Inhibited Caspase-3 Activation Research Articles

Preventive effect of yuzu and hesperidin on left ventricular remodeling and dysfunction in rat permanent left anterior descending coronary artery occlusion model.

Left ventricular (LV) remodeling, which includes ventricular dilatation and increased interstitial fibrosis after myocardial infarction (MI), is the critical process underlying the progression to heart failure. Therefore, a novel approach for preventing LV remodeling after MI is highly desirable. Yuzu is a citrus plant originating in East Asia, and has a number of cardioprotective properties such as hesperidin. However, no study has proved whether yuzu can prevent LV remodeling. The aim of this study was to determine the effects of yuzu on heart failure (HF) and its potential impact on the LV remodeling process after MI. Our in vivo study using the permanent left anterior descending coronary artery (LAD) occlusion model demonstrate that one week pre-treatment with yuzu or its major metabolite hesperidin before LAD occlusion significantly attenuated cardiac dysfunction, myocyte apoptosis and inflammation. Not only yuzu but also hesperidin inhibited caspase-3 activity, myeloperoxidase expression, α-smooth muscle actin expression, and matrix metalloproteinase-2 activity in a permanent LAD occlusion rat model. To our knowledge, our findings provide the first evidence that yuzu and hesperidin prevent MI-induced ventricular dysfunction and structural remodeling of myocardium.

Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/β-catenin signaling and suppresses lung inflammation.

Alveolar macrophages (AMs) undergo increased apoptosis during sepsis-induced acute respiratory distress syndrome (ARDS). Ghrelin exhibits an antiapoptotic effect in several cell types and protects against sepsis-induced ARDS in rats; however, the molecular mechanisms underlying this antiapoptotic effect remain poorly understood. In this study, we first examined the antiapoptotic effect of ghrelin on lipopolysaccharide (LPS)-stimulated AMs in vitro. In AMs, GH secretagogue receptor-1a (GHSR-1a), the ghrelin receptor, was expressed, and treatment of AMs with ghrelin markedly reduced LPS-induced apoptosis, mitochondrial transmembrane potential decrease, and cytochrome c release. These effects of ghrelin were mediated by GHSR-1a because a GHSR-1a-targeting small interfering RNA abolished the antiapoptotic action of ghrelin. LPS treatment activated the c-Jun N-terminal kinase (JNK) signaling pathway but inhibited the Wnt/β-catenin pathway. Interestingly, combined LPS-ghrelin treatment reduced JNK activation and increased Wnt/β-catenin activation. Furthermore, like ghrelin treatment, the addition of the JNK inhibitor SP600125 or the glycogen synthase kinase-3β inhibitor SB216763 rescued AMs from apoptosis. We also demonstrated that ghrelin altered the balance of Bcl-2-family proteins and inhibited caspase-3 activity. Next, we investigated whether ghrelin protected against septic ARDS in vivo. Sepsis was induced in male rats by performing cecal ligation and puncture; administration of ghrelin reduced sepsis-induced AMs apoptosis, pulmonary injury, protein concentrations in the bronchoalveolar lavage fluid, the lung neutrophil infiltration, and wet to dry weight ratio. However, administration of a specific ghrelin-receptor antagonist, [D-Lys-3]-GH-releasing peptide-6, abolished the beneficial effects of ghrelin. Collectively our results suggest that ghrelin exerts an antiapoptotic effect on AMs at least partly by inhibiting JNK and activating the Wnt/β-catenin pathway and thereby helps alleviate septic ARDS in rats.

The Flavonoid Apigenin Ameliorates Cisplatin-Induced Nephrotoxicity through Reduction of p53 Activation and Promotion of PI3K/Akt Pathway in Human Renal Proximal Tubular Epithelial Cells.

Apigenin is a member of the flavone subclass of flavonoids present in fruits and vegetables. Apigenin has long been considered to have various biological activities, such as antioxidant, anti-inflammatory, and antitumorigenic properties, in various cell types. Cisplatin was known to exhibit cytotoxic effect to renal cells by inducing apoptosis through activation of p53. The present study investigated the antiapoptotic effects of apigenin on the cisplatin-treated human renal proximal tubular epithelial (HK-2) cells. HK-2 cells were pretreated with apigenin (5, 10, 20 μM) for 1 h and then treated with 40 μM cisplatin for various times. Apigenin inhibited the cisplatin-induced apoptosis of HK-2 cells. Interestingly, apigenin itself exerted cytostatic activity because of its ability to induce cell cycle arrest. Apigenin inhibited caspase-3 activity and PARP cleavage in cisplatin-treated cells. Apigenin reduced cisplatin-induced phosphorylation and expression of p53, with no significant influence on production of ROS that is known to induce p53 activation. Furthermore, apigenin promoted cisplatin-induced Akt phosphorylation, suggesting that enhanced Akt activation may be involved in cytoprotection. Taken together, these results suggest that apigenin ameliorates cisplatin-induced apoptosis through reduction of p53 activation and promotion of PI3K/Akt pathway in HK-2 cells.

Mangiferin inhibits endoplasmic reticulum stress-associated thioredoxin-interacting protein/NLRP3 inflammasome activation with regulation of AMPK in endothelial cells

BackgroundEndothelial dysfunction is tightly associated with cardiovascular complications in diabetic patients. This study aims to investigate the effects of mangiferin on the regulation of endothelial homeostasis under endoplasmic reticulum stress (ER stress) conditions. ResultsHigh glucose (25mmol/L) exposure induced ER stress and promoted ROS production in endothelial cells. Mangiferin effectively inhibited ER stress-associated oxidative stress by attenuating IRE1α phosphorylation and reducing ROS production. In response to ER stress, thioredoxin-interacting protein (TXNIP) expression increased, followed by NLRP3 inflammasome activation and increased IL-1β secretion. Mangiferin treatment attenuated the expressions of TXNIP and NLRP3 and reduced IL-1β and IL-6 production, demonstrating its inhibitory effects on TXNIP/NLRP3 inflammasome activation. NLRP3 inflammasome activation is responsible for mitochondrial cell death. Mangiferin restored the loss of the mitochondrial membrane potential (Δψm) and inhibited caspase-3 activity, and thereby protected cells from high glucose-induced apoptosis. Moreover, mangiferin inhibited ET-1 secretion and restored the loss of NO production when cells were exposed to high glucose. Mangiferin enhanced AMPK phosphorylation and AMPK inhibitor compound C diminished its beneficial effects, indicating the potential role of AMPK in its action. ConclusionOur work showed the beneficial effects of mangiferin on the improvement of endothelial homeostasis and elucidated the molecular pathway through which mangiferin ameliorated endothelial dysfunction by inhibition of ER stress-associated TXNIP/NLRP3 inflammasome activation in endothelial cells. SignificanceThese findings demonstrated the beneficial effects of mangiferin on the regulation of endothelial homeostasis and indicated its potential application in the management of diabetic cardiovascular complications.

Hepatocellular Heme Oxygenase-1

Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis

Artemisinin (ART) is a well-known anti-malarial drug, and recently it is shown prospective to selectively kill cancer cells. But low potency makes it inappropriate for use as an anticancer drug. In this study, we modulated the ART-induced autophagy to increase Potency of ART as an anticancer agent. ART reduced the cell viability and colony forming ability of non-small lung carcinoma (A549) cells and it was non-toxic against normal lung (WI38) cells. ART induced autophagy at the early stage of treatment. Pre-treatment with chloroquine (CQ) and followed by ART treatment had synergistic combination index (CI) for cell death. Inhibition of autophagy by CQ pre-treatment led to accumulation of acidic vacuoles (AVOs) which acquainted with unprocessed damage mitochondria that subsequently promoted ROS generation, and resulted releases of Cyt C in cytosol that caused caspase-3 dependent apoptosis cell death in ART-treated A549 cells. Scavenging of ROS by antioxidant N-acetyl-cysteine (NAC) inhibited caspase-3 activity and rescued the cells from apoptosis. Similar effects were observed in other cancer cells SCC25 and MDA-MB-231. The appropriate manipulation of autophagy by using CQ provides a powerful strategy to increase the Potency of selective anticancer property of ART.

Abstract 1353: Protein tyrosine phosphatases PTP-1B, SHP-2 and PTEN facilitate Rb/E2F-associated apoptotic signaling

Abstract To maintain tissue homeostasis, apoptosis is functionally linked to cell proliferation via the retinoblastoma tumor suppressor (Rb)/E2F complex. Rb/E2F-associated apoptosis is a robust apoptotic response initiated by loss of constitutive Rb/E2F transcriptional repression and mediated by caspase-8. It is characterized by inactivation of focal adhesion kinase (FAK) through dephosphorylation by protein tyrosine phosphatase(s). In the current work, we report that three protein tyrosine phosphatases, PTP-1B, SHP-2, and PTEN, are involved in Rb/E2F-associated apoptosis. Induction of apoptosis in an osteoscarcoma cell line that conditionally expresses a dominant negative mutant of E2F-1 (ER-dnE2F-1) by treatment with 4-hydroxytamoxifen (4-OHT) led to a significant increase in levels of PTP-1B, SHP-2, and PTEN mRNA expression in comparison to untreated cells. Chromatin immunoprecipitation assays showed that E2F-1 specifically binds to putative E2F-1 binding sites identified within the three PTP promoters. Knockdown of SHP-2, PTEN, or PTP-1B using siRNA increased cell viability and decreased cell death in 4-OHT-treated ER-dnE2F-1 cells compared to control. Consistent with this observation, knockdown of SHP-2 or PTEN significantly inhibited caspase-8 activity in 4-OHT-treated ER-dnE2F1 cells in comparison to control while knockdown of SHP-2, PTEN, or PTP-1B significantly inhibited caspase-3 activity in 4-OHT-treated ER-dnE2F-1 cells compared to control. Combined, our results indicated PTP-1B, SHP-2, and PTEN are required to regulate Rb/E2F-associated apoptotic signal transduction. Furthermore, our findings suggest PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism. Citation Format: Liza D. Morales, Mario Capetillo, Edgar Casillas Pavon, Jun W. Shin, Alexander Garcia, Jonathan H. Lieman, Dae J. Kim. Protein tyrosine phosphatases PTP-1B, SHP-2 and PTEN facilitate Rb/E2F-associated apoptotic signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1353. doi:10.1158/1538-7445.AM2014-1353

Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5days and repeated 4 times with 1–4month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

The Effects of High Glucose Levels on Reactive Oxygen Species-Induced Apoptosis and Involved Signaling in Human Vascular Endothelial Cells

This study investigated the effects of high glucose levels on human vascular endothelial cells and the expression of apoptosis-associated signaling molecules. Cell proliferation of human umbilical vein endothelial cells (HUVECs) was analyzed by colorimetric assay and cell number counting. Apoptosis was measured by Annexin V/FITC staining and flow cytometry. Gene knockdown was established by transfection of synthesized small interfering RNA. Caspase-3 activation was inhibited by a caspase-3 inhibitor. Protein expression of signaling molecules was measured by Western blot. Glucose significantly decreased HUVEC viability, induced cell apoptosis, and elevated levels of intracellular reactive oxygen species in a concentration and time-dependent manner. Glucose significantly upregulated the Nox4 isoform of nicotinamide adenine dinucleotide phosphate oxidase and phosphatase and tensin homolog (PTEN) protein expression, increased PTEN phosphorylation, and activated caspase-3 in a concentration-dependent manner. Silencing Nox4 and PTEN gene expression and inhibiting caspase-3 activation significantly protected HUVECs from glucose-induced cell apoptosis. Silencing Nox4 significantly normalized the levels of reactive oxygen species in glucose-treated cells; 20 mM glucose obviously upregulated Nox4, PTEN, phosphor-PTEN, and Bax levels, but significantly reduced integrin-linked kinase (ILK) activity, Bcl-2 (B cell lymphoma 2) expression, and protein kinase B (Akt) phosphorylation at serine 473. High glucose levels can reduce cell viability and induce apoptosis in HUVECs through Nox4-produced reactive oxygen species. Elevated levels of reactive oxygen species decreased Bcl-2 expression and increased PTEN expression and phosphorylation, which lead to the subsequent inhibition of ILK-Akt signaling, elevation of Bax expression, and activation of caspase-3.

SIRT2 mediates oxidative stress-induced apoptosis of differentiated PC12 cells.

Sirtuin 2 (SIRT2) is a member of the sirtuin family. Previous studies have suggested that SIRT2 mediates the cell death in models of Parkinson's disease and Huntington's disease. However, the role of SIRT2 in oxidative stress-induced cell death has remained unclear. In this study, we investigated the roles of SIRT2 in oxidative stress-induced cell death using differentiated PC12 cells as a cell model. We found that H2O2 induced a significant increase in the SIRT2 level in the cells. Both SIRT2 silencing and the SIRT2 inhibitor AGK2 significantly decreased H2O2-induced apoptosis, partially by inhibiting caspase-3 activation. We further found that silencing of SIRT2 led to decreased reactive oxygen species levels in the H2O2-treated cells. Collectively, our observations have suggested that SIRT2 plays a significant role in oxidative stress-induced cell death.

Vascular endothelial growth factor A improves quality of matured porcine oocytes and developing parthenotes

Vascular endothelial growth factor is a multipotent angiogenic factor implicated in cell survival and proliferation. The objective was to determine effects of exogenous recombinant human VEGFA (or VEGFA165) in culture media on porcine oocyte maturation and parthenote development. Adding 5 ng/mL VEGFA to the culture medium improved the maturation rate of denuded oocytes (P < 0.05), although 5, 50, or 500 ng/mL did not significantly affect nuclear maturation of oocytes. Parthenotes from oocytes cultured either in in vitro maturation or in vitro culture medium supplemented with 5 or 50 ng/mL VEGFA had an improved blastocyst rate and increased total numbers of cells (P < 0.05). Moreover, those treated with 5 ng/mL of VEGFA had a higher hatched blastocyst rate (average of 121 cells per blastocyst). All VEGFA-treated oocytes had reduced apoptotic indices (P < 0.05), except for those with a higher dose (500 ng/mL) of VEGFA which had more apoptotic cells (P < 0.05). Adding 5 ng/mL VEGFA to oocytes during the last 22 h of in vitro maturation improved (P < 0.05) blastocyst rates and total numbers of cells, with reduced apoptosis indices similar to that of long-term (44 h) culture. Furthermore, Axitinib (VEGFR inhibitor) reversed the effects of VEGFA on parthenote development (P < 0.05). Follicular fluids from medium (2–6 mm) to large (>6 mm) follicles contained 5.3 and 7.0 ng/mL vascular endothelial growth factor protein, respectively, higher (P < 0.05) than concentrations in small (<2 mm) follicles (0.4 ng/mL). Also, VEGFA and its receptor (VEGFR-2) were detected (immunohistochemistry) in growing follicles and developing blastocysts. In addition, VEGFA inhibited caspase-3 activation in matured oocytes (P < 0.05). In conclusion, this is apparently the first report that VEGFA has proliferative and cytoprotective roles in maturing porcine oocytes and parthenotes. Furthermore, an optimal VEGFA concentration promoted porcine oocyte maturation and subsequent development.

Hydrogen Sulfide Ameliorates High-Glucose Toxicity in Rat Peritoneal Mesothelial Cells by Attenuating Oxidative Stress

Background/Aims: Continuous exposure of the peritoneal membrane to high-glucose (HG) peritoneal dialysis fluids (PDFs) can produce peritoneal mesothelial cells (PMCs) injury. It has been demonstrated that hydrogen sulfide (H₂S), the third endogenous gaseous mediator identified after nitric oxide and carbon monoxide, exhibits a potent protective effect on cell activity. We studied the toxic effects of HG PDFs and their reversal by H₂S on cultures of rat PMCs. Methods: Synchronized confluent rat PMCs were incubated with 2.5% glucose PDFs with or without NaHS, an H₂S donor. Cell viability was assessed by methyl thiazolyl tetrazolium assay and flow cytometry. The level of phospho-p38 mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. p53, Bax and Bcl-2 mRNA expressions by rat PMCs were detected by real-time PCR. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and caspase-3 activity were measured. Results: Exposure of rat PMCs to 2.5% glucose PDFs for 24 h resulted in a significant induction of apoptosis, which was attenuated by NaHS. NaHS also restored the 2.5% glucose PDF-induced increase in phospho-p38 MAPK (indices of cellular toxicity). Further investigation of the apoptotic mechanisms in rat PMCs demonstrated that HG activated caspase-3 and upregulated Bax, while it downregulated Bcl-2. All the above responses were prevented by pretreatment with NaHS. Moreover, NaHS reversed the 2.5% glucose PDF-induced increase in ROS generation and decrease in SOD activity. Conclusions: These findings suggest that HG PDFs significantly inhibit rat PMC viability, leading to peritoneal injury. H₂S exhibits a potent anti-apoptotic ability by attenuating oxidative stress and inhibiting caspase-3 activation, which in turn restores peritoneal injury.

Protective effects of a wheat germ peptide (RVF) against H2O 2-induced oxidative stress in human neuroblastoma cells.

RVF (Arg-Val-Phe), a peptide derived from wheat germ, shows antioxidant properties. Here, the neuroprotective efficacies of RVF were investigated in human neuroblastoma cells (SH-SY5Y) that were pretreated with RVF (150-250 μM, 4 h) and exposed to H2O2 (200 μM). RVF increased viable cell numbers by 37 % and reduced the release of lactate dehydrogenase. Pretreatment with RVF also inhibited H2O2-induced accumulation of reactive oxygen species and maintained the mitochondrial transmembrane potential as well as preventing intracellular Ca(2+) dysregulation during H2O2 exposure. Furthermore, pretreatment with RVF increased the Bcl-2/Bax ratio and blocked cleavage poly(ADP-ribose) polymerase by inhibiting caspase-3 activation, thus decreasing apoptosis.

Walnuts (Seeds of Juglandis sinensis L.) Protect Human Epidermal Keratinocytes against UVB-Induced Mitochondria-Mediated Apoptosis through Upregulation of ROS Elimination Pathways

Purpose: Ultraviolet (UV) light from sunlight is an important environmental factor causing hazardous health effects, including various skin disorders. UV irradiation downregulates reactive oxygen species (ROS) elimination pathways, thereby promoting the production of ROS, which are implicated in mitochondria-mediated apoptosis. Walnuts, the seeds of Juglandissinensis L., are a highly nutritious food and have been shown to have a number of pharmacological activities. To our knowledge, no study on the protective effects of walnuts on human epidermal keratinocytes has been reported previously. Here, we investigated the protective effects of walnuts against UVB (50 mJ/cm²) -induced mitochondria-mediated apoptosis. Procedures and Results: Walnuts significantly and dose-dependently reduced UVB-induced apoptotic toxicity by lactate dehydrogenase assay kit. Walnuts decreased mitochondrial dysfunction, B-cell lymphoma 2 (Bcl-2)-associated X (Bax) protein levels, and cytochrome c release from mitochondria, while increasing Bcl-2 protein levels using immunofluorescence, Western blot, or kit analysis. Moreover, walnuts inhibited caspase-3 activity, indicating an inhibition of the apoptotic cascade, and induced the expression of heme oxygenase and NAD(P)H dehydrogenase via NF-E2-related factor-2 activation using immunofluorescence or Western blot analysis. Conclusion: Together, these results demonstrate that walnuts can protect human epidermal keratinocytes against UVB-induced mitochondria-mediated apoptosis by regulating ROS elimination pathways.

Group III mGlu receptor agonist, ACPT-I, exerts potential neuroprotective effects in vitro and in vivo.

Many evidence suggest that metabotropic glutamate receptors (mGluRs) may modulate glutamatergic transmission, hence, these receptors are regarded as potential targets for neuroprotective drugs. Since group III mGlu receptor agonists are known to reduce glutamatergic transmission by inhibiting glutamate release, we decided to investigate the neuroprotective potential of the group III mGlu receptor agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-I) against kainate (KA)-induced excitotoxicity in vitro and in vivo. In primary neuronal cell cultures ACPT-I (1–200 μM), applied 30 min–3 h after starting the exposure to KA (150 μM), significantly attenuated the KA-induced LDH release, increased cell viability, and inhibited caspase-3 activity both in cortical and hippocampal cell cultures. The effects were dose-, time- and structure-dependent. The neuroprotective effects of ACPT-I were reversed by (RS)-alpha-cyclopropyl-4-phosphonophenyl glycine, a group III mGluR antagonist. In the in vivo studies, KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region and the size of degeneration was examined by stereological counting of surviving neurons in the CA pyramidal layer. It was found that ACPT-I (7.5 or 15 nmol/1 μl), injected into the dorsal hippocampus 30 min, 1 or 3 h after KA in dose-dependent manner prevented the KA-induced neuronal damage. Moreover, in vivo microdialysis studies in the rat hippocampus showed that ACPT-I (200 μM) given simultaneously with KA (50 μM) significantly diminished the KA-induced glutamate release in the hippocampus. This mechanism seems to play a role in mediating the neuroprotective effect of ACPT-I.

Valproic Acid Treatment Attenuates Caspase-3 Activation and Improves Survival After Lethal Burn Injury in a Rodent Model

Burn injury may result in multiple organ dysfunction partially because of apoptotic cell death. The authors have previously shown that valproic acid (VPA) improves survival in a dog burn model. The aim of this study is to examine whether a VPA improves survival in a rodent burn model and whether this was because of inhibition of cell apoptosis. Rats were subjected to third-degree 55% TBSA burns and randomized to treatment with a VPA (300 mg/kg) or normal saline. One group of animals was monitored for 12 hours for survival analysis; another group was killed at 6 hours after injury, and brains, hearts, and blood samples were harvested for examination. Plasma creatine kinase (CK)-MB activities and neuron-specific enolase (NSE) levels were measured to evaluate the cardiac and brain damages. The effects of a VPA on acetylation of histone H3 and caspase-3 activation were also evaluated. Major burn injury resulted in a significant decrease in the acetylation of histone H3, and there was an increase in plasma CK-MB activities, NSE concentrations, and tissue levels of activated caspase-3. A VPA treatment significantly increased the acetylation of histone H3 and survival of the animals after major burn injury. In addition, a VPA treatment significantly attenuated the plasma CK-MB activities, an NSE concentrations, and inhibited caspase-3 activation after major burn injury. These results indicate that a VPA can attenuate cardiac and brain injury, and can improve survival in a rodent model of lethal burn injury. These protective effects may be mediated in part through the inhibition of caspase-3 activation.

Nicotine promotes cell proliferation and induces resistance to cisplatin by α7 nicotinic acetylcholine receptor-mediated activation in Raw264.7 and El4 cells

Although nicotine is a risk factor for carcinogenesis and atherosclerosis, epidemiological data indicate that nicotine has therapeutic benefits in treating Alzheimer's disease. Our previous studies also showed that nicotine-treated dendritic cells have potential antitumor effects. Hence, the precise effects of nicotine on the biological characterizations of cells are controversial. The aim of the present study was to assess the roles of α7 nicotinic acetylcholine receptors (nAChRs), Erk1/2-p38-JNK and PI3K-Akt pathway in nicotine-mediated proliferation and anti-apoptosis effects. The results firstly showed that nicotine treatment clearly augmented cell viability and upregulated PCNA expression in both Raw264.7 and El4 cells. Meanwhile, nicotine afforded protection against cisplatin-induced toxicity through inhibiting caspase-3 activation and upregulating anti-apoptotic protein expression. Further exploration demonstrated that nicotine efficiently abolished cisplatin-promoted mitochondria translocation of Bax and the release of cytochrome c. The pretreatment of α-bungarotoxin and tubocurarine chloride significantly attenuated nicotine-augmented cell viability, abolished caspase-3 activation and α7 nAChR upregulation. Both Erk-JNK-p38 and PI3K-Akt signaling pathways could be activated by nicotine treatment in Raw264.7 and El4 cells. Notably, when Erk-JNK and PI3K-Akt activities were inhibited, nicotine-augmented cell proliferation and anti-apoptotic effects were abolished accordingly. The results presented here indicate that nicotine could achieve α7 nAChR-mediated proliferation and anti-apoptotic effects by activating Erk-JNK and PI3K-Akt pathways respectively, providing potential therapeutic molecules to deal with smoking-associated human diseases.

Antiviral activity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is an acute infectious disease. The prevalence of PRRS has made swine industry suffered huge financial losses. Matrine, a natural compound, has been demonstrated to possess anti-PRRSV activity in Marc-145 cells. However, the underlying molecular mechanisms were still unknown. The main objective of our study was to discuss the effect of Matrine on PRRSV N protein expression and PRRSV induced apoptosis. Indirect immunofluorescence assay (IFA) and Western blot were used to assess the effect of Matrine on N protein expression. Apoptosis was analyzed by fluorescence staining. In addition, the effect of Matrine on caspase-3 activation was investigated by Western blot. Indirect immunofluorescence assay and Western blot analysis demonstrated that Matrine could inhibit N protein expression in Marc-145 cells. And Matrine was found to be able to impair PRRSV-induced apoptosis by inhibiting caspase-3 activation.

Fatty Acid Synthase Inhibitor C75 Ameliorates Experimental Colitis

Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic α-methylene-γ-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or dimethyl sulfoxide (DMSO) (vehicle) was administered intraperitoneally from d 2 to 6. Clinical parameters were monitored daily. Mice were euthanized on d 8 for histological evaluation and measurements of colon length, chemokine, cytokine and inflammatory mediator expression. C75 significantly reduced body weight loss from 23% to 15% on d 8, compared with the vehicle group. The fecal bleeding, diarrhea and colon histological damage scores in the C75-treated group were significantly lower than scores in the vehicle animals. Colon shortening was significantly improved after C75 treatment. C75 protected colon tissues from DSS-induced apoptosis by inhibiting caspase-3 activity. Macrophage inflammatory protein 2, keratinocyte-derived chemokine, myeloperoxidase activity and proinflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β and IL-6) in the colon were significantly downregulated in the C75-treated group, compared with the vehicle group. Treatment with C75 in colitis mice inhibited the elevation of FASN, cyclooxygenase-2 and inducible nitric oxide synthase expression as well as IκB degradation in colon tissues. C75 administration alleviates the severity of colon damage and inhibits the activation of inflammatory pathways in DSS-induced colitis. Thus, inhibition of FASN may represent an attractive therapeutic potential for treating IBD.

Flotillin-2 Modulates Fas Signaling Mediated Apoptosis after Hyperoxia in Lung Epithelial Cells

Lipid rafts are subdomains of the cell membrane with distinct protein composition and high concentrations of cholesterol and glycosphingolipids. Raft proteins are thought to mediate diverse cellular processes including signal transduction. However, its cellular mechanisms remain unclear. Caveolin-1 (cav-1, marker protein of caveolae) has been thought as a switchboard between extracellular matrix (ECM) stimuli and intracellular signals. Flotillin-2/reggie-1(Flot-2) is another ubiquitously expressed raft protein which defines non-caveolar raft microdomains (planar raft). Its cellular function is largely uncharacterized. Our novel studies demonstrated that Flot-2, in conjunction with cav-1, played important functions on controlling cell death via regulating Fas pathways. Using Beas2B epithelial cells, we found that in contrast to cav-1, Flot-2 conferred cytoprotection via preventing Fas mediated death-inducing signaling complex (DISC) formation, subsequently suppressed caspase-8 mediated extrinsic apoptosis. Moreover, Flot-2 reduced the mitochondria mediated intrinsic apoptosis by regulating the Bcl-2 family and suppressing cytochrome C release from mitochondria to cytosol. Flot-2 further modulated the common apoptosis pathway and inhibited caspase-3 activation via up-regulating the members in the inhibitor of apoptosis (IAP) family. Last, Flot-2 interacted with cav-1 and limited its expression. Taken together, we found that Flot-2 protected cells from Fas induced apoptosis and counterbalanced the pro-apoptotic effects of cav-1. Thus, Flot-2 played crucial functions in cellular homeostasis and cell survival, suggesting a differential role of individual raft proteins.