ABSTRACT
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 SQSinduced 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, SQSinduced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV12, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCεdependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV12 and S67A mutation of AE3 eradicated the SQSinduced increase of AE3 activity, reversed the inhibitory effect of SQS on H/Rinduced elevation of [Cl]i, Ca2+ overload and generation of reactive oxygen species, and eliminated SQSinduced cardioprotection. In conclusion, PKCεdependent 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.
Subject(s)
Bicarbonates/metabolism , Chloride-Bicarbonate Antiporters/metabolism , Chlorides/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Oxygen/metabolism , Saponins/pharmacology , Animals , Biological Transport/drug effects , Calcium/metabolism , Cardiotonic Agents/pharmacology , Cell Hypoxia/drug effects , Cell Survival/drug effects , Creatine Kinase/metabolism , Enzyme Activation/drug effects , HEK293 Cells , Humans , L-Lactate Dehydrogenase/metabolism , Myocytes, Cardiac/drug effects , Phosphorylation/drug effects , Phosphoserine/metabolism , Protein Kinase C-epsilon/metabolism , Rats , Reactive Oxygen Species/metabolismABSTRACT
Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.
Subject(s)
Cardiotonic Agents/pharmacology , Chlorides/chemistry , Cytoplasm/chemistry , Mitochondrial Membrane Transport Proteins/antagonists & inhibitors , Saponins/pharmacology , Animals , Atractyloside/pharmacology , Cell Line , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Membrane Transport Proteins/drug effects , Mitochondrial Permeability Transition Pore , Myocytes, Cardiac/drug effects , Rats , Reactive Oxygen Species/metabolism , Reperfusion Injury/physiopathologyABSTRACT
DJ1 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 DJ1 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 SpragueDawley rats to IPC (3 cycles of 5min coronary occlusion/5min reperfusion) 24 h prior to I/R (30min coronary occlusion/120min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ1 in situ. Lactate dehydrogenase (LDH) and creatine kinaseMB (CKMB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ1 protein. In addition, IPC reduced LDH and CKMB 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 DJ1 attenuated the IPCinduced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/Rinduced oxidative stress. The present study therefore provided novel evidence that DJ1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ1 is required for IPC to inhibit I/Rinduced oxidative stress.
Subject(s)
Ischemic Preconditioning, Myocardial , Myocardial Reperfusion Injury/pathology , Myocardium/pathology , Oxidative Stress , Protein Deglycase DJ-1/genetics , Animals , Ischemic Preconditioning, Myocardial/methods , Male , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocardium/metabolism , Protein Deglycase DJ-1/metabolism , RNA Interference , RNA, Small Interfering/genetics , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolismABSTRACT
Sasanquasaponin (SQS) is an active component of Camellia oleifera Abel. A recent study by our group demonstrated that SQS was able to inhibit ischemia/reperfusioninduced 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 pretreated with or without SQS or SQS plus εV12, 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/Rinduced elevation of [Cl]i, paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation. However, when the cardiomyocytes were treated with εV12 prior to SQS preconditioning, the cardioprotection induced by SQS was reduced and the inhibitory effects of SQS on sI/Rinduced elevation of [Cl]i, production of ROS and loss of mitochondrial membrane potential were also attenuated. These findings indicated that SQS may inhibit sI/Rinduced elevation of [Cl]i through the PKCε signaling pathway to elicit cardioprotection in cultured cardiomyocytes.
Subject(s)
Cardiotonic Agents/pharmacology , Chlorides/metabolism , Protein Kinase C-epsilon/metabolism , Saponins/pharmacology , Signal Transduction/drug effects , Animals , Apoptosis/drug effects , Blotting, Western , Cell Hypoxia , Cells, Cultured , Membrane Potential, Mitochondrial/drug effects , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolismABSTRACT
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 antioxidant enzymes and attenuate hypoxia/reoxygenation (H/R)induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2like 2 (Nrf2) is an essential transcription factor that regulates the expression of several antioxidant genes via binding to the antioxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of antioxidative enzymes by DJ1 and contributes to the protective functions of DJ1 against H/Rinduced oxidative stress in H9c2 cells. The results demonstrated that DJ1overexpressing H9c2 cells exhibited antioxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/Rinduced oxidative stress compared with native cells, whereas DJ1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ1mediated antioxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, AREbinding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ1mediated induction of antioxidant 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.
Subject(s)
Microtubule-Associated Proteins/physiology , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Reperfusion Injury/metabolism , Animals , Catalase/genetics , Catalase/metabolism , Cell Hypoxia , Cell Line , Enzyme Induction , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Protein Deglycase DJ-1 , Rats , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
We have recently shown that DJ-1 is implicated in the delayed cardioprotective effect of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R) injury as an endogenous protective protein. This study aims to further investigate the underlying mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress. Using a well-characterized cellular model of HPC from rat heart-derived H9c2 cells, we found that HPC promoted nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein-1 (Keap1) dissociation and resulted in increased nuclear translocation, antioxidant response element-binding, and transcriptional activity of Nrf2 24 hours after HPC, with subsequent upregulation of manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO-1), which provided delayed protection against H/R-induced oxidative stress in normal H9c2 cells. However, the aforementioned effects of HPC were abolished in DJ-1-knockdown H9c2 cells, which were restored by restoration of DJ-1 expression. Importantly, we showed that inhibition of the Nrf2 pathway in H9c2 cells mimicked the effects of DJ-1 knockdown and abolished HPC-derived induction of antioxidative enzymes (MnSOD and HO-1) and the delayed cardioprotection. In addition, inhibition of Nrf2 also reversed the effects of restored DJ-1 expression on induction of antioxidative enzymes and delayed cardioprotection by HPC in DJ-1-knockdown H9c2 cells. Taken together, this work revealed that activation of Nrf2 pathway and subsequent upregulation of antioxidative enzymes could be a critical mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress in H9c2 cells.
Subject(s)
Antioxidants/metabolism , Microtubule-Associated Proteins/physiology , Myocytes, Cardiac/metabolism , NF-E2-Related Factor 2/metabolism , Up-Regulation/physiology , Animals , Cell Hypoxia/physiology , Cell Line , Gene Knockdown Techniques/methods , Humans , Ischemic Preconditioning, Myocardial/methods , Protein Deglycase DJ-1 , Rats , Signal Transduction/physiologyABSTRACT
It has been well demonstrated that hypoxic preconditioning (HPC) can attenuate hypoxia/reoxygenation (H/R)-induced oxidant stress and elicit delayed cardioprotection by upregulating the expression of multiple antioxidative enzymes such as heme oxygenase-1 (HO-1), manganese superoxide dismutase (MnSOD) and so on. However, the underlying mechanisms of HPC-induced upregulation of antioxidative enzymes are not fully understood. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential transcription factor that regulates expression of several antioxidant genes via binding to the antioxidant response element (ARE) and plays a crucial role in cellular defence against oxidative stress. Here, we wondered whether activation of the Nrf2-ARE pathway is responsible for the induction of antioxidative enzymes by HPC and contributes to the delayed cardioprotection of HPC. Cellular model of HPC from rat heart-derived H9c2 cells was induced 24 h prior to H/R. The results showed that HPC efficiently attenuated H/R-induced viability loss and lactate dehydrogenase leakage. In addition, HPC increased nuclear translocation and ARE binding of Nrf2 during the late phase, upregulated the expression of antioxidative enzymes (HO-1 and MnSOD), inhibited H/R-induced oxidant stress. However, when Nrf2 was specifically knocked down by siRNA, the induction of antioxidative enzymes by HPC was completely abolished and, as a result, the inhibitory effect of HPC on H/R-induced oxidant stress was reversed, and the delayed cardioprotection induced by HPC was also abolished. These results suggest that HPC upregulates antioxidative enzymes through activating the Nrf2-ARE pathway and confers delayed cardioprotection against H/R-induced oxidative stress.
Subject(s)
Antioxidants/metabolism , Cardiotonic Agents/metabolism , Ischemic Preconditioning, Myocardial , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , NF-E2-Related Factor 2/metabolism , Up-Regulation , Animals , Antioxidant Response Elements/genetics , Cell Hypoxia , Cell Line , Cell Nucleus/metabolism , Gene Knockdown Techniques , Protein Binding , Protein Transport , Rats , Signal Transduction , Stress, PhysiologicalABSTRACT
AIM: To investigate the protective effects of preconditioning human umbilical vein endothelial cells (HUVECs) with Polygonum multiflorum stilbeneglycoside (PMS) under anoxia/reoxygenation (A/R), and the mechanism of protection. METHODS: Prior to A/R, HUVECs were incubated with PMS (0.6 x 10(-11), 1.2 x 10(-11), or 2.4 x 10(-11) mol/L) for 3 h. Cell injury was subsequently evaluated by measuring cell viability with an MTT assay and lactate dehydrogenase (LDH) release, whereas lipid peroxidation was assayed by measuring malondialdehyde (MDA) content. Antioxidant capacity was quantified by superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Nitric oxide (NO) production was determined by nitrite accumulation. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein expression was detected by Western blotting. Guanylate cyclase activity and cyclic GMP (cGMP) activity were assessed by an enzyme immunoassay kit. RESULTS: PMS incubation attenuated A/R-induced injury in a concentration-dependent manner, as evidenced by a decrease in LDH activity and an increase in cell viability. PMS exerted its protective effect by inhibiting the A/R-mediated elevation of MDA content, as well as by promoting the recovery of SOD and GSH-Px activities. Additionally, PMS incubation enhanced NO and cGMP formation by increasing iNOS expression and guanylate cyclase activity. The protective effects of PMS were markedly attenuated by NOS inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ or PKG inhibitor KT5823. CONCLUSION: PMS preincubation resulted in the enhancement of antioxidant activity and anti-lipid peroxidation. The NO/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway was involved in the effect of PMS on HUVECs.
Subject(s)
Antioxidants/therapeutic use , Drugs, Chinese Herbal/therapeutic use , Endothelial Cells/drug effects , Glycosides/therapeutic use , Polygonum/chemistry , Reperfusion Injury/drug therapy , Stilbenes/therapeutic use , Cell Survival/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Glutathione Peroxidase/metabolism , Guanylate Cyclase/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Lipid Peroxidation/drug effects , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Superoxide Dismutase/metabolism , Umbilical Veins/cytologyABSTRACT
Development of intracellular calcium overload is an important pathophysiological factor in myocardial ischemia/reperfusion or anoxia/reoxygenation injury. Recent studies have shown that Sodium Ferulate (SF) stimulates nitric oxide (NO) production and exerts a cardioprotective effect in the ischemia-reperfused heart. However, it has not been determined whether the cardioprotection of SF is associated with suppression of Ca(2+) overload via NO/cyclic GMP (cGMP)/cGMP-dependent protein kinase (PKG) pathway. In this work, after cardiomyocytes were incubated with 100, 200, 400, or 800 microM SF for 3 h, anoxia/reoxygenation injury was induced and intracellular Ca(2+) concentration, NO synthase (NOS) activity, guanylate cyclase activity, NO, and cGMP formation were measured appropriately. The results showed that treatment with SF concentration-dependently inhibited calcium overload induced by anoxia/reoxygenation. We also demonstrated that SF (100-800 microM) concentration dependently enhanced NO and cGMP formation through increasing NOS activity and guanylate cyclase activity in the cardiomyocytes. On the contrary, inhibition of calcium overload by SF was markedly attenuated by addition of an NOS inhibitor, an NO scavenger, an soluble guanylate cyclase inhibitor, and a PKG inhibitor: N(G)-nitro-l-arginine methyl ester (L-NAME, 100 microM), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (c-PTIO, 1.0 microM), 1H-[1, 2, 4] oxadiazolo [4, 3-alpha] quinoxalin-1-one (ODQ, 20 microM) and KT5823 (0.2 microM), respectively. Our findings indicate that SF significantly attenuates anoxia/reoxygenation-induced Ca(2+) overload and improves cell survival in cultured cardiomyocytes through NO/cGMP/PKG signal pathway.
Subject(s)
Calcium/metabolism , Coumaric Acids/pharmacology , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclic GMP/metabolism , Hypoxia/complications , Myocytes, Cardiac/drug effects , Nitric Oxide/metabolism , Oxygen/metabolism , Animals , Animals, Newborn , Cyclic GMP/biosynthesis , Gene Expression Regulation/drug effects , Guanylate Cyclase/chemistry , Guanylate Cyclase/metabolism , Myocytes, Cardiac/metabolism , Nitric Oxide Synthase/metabolism , Rats , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Signal Transduction/drug effects , SolubilityABSTRACT
Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.