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Sasanquasaponin (SQS) is an active component of Camellia oleifera Abel. A recent study by our group demonstrated that SQS was able to inhibit ischemia/reperfusion-induced elevation of the intracellular chloride ion concentration ([Cl-]i) and exerted cardioprotective effects; however, the underlying intracellular signal transduction mechanisms have yet to be elucidated. As protein kinase C ε (PKCε) is able to mediate Cl- homeostasis, the present study investigated its possible involvement in the effects of SQS on cardiomyocytes subjected to ischemia/reperfusion injury. Cardiomyocytes were pre-treated with or without SQS or SQS plus εV1-2, a selective PKCε inhibitor, followed by simulated ischemia/reperfusion (sI/R). The effects on cell viability, PKCε phosphorylation levels, [Cl-]i, mitochondrial membrane potential and reactive oxygen species (ROS) production were assessed using an MTS assay, western blot analysis, colorimetric assays and flow cytometry. The results revealed that treatment with SQS prior to sI/R increased the viability of cardiomyocytes, and efficiently attenuated lactate dehydrogenase and creatine phosphokinase release induced by sI/R. In addition, SQS promoted PKCε phosphorylation and inhibited sI/R-induced elevation of [Cl-]i, paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation. However, when the cardiomyocytes were treated with εV1-2 prior to SQS pre-conditioning, the cardioprotection induced by SQS was reduced and the inhibitory effects of SQS on sI/R-induced elevation of [Cl-]i, production of ROS and loss of mitochondrial membrane potential were also attenuated. These findings indicated that SQS may inhibit sI/R-induced elevation of [Cl-]i through the PKCε signaling pathway to elicit cardioprotection in cultured cardiomyocytes. [ABSTRACT FROM AUTHOR]

DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain anti-oxidant enzymes and attenuate hypoxia/re-oxygenation (H/R)-induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2-like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti-oxidant genes via binding to the anti-oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti-oxidative enzymes by DJ-1 and contributes to the protective functions of DJ-1 against H/R-induced oxidative stress in H9c2 cells. The results demonstrated that DJ-1-overexpressing H9c2 cells exhibited anti-oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R-induced oxidative stress compared with native cells, whereas DJ-1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ-1-mediated anti-oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ-1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE-binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ-1-mediated induction of anti-oxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2 cells. [ABSTRACT FROM AUTHOR]