Danshen–Gegen decoction protects against hypoxia/reoxygenation-induced apoptosis by inhibiting mitochondrial permeability transition via the redox-sensitive ERK/Nrf2 and PKCɛ/mK_ATP pathways in H9c2 cardiomyocytes

Abstract 

Danshen–Gegen (DG) Decoction, an herbal formulation containing Radix Salviae miltiorrhizae and Radix Puerariae lobatae, has been used for the treatment of coronary artery disease in Chinese medicine. In the present study, the involvement of ERK- and PKCɛ-mediated pathways in the cytoprotection against apoptosis afforded by DG pretreatment was investigated in H9c2 cardiomyocytes. Pretreatment with a methanol extract of aqueous DG decoction protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated the enhancement of cellular reduced glutathione and a reduced sensitivity to Ca²⁺-induced mitochondrial permeability transition. DG extract increased the production of cytochrome P-450 (CYP)-dependent reactive oxygen species (ROS) in H9c2 cardiomyocytes, which was accompanied by the concomitant activation of ERK1/2 and PKCɛ. The DG-induced ERK1/2 activation was followed by the translocation of Nrf2 from the cytosol to the mitochondria accompanied by an increase in the expression of glutathione-related antioxidant proteins. In addition, the increased expression of hemeoxygenase-1 was associated with the activation of Akt and BAD, indicative of anti-apoptotic activity. In conclusion, DG treatment activated both ERK/Nrf2 and PKCɛ pathways, presumably by ROS arising from CYP-catalyzed processes, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.

Abbreviations: ABT, 1-aminotriazole, Akt, alpha-serine threonine kinase, BAD, Bcl-2 associated death promoter, CYP, cytochrome P-450, ERK, extracellular signal regulated kinase, DCFDA, 2′-7′-dichlorofluorescein diacetate, DG, Danshen–Gegen, DMTU, dimethylthiouracil, GCL, γ-glutamyl cysteine ligase, GCLm, modulatory subunit of GCL, G6DPH, glucose 6-phosphate dehydrogenase, GR, glutathione reductase, GSH, reduced glutathione, 5-HD, 5-hydroxydecanoate, HO-1, hemeoxygenase 1, mK_ATP, mitochondrial ATP-dependent potassium channel, MPT, mitochondrial permeability transition, Nrf2, nuclear factor eryhtroid 2-related factor 2, PBS, phosphate-buffered saline, PI, propidium iodide, PKCɛ, protein kinase C-epsilon, ROS, Reactive oxygen species

Keywords: Salviae miltiorrhizae, Puerariae lobatae, Extracellular signal-regulated protein kinase (ERK), Protein kinase C-epsilon (PKCɛ), Mitochondrial permeability transition, Apoptosis, H9c2