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Anion exchanger 3 (AE3) is known to serve crucial roles in maintaining intracellular chloride homeostasis by facilitating the reversible electroneutral exchange of Cl‑ for HCO3‑ across the plasma membrane. Our previous studies reported that sasanquasaponin (SQS) can inhibit hypoxia/reoxygenation (H/R)‑induced elevation of intracellular Cl‑ concentration ([Cl‑]i) and elicit cardioprotection by favoring Cl‑/HCO3 ‑ exchange of AE3. However, the molecular basis for SQS‑induced increase of Cl‑/HCO3 ‑ exchange of AE3 remains unclear. The present study demonstrated that SQS activates protein kinase Cε (PKCε) and stimulates the phosphorylation of AE3 in H9c2 cells. Notably, SQS‑induced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV1‑2, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCε‑dependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV1‑2 and S67A mutation of AE3 eradicated the SQS‑induced increase of AE3 activity, reversed the inhibitory effect of SQS on H/R‑induced elevation of ([Cl‑]i), Ca2+ overload and generation of reactive oxygen species, and eliminated SQS‑induced cardioprotection. In conclusion, PKCε‑dependent phosphorylation of serine 67 on AE3 may be responsible for the increase of Cl‑/HCO3 ‑ exchange of AE3 and intracellular chloride efflux by SQS, and contributes to the cardioprotection of SQS against H/R in H9c2 cells. [ABSTRACT FROM AUTHOR]

DJ-1 protein, as a multifunctional intracellular protein, has been demonstrated to serve a critical role in regulating cell survival and oxidative stress. To provide in vivo evidence that DJ-1 is involved in the delayed cardioprotection induced by ischemic preconditioning (IPC) against oxidative stress caused by ischemia/reperfusion (I/R), the present study subjected male Sprague-Dawley rats to IPC (3 cycles of 5-min coronary occlusion/5-min reperfusion) 24 h prior to I/R (30-min coronary occlusion/120-min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ-1 in situ. Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ-1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ-1 protein. In addition, IPC reduced LDH and CK-MB release, attenuated myocardial infarct size, improved cardiac function following I/R, and inhibited the elevation of ROS and MDA and the decrease in activities of the antioxidant enzymes SOD, CAT and GPx. However, in situ knockdown of DJ-1 attenuated the IPC-induced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/R-induced oxidative stress. The present study therefore provided novel evidence that DJ-1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ-1 is required for IPC to inhibit I/R-induced oxidative stress. [ABSTRACT FROM AUTHOR]

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]

An acquired resistance to platinum-based drugs has emerged as a significant impediment to effective ovarian cancer therapy. The present study explored the anticancer mechanisms of triptolide (TPL) in SKOV3PT platinum-resistant human ovarian cancer cells and observed that TPL activated caspase 3 and induced the dose-dependent apoptosis of the SKOV3PT cells. Furthermore, TPL inhibited complex I of the mitochondrial respiratory chain (MRC) followed by an increase of reactive oxygen species (ROS), which further inhibited nuclear factor NF-KB activation and resulted in the downregulation of anti-apoptotic proteins, Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP). Notably, the pre-treatment with N-acetyl-L-cysteine (NAC) abolished the TPL-induced ROS generation, NF-KB inhibition and cell apoptosis, but did not affect the inhibitory effect of TPL on complex I activity. These results suggested that TPL negatively regulated the NF-KB pathway through mitochondria-derived ROS accumulation, promoting the apoptosis of the SKOV3PT cells. Furthermore, TPL synergistically enhanced the cytotoxicity of cisplatin against platinum-resistant ovarian cancer cells. Collectively, these findings suggest that TPL is able to overcome chemoresistance and that it may be an effective treatment for platinum-resistant ovarian cancer, either alone or as an adjuvant therapy. [ABSTRACT FROM AUTHOR]