟激酶（PI3K）和丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）/MAPK激酶（MEK）抑制剂LY294002与PD98059干预后，DHI对H/R损伤心肌细胞存活的影响。采用荧光探针Rh123检测DHI对H/R损伤心肌细胞线粒体膜电位（Δφm）的影响。蛋白免疫印迹（Western blot）法检测LY294002和PD98059干预后DHI对H/R损伤的心肌细胞PI3K及MEK下游丝氨酸/苏氨酸激酶（Akt）和ERK1/2蛋白表达及活性水平的影响。应用海马生物能量检测仪测定正常与H/R损伤条件下DHI对线粒体能量代谢的影响。[结果] DHI能够明显逆转H/R引起的细胞活力和线粒体膜电位下降。阻断剂LY294002和PD98059干预后，DHI对心肌细胞活力和线粒体膜电位的改善作用明显减弱。DHI能够显著增加H/R损伤的心肌细胞Akt和ERK蛋白磷酸化水平，且这种促进作用能够被抑制剂LY294002和PD98059抑制。常氧条件下，DHI不影响心肌细胞线粒体能量代谢。H/R条件下，DHI能够显著抑制心肌细胞线粒体基础耗氧率与最大耗氧率下降，增加线粒体能量储备能力。[结论] DHI能够激活Akt和ERK1/2，抑制H/R造成的线粒体能量代谢障碍，维持线粒体储备能力，缓解H/R诱导的心肌细胞损伤。

Abstract:

[Objective] To study the regulation mechanism of Danhong injection (DHI) on mitochondrial function of cardiomyocytes damaged by hypoxia/reoxygenation (H/R).[Methods] Establishment of H/R injury model of primary myocardial cells. MTT assay was used to detect the effects of DHI on the survival of myocardial cells injured by H/R after intervention with phosphatidylinositol-3-hydroxykinase (PI3K) and MAPK/ERK kinase (MEK) inhibitor LY294002 and PD98059. The effect of DHI on mitochondrial membrane potential (Δφm) in myocardial cells injured by H/R was detected by fluorescent probe Rh123. Western blot was used to detect the effects of DHI on the expression and activation levels of Akt and ERK1/2 proteins,as the downstream proteins of PI3K and MEK,in H/R damaged cardiomyocytes after LY294002 and PD98059 intervention. The effects of DHI on mitochondrial energy metabolism under normal and H/R injury were detected by Seahorse bioenergy detector.[Results] DHI can significantly reverse the cell viability and mitochondrial membrane potential decline caused by H/R. After the intervention of blockers LY294002 and PD98059,the improvement effect of DHI on myocardial cells viability and mitochondrial membrane potential was significantly reduced. DHI significantly increased the phosphorylation levels of Akt and ERK proteins in H/R-damaged cardiomyocytes,and this promotion was inhibited by blockers LY294002 and PD98059. Under normal oxygen condition,DHI did not affect the mitochondrial energy metabolism of cardiac myocytes. However,under H/R conditions,DHI could significantly inhibit the decrease of basal and maximum oxygen consumption rate of cardiomyocytes,and increase the mitochondrial reserve capacity.[Conclusion] DHI could activate Akt and ERK1/2,inhibit mitochondrial energy metabolism disorder,maintain mitochondrial reserve capacity,and alleviate H/R-induced cardiomyocyte damage.