Reduced Lactate Dehydrogenase Release Research Articles

Dynamin-related protein Drp1 and mitochondria are important for Shigella flexneri infection

Shigella infection in epithelial cells induces cell death which is accompanied by mitochondrial dysfunction. In this study the role of the mitochondrial fission protein, Drp1 during Shigella infection in HeLa cells was examined. Significant lactate dehydrogenase (LDH) release was detected in the culture supernatant when HeLa cells were infected with Shigella at a high multiplicity of infection. Drp1 inhibition with Mdivi-1 and siRNA knockdown significantly reduced LDH release. HeLa cell death was also accompanied by mitochondrial fragmentation. Tubular mitochondrial networks were partially restored when Drp1 was depleted with either siRNA or inhibited with Mdivi-1. Surprisingly either Mdivi-1 treatment or Drp1 siRNA-depletion of HeLa cells also reduced Shigella plaque formation. The effect of Mdivi-1 on Shigella infection was assessed using the murine Sereny model, however it had no impact on ocular inflammation. Overall our results suggest that Drp1 and the mitochondria play important roles during Shigella infection.

Hypothermia protects human neurons.

Background and AimsHypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury.MethodsHuman embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2O2-induced oxidative stress were used to induce relevant injury.ResultsHypothermia to 33°C protected these human neurons against H2O2-induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8–71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5–56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3–71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4–52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2O2-induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57–43·1).ConclusionWe conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2O2-induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.

Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion.

BackgroundConsecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.MethodsIsolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion. Reperfusion injury was assessed by measuring haemodynamic function, lactate dehydrogenase (LDH) release and infarct size. Protein kinase C (PKC) activity and glycogen content were measured in hearts after the treatment. In a separate group of hearts, Cyclosporine A (CsA), a mitochondria permeability transition pore (MPTP) inhibitor, was added with Iso/Ade. Failing hearts extracted after 16 weeks of ligation of left coronary artery in 2 months old rats were also subjected to Iso/Ade treatment followed by ischaemia/reperfusion.ResultsRecovery of the rate pressure product (RPP) in Iso/Ade-treated hearts was significantly higher than in controls. Thus in Iso/Ade treated hearts with 5 nM Iso and no washout period, RPP recovery was 76.3 ± 6.9% of initial value vs. 28.5 ± 5.2% in controls. This was associated with a 3 fold reduction in LDH release irrespective to the duration of the washout period. Hearts with no washout of the drugs (Ade) had least infarct size, highest PKC activity and also showed reduced glycogen content. Cardioprotection with CsA was not additive to the effect of Iso/Ade treatment. Iso/Ade treatment conferred significant protection to failing hearts. Thus, RPP recovery in failing hearts subjected to the treatment was 69.0 ± 16.3% while in Control hearts 19.7 ± 4.0%. LDH release in these hearts was also 3 fold lower compared to Control.ConclusionsConsecutive Iso/Ade treatment of normal heart can be effective at clinically-relevant doses and this effect appears to be mediated by glycogen depletion and inhibition of MPTP. This intervention protects clinically relevant failing heart model making it a promising candidate for clinical use.

L-Ascorbic acid and α-Tocopherol to protect against arsenic trioxide induced oxidative stress in H9c2 cardiomyocytes

Arsenic has been used as a therapeutic agent against various diseases right from the historical periods. Arsenic trioxide (As2O3), a first line chemotherapeutic against Acute Promyelocytic Leukemia (APL), is well known for its cardiotoxicity. We hypothesized that combination of drug and protective molecules can be used for reducing the side effects of chemotherapeutics. The present study evaluates the protective role of L- ascorbic acid (L-AA) and α-Tocopherol (α-TOC) against oxidative stress in H9c2 cardiomyocytes induced by As2O3. Clinically relevant concentration of arsenic trioxide induces cytotoxicity in cardiac cells. Treatment with antioxidant vitamins significantly reduced the cell death and apoptosis in H9c2 cells. The activity of antioxidant enzymes such as catalase and the concentration of reduced glutathione, which was lowered due to As 2O3 treatment, were found to be increased in cells co treated with L-AA and α-TOC. The treatment with antioxidant vitamins also reduced lactate dehydrogenase release in cells. The overall results clearly indicate the protective potential of L-AA and α-TOC against arsenic induced chemotherapeutic stress on cardiomyocytes. Keywords: Arsenic trioxide, Cardiotoxicity, H9c2 cardiomyocytes, L-Ascorbic acid, Oxidative stress, α- Tocopherol

Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation

BackgroundIt has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity,...

GW24-e1805 Involvement of mitochondrial ATP-sensitive potassium channels and mitochondrial permeability transition pores in atorvastatin preconditioning-induced protection in isolated rat hearts

ObjectivesTo evaluate the cardioprotective effect of atorvastatin (Ator) via modulation of mitochondrial permeability transition pore (mPTP) through mitochondrial ATP-sensitive potassium channels (mitoKATP channels).MethodsSprague-Dawley rat hearts were Langendorff-perfused with Krebs-Henseleit (K-H)...

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

Sirt1 Mediates Hyperbaric Oxygen Preconditioning-Induced Ischemic Tolerance in Rat Brain

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen–glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

Abstract 9485: Translocation of miRNA from Mesenchymal Stem Cells Protects Cardiomyocytes against Ischemic Injury in vitro

microRNAs (miR) play an important role in cell survival. In this study, we investigated whether mesenchymal stem cells (MSC) mediated cardioprotection was associated with the regulation of miR as well as their targeted proteins. Methods: MSC were harvested from rat bone marrow. Cardiomyocytes (CM) were obtained from neonatal rat ventricles. In vitro cardioprotection by MSC was assayed by lactate dehydrogenase (LDH) release, Annexin-V staining, and DNA fragmentation following cells exposure to hypoxia for 48 hours. The expression of miR in MSC and CM was assessed by quantitative real-time PCR. miR targeted proteins were evaluated using a luciferase reporter assay and western blotting. To further investigate the role of miR, pre-miR precursor molecules were introduced into MSC using Lipofectamine 2000. Results: miR-221 was expressed 12-fold more in MSC than in CM (p<0.05). The p53-upregulated modulator of apoptosis (PUMA), one of miR-221 targeted proteins, was lower in MSC than in CM (Fig. A). When co-cultured with CM in a dual-chamber system, MSC markedly reduced LDH release and the number of annoxin-V positive CM, and increased CM survival in hypoxic culture. Transfection of miR-221 into MSC significantly increased MSC mediated cardioprotection compared to MSC transfected with negative control miR. In CM co-cultures with MSC, miR-221 transferred from MSC to CM was confirmed by using fluorescence in situ hybridization (FISH) and immunostaining. Moreover, less PUMA protein was found in CM co-cultured with MSC than in CM cultured alone (Fig. B). Conclusions: Cardioprotective effects of MSC are putatively associated with intercellular translocation of specific miR and regulation of their targeted proteins such as PUMA in CM.

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.

Melatonin prevents ultraviolet radiation‐induced alterations in plasma membrane potential and intracellular pH in human keratinocytes

Melatonin exhibits protective effects against ultraviolet radiation (UVR) via modulation of proinflammatory mediators and its free radical scavenging capacity. To date, several reports presented protective mechanisms of this agent against UVR-induced alterations in mitochondria and nuclei. This investigation evaluates the potent preventing action of melatonin regarding early-stage UVR-mediated perturbations in plasma membrane potential (mbΔψ) and intracellular (cytosolic) pH (pH i) analyzed by flow cytometry. Experiments were carried out in a dose- and time-dependent manner using human keratinocytes [HaCaT and normal human epidermal keratinocytes (NHEK)]. First investigations, which used viability/cytotoxicity assays, showed the gradual mortality with increasing UVR doses and cultivation time. Pre-incubation with melatonin (10(-3) m) prior to UVR exposure reduced lactate dehydrogenase release by 30% (HaCaT) and 28% (NHEK) at the dose of 50 mJ/cm(2) after 48 hr (P < 0.001). Furthermore, UVR caused hyperpolarization of mbΔψ immediately (0 hr) after irradiation (25 or 50 mJ/cm(2)). At the dose of 50 mJ/cm(2), cells cultivated for 48 hr manifested a marked increase in mbΔψ by 112% (HaCaT) and 123% (NHEK). The presence of melatonin significantly protected the cells by 12% (HaCaT) and 14% (NHEK) (P < 0.001). Simultaneously, 50 mJ/cm(2) induced dramatic acidification reaching after 24 hr the level of 6.40 (without melatonin), 6.56 (with melatonin) for HaCaT and 6.11 (without melatonin), 6.43 (with melatonin) for NHEK. The results presented provide information about the protective mechanisms of melatonin itself on one hand and, combined with data reported so far, confirm the potent antiapoptotic action of melatonin.

Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice

Oxidative stress and glucose affect the expression of various genes that contribute to both reactive oxygen species generation and antioxidant systems. However, systemic alteration of oxidative stress-related gene expression in normal brains and in brains with a high-glucose status after ischemic–reperfusion has not been explored. Using a polymerase chain reaction array system, we demonstrate that thioredoxin-interacting protein (Txnip) is induced by both oxidative stress and glucose. We found that Txnip mRNA is induced by ischemic–reperfusion injury and that Txnip is located in the cytoplasm of neurons. Moreover, in vitro oxygen–glucose deprivation (OGD) and subsequent reoxygenation without glucose and in vivo administration of 3-nitropropionic acid also promoted an increase in Txnip in a time-dependent manner, indicating that oxidative stress without glucose can induce Txnip expression in the brain. However, calcium channel blockers inhibit induction of Txnip after OGD and reoxygenation. Using the polymerase chain reaction array with ischemic and hyperglycemic-ischemic samples, we confirmed that enhanced expression of Txnip was observed in hyperglycemic-ischemic brains after middle cerebral artery occlusion. Finally, transfection of Txnip small interfering RNA into primary neurons reduced lactate dehydrogenase release after OGD and reoxygenation. This is the first report showing that Txnip expression is induced in neurons after oxidative or glucose stress under either ischemic or hyperglycemic-ischemic conditions, and that Txnip is proapoptotic under these conditions.

Protective Effect of Processed Panax ginseng, Sun Ginseng on UVB-irradiated Human Skin Keratinocyte and Human Dermal Fibroblast

In this study, we investigated the protective effects of processed Panax ginseng, sun ginseng (SG) against the UVB-irradiation on epidermal keratinocytes and dermal fibroblasts. Pretreatment of SG in HaCaT keratinocytes and human dermal fibroblasts reduced UVB-induced cell damage as seen by reduced lactate dehydrogenase release. We also found that SG restored the UVB-induced decrease in anti-apoptotic gene expression (bcl-2 and bcl-xL) in these cells, indicating that SG has an anti-apoptotic effect and thus can protect cells from cell death caused by strong UVB radiation. In addition, SG inhibited the excessive expression of c-jun and c-fos gene by the UVB in HeCaT cells and human dermal fibroblasts. We also demonstrated that SG may exert an anti-inflammatory activity by reducing the nitric oxide production and inducible nitric oxide synthase mRNA synthesis in HaCaT keratinocytes and human dermal fibroblasts. This was further supported by its inhibitory effects on the elevated cyclooxygenase-2 and tumor necrosis factor-α transcription which was induced by UVB-irradiation in HaCaT cells. In addition, SG may have anti-aging property in terms of induction of procollagen gene expression and inhibition of the matrix metalloprotease-1 gene expression caused by UVBexposure. These findings suggest that SG can be a potential agent that may protect against the dermal cell damage caused by UVB.

Neuroprotective effect of fucoidan on H2O2-induced apoptosis in PC12 cells via activation of PI3K/Akt pathway.

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.

Protective Effect of Schisandrin B Against Cyclosporine A-Induced NephrotoxicityIn VitroandIn Vivo

Schisandrin B (Sch B) is an active ingredient of the fruit of Schisandra chinensis. It has many therapeutic effects arising from its tonic, sedative, antitussive and antiaging activities and is also used in the treatment of viral and chemical hepatitis. The aim of this study was to investigate the protective effects of Sch B on cyclosporine A (CsA)-induced nephrotoxicity in mice and HK-2 cells (a human proximal tubular epithelial cell line). After gavage with Sch B (20 mg/kg) or olive oil (vehicle), mice received CsA (30 mg/kg) by subcutaneous injection once daily for four weeks. Renal function, histopathology, and tissue glutathione (GSH) and malondialdehyde (MDA) levels were evaluated after the last treatment. The effects of Sch B on CsA-induced oxidative damage in HK-2 cells were investigated by measuring cell viability, the release of lactate dehydrogenase (LDH), the level of reactive oxygen species (ROS), and the cellular GSH and ATP concentrations. Cellular apoptosis was assessed by flow cytometry. Treatment with Sch B in CsA-treated mice significantly suppressed the elevation of blood urea nitrogen (BUN) and serum creatinine levels and attenuated the histopathological changes. Additionally, Sch B also decreased renal MDA levels and increased GSH levels in CsA-treated mice. Using an in vitro model, Sch B (2.5, 5 and 10 μM) significantly increased the cell viability and reduced LDH release and apoptosis induced by CsA (10 μM) in HK-2 cells. Furthermore, Sch B increased the intracellular GSH and ATP levels and attenuated CsA-induced ROS generation. In conclusion, Sch B appears to protect against CsA-induced nephrotoxicity by decreasing oxidative stress and cell death.

ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O(2)), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion

Intermittent hypobaric hypoxia (IHH) protects hearts against ischemia-reperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ± 5.3% in IHH vs. 42.1 ± 3.8% in the normoxic control, P < 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K(+) channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3β, as well as translocation of PKC-ε were not affected by applying H(2)O(2) (20 μmol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHH-induced cardioprotection depends on elevated ROS production during early reperfusion.

Hyperthermia conditioned astrocyte-cultured medium protects neurons from ischemic injury by the up-regulation of HIF-1 alpha and the increased anti-apoptotic ability

It has been demonstrated that conditioned medium from astrocytes challenged by in vitro ischemia (oxygen–glucose deprivation, OGD) improved neuronal survival. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized that hyperthermia-conditioned astrocyte-cultured medium (ACM) might also have protective effect on neurons against ischemic injury. The cultured-media, named 38ACM and 40ACM respectively, were collected after astrocytes had been incubated at 38 °C or 40 °C for 6 h, followed by incubation at 37 °C for 24 h. It was found that ischemia for 6 h induced a significant reduction in the number of neuronal cells and cell-viability, and an increase in lactate dehydrogenase (LDH) release and the percentage of apoptotic nuclei in neurons. Pre-treatment with 38ACM or 40ACM for 24 h significantly diminished ischemia injury, enhanced cell viability, reduced LDH release and reversed apoptosis. Western blot analysis showed that treatment with 38ACM or 40ACM for 24 h led to a significant increase in hypoxia-inducible factor-1 (HIF-1) alpha expression. The EMSA demonstrated that the ACM increased the binding activity of HIF-1 in ischemic neurons. The data implied that hyperthermia-conditioned ACM protects neurons from ischemic injury by up-regulating HIF-1 alpha, and the increased binding activity of HIF-1 and anti-apoptotic ability.