Attenuated Caspase-3 Activation Research Articles

Neuroprotective effects of polyacrylic acid (PAA) conjugated cerium oxide against hydrogen peroxide- and 6-OHDA-induced SH-SY5Y cell damage

Cerium oxide nanoparticles have been widely investigated against neurodegenerative diseases due to their antioxidant properties that aid in quenching reactive oxygen species. In this study, polyacrylic acid conjugated cerium oxide (PAA-CeO) nanoparticles were synthesized in a 50–60 nm size range with a zeta potential of − 35 mV. X-ray photoelectron spectroscopy analysis revealed a mixed valence state of Ce4+ and Ce3+. PAA-CeO nanoparticles were safe for undifferentiated (UN-) and retinoic acid-differentiated (RA-) human neuroblastoma SH-SY5Y cells and reduced the extent of cell damage evoked by hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA). In the H2O2 model of cell damage PAA-CeO did not affect the caspase-3 activity (apoptosis marker) but attenuated the number of propidium iodide-positive cells (necrosis marker). In the 6-OHDA model, nanoparticles profoundly reduced necrotic changes and partially attenuated caspase-3 activity. However, we did not observe any impact of PAA-CeO on intracellular ROS formation induced by H2O2. Further, the flow cytometry analysis of fluorescein isothiocyanate-labeled PAA-CeO revealed a time- and concentration-dependent cellular uptake of nanoparticles. The results point to the neuroprotective potential of PAA-CeO nanoparticles against neuronal cell damage induced by H2O2 and 6-OHDA, which are in both models associated with the inhibition of necrotic processes and the model-dependent attenuation of activity of executor apoptotic protease, caspase-3 (6-OHDA model) but not with the direct inhibition of ROS (H2O2 model).

Targeting Nrf2/HO-1 signaling by crocin: Role in attenuation of AA-induced ulcerative colitis in rats

Ulcerative colitis (UC) is usually a mildly active disease; however, it can be associated with serious systemic complications. The current study was undertaken to evaluate the anti-ulcerogenic and coloprotective properties of crocin; the major biologically active ingredient of saffron against experimentally-induced UC, by intracolonic instillation of AA (AA). Rats received either felodipine (3 mg/kg) or crocin (20 mg/kg) orally for 8 successive days as protective and curative therapies. Either as a protective or as a curative remedy, crocin significantly reversed AA-induced colonic damage. Intracolonic AA-induced a significant functional, biochemical and inflammatory colon injury. On the other hand, crocin significantly attenuated AA-induced oxidative, inflammatory and apoptotic activity. Crocin significantly enhanced colon anti-oxidant defenses and reduced colon tumor necrosis factor-α (TNF-α) and Ca+2 contents with down-regulation of the inflammatory response and oxidative load. The anti-ulcerogenic effect of crocin appears to be Ca+2 dependent. Most importantly, crocin enhanced colon Nuclear Factor, Erythroid-Derived 2 like Protein 2 (Nrf2) content and Heme Oxygenase-1 (HO-1) activity and attenuated caspase-3 activity. In conclusion; crocin demonstrated anti-ulcerogenic and coloprotective effect mediated primarily by its antioxidant, anti-inflammatory and anti-apoptotic properties. The therapeutic impact is mediated primarily via enhancement of colon Nrf2 content by 211% in protective protocol and by 350% in curative and HO-1 signaling by 49% in protective protocol and, 288% in the curative protocol. Enhancement of Nrf2 and HO-1 signaling and down-regulation of caspase-3 activity are believed to underly the observed therapeutic effect.

Telmisartan attenuates hydrogen peroxide-induced apoptosis in differentiated PC12 cells.

The present study investigated the protective actions of telmisartan, an angiotensin II type 1 receptor blocker (ARBs), against the cell apoptosis induced by exposure to hydrogen peroxide (H2O2) in differentiated PC12 cells. Preincubation of PC12 cells with telmisartan prevented H2O2-induced cytotoxicity as indicated by increased MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide) reduction, decreased lactate dehydrogenase (LDH) release, and improved morphological changes. Hoechst 33,258 staining showed that telmisartan markedly reduced shrunken nuclei of the cells, and Western blot analysis indicated that telmisartan significantly attenuated caspase-3 activity, as indicated by decreased ratio of cleaved Caspase-3 to its precursor and increased ratio of Bcl-2/Bax. The present findings showed that telmisartan protected against cellular oxidative damages by inhibiting apoptotic response.

Pterostilbene protects against acute renal ischemia reperfusion injury and inhibits oxidative stress, inducible nitric oxide synthase expression and inflammation in rats via the Toll-like receptor 4/nuclear factor-κB signaling pathway.

Previous studies have demonstrated that pterostilbene (Pter) prevents oxidative stress, suppresses cell growth and exhibits anti-fungal and anti-inflammatory effects. Pter is used to treat a number of clinical diseases, including Alzheimer's disease, various malignancies and hypercholesteremia. The aim of the present study was to investigate whether Pter protects against acute renal ischemia reperfusion injury (IRI) and inhibits oxidative stress, inducible nitric oxide synthase (iNOS) expression and inflammation in rats. A total of 40 adult male Sprague Dawley rats were divided into the following 5 groups at random: Control group, where rats were not subjected to renal IRI; IRI group, where rats were subjected to renal IRI; Pter 10 group, where rats underwent renal IRI and were treated with 10 mg/kg Pter; Pter 20 group, where rats underwent renal IRI and were treated with 20 mg/kg Pter; Pter 30 group, where rats underwent renal IRI and were treated with 30 mg/kg Pter. The results demonstrated that Pter treatment improved renal function following acute renal IRI. Compared with the untreated renal IRI group, myeloperoxidase, iNOS, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α expression levels were significantly decreased (P<0.01), whereas IL-10 expression levels were significantly increased (P<0.01) following treatment with Pter in acute renal IRI rats. In addition, Pter significantly attenuated caspase-3 activity and the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway induced by acute renal IRI (P<0.01). These results provide evidence to suggest that administration of Pter may protect against acute renal IRI and inhibit oxidative stress, iNOS expression and inflammation in rats via the TLR4/NF-κB signaling pathway.

Rhus verniciflua Stokes Extract and Its Flavonoids Protect PC-12 Cells against H2O2-Induced Cytotoxicity.

Rhus verniciflua Stokes (RVS), an herbal medicine found in East Asia, was extracted and further fractionated to investigate its antioxidant capacity and neuroprotective effects. The RVS ethyl acetate (EtOAc) fraction had the highest level of total phenolics and antioxidant capacity among all solvent fractions tested. Pretreatment of PC-12 cells with the EtOAc fraction effectively attenuated H2O2-induced oxidative damage. Furthermore, the EtOAc fraction significantly attenuated caspase-3 activity, resulting in inhibition of H2O2-induced apoptosis. We identified and quantified fustin, sulfuretin, and butein in the EtOAc fraction using accurate mass quadrupole time-of-flight mass spectrometry and reversed-phase high-performance liquid chromatography. The intracellular antioxidant capacity and superoxide dismutase (SOD) activity were significantly increased in PC-12 cells treated with the EtOAc fraction and with individual flavonoids. When cells were pretreated with the EtOAc fraction or individual flavonoids and then co-incubated with diethyldithiocarbamic acid (an inhibitor of SOD activity), cell viability against H2O2-induced oxidative stress was attenuated. These results suggest that the RVS EtOAc fraction and its flavonoid constituents protect PC-12 cells against H2O2-induced neurotoxicity through their antioxidant properties.

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis.

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases.

PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure

Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A2 (sPLA2-IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA2-IIA herein. The effects of PX-18, an inhibitor of both sPLA2-IIA and apoptosis, on residual endothelium and the presence of sPLA2-IIA were studied in human saphenous vein segments (n=6) perfused at arterial blood pressure with autologous blood for 6hrs. The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA2-IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA2-IIA. In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA2-IIA inhibition.

Inhibition of Prolyl Hydroxylase Attenuates Fas Ligand-Induced Apoptosis and Lung Injury in Mice.

Alveolar epithelial injury and increased alveolar permeability are hallmarks of acute respiratory distress syndrome. Apoptosis of lung epithelial cells via the Fas/Fas ligand (FasL) pathway plays a critical role in alveolar epithelial injury. Activation of hypoxia-inducible factor (HIF)-1 by inhibition of prolyl hydroxylase domain proteins (PHDs) is a possible therapeutic approach to attenuate apoptosis and organ injury. Here, we investigated whether treatment with dimethyloxalylglycine (DMOG), an inhibitor of PHDs, could attenuate Fas/FasL-dependent apoptosis in lung epithelial cells and lung injury. DMOG increased HIF-1α protein expression in vitro in MLE-12 cells, a murine alveolar epithelial cell line. Treatment of MLE-12 cells with DMOG significantly suppressed cell surface expression of Fas and attenuated FasL-induced caspase-3 activation and apoptotic cell death. Inhibition of the HIF-1 pathway by echinomycin or small interfering RNA transfection abolished these antiapoptotic effects of DMOG. Moreover, intraperitoneal injection of DMOG in mice increased HIF-1α expression and decreased Fas expression in lung tissues. DMOG treatment significantly attenuated caspase-3 activation, apoptotic cell death in lung tissue, and the increase in alveolar permeability in mice instilled intratracheally with FasL. In addition, inflammatory responses and histopathological changes were also significantly attenuated by DMOG treatment. In conclusion, inhibition of PHDs protects lung epithelial cells from Fas/FasL-dependent apoptosis through HIF-1 activation and attenuates lung injury in mice.

MTOR inactivation in myocardium from infant mice rapidly leads to dilated cardiomyopathy due to translation defects and p53/JNK-mediated apoptosis

Mechanistic target of rapamycin (mTOR) is a central regulator of cell growth, proliferation, survival and metabolism, as part of mTOR complex 1 (mTORC1) and mTORC2. While partial inhibition of mTORC1 using rapamycin was shown to be cardioprotective, genetic studies in mouse models revealed that mTOR is essential for embryonic heart development and cardiac function in adults. However, the physiological role of mTOR during postnatal cardiac maturation is not fully elucidated. We have therefore generated a mouse model in which cardiac mTOR was inactivated at an early postnatal stage. Mutant mTORcmKO mice rapidly developed a dilated cardiomyopathy associated with cardiomyocyte growth defects, apoptosis and fibrosis, and died during their third week. Here, we show that reduced cardiomyocyte growth results from impaired protein translation efficiency through both 4E-BP1-dependent and -independent mechanisms. In addition, infant mTORcmKO hearts displayed markedly increased apoptosis linked to stretch-induced ANKRD1 (Ankyrin repeat-domain containing protein 1) up-regulation, JNK kinase activation and p53 accumulation. Pharmacological inhibition of p53 with pifithrin-α attenuated caspase-3 activation. Cardiomyocyte death did not result from activation of the MST1/Hippo pro-apoptotic pathway as reported in adult rictor/mTORC2 KO hearts. As well, mTORcmKO hearts showed a strong downregulation of myoglobin content, thereby leading to a hypoxic environment. Nevertheless, they lacked a HIF1α-mediated adaptive response, as mTOR is required for hypoxia-induced HIF-1α activation. Altogether, our results demonstrate that mTOR is critically required for cardiomyocyte growth, viability and oxygen supply in early postnatal myocardium and provide insight into the molecular mechanisms involved in apoptosis of mTOR-depleted cardiomyocytes.

Anti-oxidative and anti-inflammatory effects of cinnamaldehyde on protecting high glucose-induced damage in cultured dorsal root ganglion neurons of rats.

To examine the mechanism underlying the beneficial role of cinnamaldehyde on oxidative damage and apoptosis in high glucose (HG)-induced dorsal root ganglion (DRG) neurons in vitro. HG-treated DRG neurons were developed as an in vitro model of diabetic neuropathy. The neurons were randomly divided into five groups: the control group, the HG group and the HG groups treated with 25, 50 and 100 nmol/L cinnamaldehyde, respectively. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis rate was evaluated by the in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. The intracellular level of reactive oxygen species (ROS) was measured with flow cytometry. Expression of nuclear factor-kappa B (NF-κB), inhibitor of κB (IκB), phosphorylated IκB (p-IκB), tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and caspase-3 were determined by western blotting and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were also measured by western blotting. Cinnamaldehyde reduced HG-induced loss of viability, apoptosis and intracellular generation of ROS in the DRG neurons via inhibiting NF-κB activity. The western blot assay results showed that the HG-induced elevated expressions of NF-κB, IκB and p-IκB were remarkably reduced by cinnamaldehyde treatment in a dose-dependent manner (P <0.01). The HG-induced over-expression of NF-κB p65 mRNA was remarkably attenuated after cinnamaldehyde treatment in a dose-dependent manner (P <0.01). However, the expressions of Nrf2 and HO-1 were not upregulated. Treatment with cinnamaldehyde not only attenuated caspase-3 activation and the caspase cleavage cascade in DRG neurons, but also lowered the elevated IL-6, TNF-α, cyclo-oxygenase and inducible nitric oxide synthase levels, indicating a reduction in inflammatory damage. Cinnamaldehyde protected DRG neurons from the deleterious effects of HG through inactivation of NF-κB pathway but not through activation of Nrf2/HO-1. And thus cinnamaldehyde may have potential application as a treatment for DPN.

Analyzing the anti-ischemia-reperfusion injury effects of ginsenoside Rb1 mediated through the inhibition of p38α MAPK.

Recent studies have demonstrated that ginsenoside Rb1 protects the myocardium from ischemia-reperfusion (I/R) injury. However, the precise mechanisms for this protection have not been determined. This study aimed to determine whether the attenuation of I/R-induced myocardial injury by ginsenoside Rb1 (GS Rb1) is due to inhibition of p38α mitogen-activated protein kinase (MAPK). Sprague-Dawley rats were distributed among 6 treatment groups: sham group; I/R group; p38 MAPK inhibitor SB203580 group (SB + I/R); GS Rb1 group (GS + I/R); p38 MAPK agonist anisomycin group (Ani + I/R); and the GS Rb1 + Ani group (GS + Ani + I/R). All of the anaesthetized rats, except those in the sham group, underwent an open-chest procedure that involved 30 min of myocardial ischemia followed by 2 h of reperfusion. Myocardial infarction size (MIS), caspase-3 activity, and levels of the cytokine tumor necrosis factor alpha (TNF-α) in the myocardium were monitored. The expressions of p38α MAPK, caspase-3, and TNF-α in the myocardium were assayed. GS Rb1 reduced MIS and attenuated caspase-3 activity and the levels of TNF-α in the myocardium. Protein expression of total p38α MAPK was not significantly altered. In the Ani + I/R and I/R groups, the levels of phospho-p38α MAPK were significantly increased compared with the sham group, and these increased levels were reduced with GS Rb1. Hemodynamic parameters were not significantly different between the GS + I/R and SB + I/R groups. GS Rb1 exerts an anti-apoptotic effect that protects against I/R injury by inhibiting p38α MAPK phosphorylation, suggesting that GS Rb1-mediated protection requires the inhibition of p38α MAPK.

Voluntary Exercise Preconditioning Activates Multiple Antiapoptotic Mechanisms and Improves Neurological Recovery after Experimental Traumatic Brain Injury.

Physical activity can attenuate neuronal loss, reduce neuroinflammation, and facilitate recovery after brain injury. However, little is known about the mechanisms of exercise-induced neuroprotection after traumatic brain injury (TBI) or its modulation of post-traumatic neuronal cell death. Voluntary exercise, using a running wheel, was conducted for 4 weeks immediately preceding (preconditioning) moderate-level controlled cortical impact (CCI), a well-established experimental TBI model in mice. Compared to nonexercised controls, exercise preconditioning (pre-exercise) improved recovery of sensorimotor performance in the beam walk task, as well as cognitive/affective functions in the Morris water maze, novel object recognition, and tail-suspension tests. Further, pre-exercise reduced lesion size, attenuated neuronal loss in the hippocampus, cortex, and thalamus, and decreased microglial activation in the cortex. In addition, exercise preconditioning activated the brain-derived neurotrophic factor pathway before trauma and amplified the injury-dependent increase in heat shock protein 70 expression, thus attenuating key apoptotic pathways. The latter include reduction in CCI-induced up-regulation of proapoptotic B-cell lymphoma 2 (Bcl-2)-homology 3-only Bcl-2 family molecules (Bid, Puma), decreased mitochondria permeabilization with attenuated release of cytochrome c and apoptosis-inducing factor (AIF), reduced AIF translocation to the nucleus, and attenuated caspase activation. Given these neuroprotective actions, voluntary physical exercise may serve to limit the consequences of TBI.

Deciphering of mitochondrial cardiolipin oxidative signaling in cerebral ischemia-reperfusion.

It is believed that biosynthesis of lipid mediators in the central nervous system after cerebral ischemia-reperfusion starts with phospholipid hydrolysis by calcium-dependent phospholipases and is followed by oxygenation of released fatty acids (FAs). Here, we report an alternative pathway whereby cereberal ischemia-reperfusion triggered oxygenation of a mitochondria-specific phospholipid, cardiolipin (CL), is followed by its hydrolysis to yield monolyso-CLs and oxygenated derivatives of fatty (linoleic) acids. We used a model of global cerebral ischemia-reperfusion characterized by 9 minutes of asphyxia leading to asystole followed by cardiopulmonary resuscitation in postnatal day 17 rats. Global ischemia and cardiopulmonary resuscitation resulted in: (1) selective oxidation and hydrolysis of CLs, (2) accumulation of lyso-CLs and oxygenated free FAs, (3) activation of caspase 3/7 in the brain, and (4) motor and cognitive dysfunction. On the basis of these findings, we used a mitochondria targeted nitroxide electron scavenger, which prevented CL oxidation and subsequent hydrolysis, attenuated caspase activation, and improved neurocognitive outcome when administered after cardiac arrest. These data show that calcium-independent CL oxidation and subsequent hydrolysis represent a previously unidentified pathogenic mechanism of brain injury incurred by ischemia-reperfusion and a clinically relevant therapeutic target.

G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia–ischemia in rats

Granulocyte-colony stimulating factor (G-CSF), a growth factor, has known neuroprotective effects in a variety of experimental brain injury models. Herein we show that G-CSF administration attenuates neuronal apoptosis after neonatal hypoxia–ischemia (HI) via glycogen synthase kinase-3β (GSK-3β) inhibition. Ten day old Sprague–Dawley rat pups (n=157) were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3β siRNA (4μL/pup), G-CSF (50μg/kg), G-CSF combined with 0.1 or 0.4nM G-CSF receptor (G-CSFR) siRNA, phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (86ng/pup), or DMSO (vehicle for Wortmannin). Pups were euthanized 48h post-HI to quantify brain infarct volume. G-CSFR, activated Akt (p-Akt), activated GSK-3β (p-GSK-3β), Cleaved Caspase-3 (CC3), Bcl-2, and Bax were quantified using Western blot analysis and the localizations of each was visualized via immunofluorescence staining. Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Our results showed p-GSK-3β increased after HI until its peak at 48h post-ictus, and both GSK-3β siRNA and G-CSF administration reduced p-GSK-3β expression, as well as infarct volume. p-GSK-3β and CC3 were generally co-localized in neurons. Furthermore, G-CSF increased p-Akt expression and the Bcl-2/Bax ratio and also decreased p-GSK-3β and CC3 expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3β siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3β activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin.

Impact of recombinant globular adiponectin on early warm ischemia-reperfusion injury in rat bile duct after liver transplantation.

Adiponectin (APN) is an adipocyte protein with anti-diabetic properties, which has been recently revealed to have anti-inflammatory activity in organ ischemia- reperfusion injury (IRI). However, little is known about its function in bile duct IRI after liver transplantation. Therefore, we investigated whether APN affects early warm IRI in rat bile duct using a liver autologous transplantation model. In our study, rats were randomly divided into three experimental groups: a sham group, a IRI group, and a APN group. The serum enzyme levels and BDISS scores of bile duct histology associated with bile duct injury, decreased after administration of APN. Subsequently, the expression of proinflammatory cytokines, such as tumor necrosis factor(TNF-α),.interleukin-6(IL-6) and myeloperoxidase (MPO) decreased. Furthermore, pretreatment with APN suppressed the activation of nuclear factor-kappa B (NF-κB) (p65), a transcription factor involved in inflammatory reactions, compared to other two groups. Administration of APN also downregulated the expression of Fas protein and attenuated caspase-3 activity to decrease bile duct apoptosis. Our results illustrate that APN protects the rat bile duct against early warm IRI by suppressing the inflammatory response and hepatocyte apoptosis, and NF-κB (p65) plays an important role in this process.

Therapeutic effects of pharmacologically induced hypothermia against traumatic brain injury in mice.

Preclinical and clinical studies have shown therapeutic potential of mild-to-moderate hypothermia for treatments of stroke and traumatic brain injury (TBI). Physical cooling in humans, however, is usually slow, cumbersome, and necessitates sedation that prevents early application in clinical settings and causes several side effects. Our recent study showed that pharmacologically induced hypothermia (PIH) using a novel neurotensin receptor 1 (NTR1) agonist, HPI-201 (also known as ABS-201), is efficient and effective in inducing therapeutic hypothermia and protecting the brain from ischemic and hemorrhagic stroke in mice. The present investigation tested another second-generation NTR1 agonist, HPI-363, for its hypothermic and protective effect against TBI. Adult male mice were subjected to controlled cortical impact (CCI) (velocity=3 m/sec, depth=1.0 mm, contact time=150 msec) to the exposed cortex. Intraperitoneal administration of HPI-363 (0.3 mg/kg) reduced body temperature by 3-5°C within 30-60 min without triggering a shivering defensive reaction. An additional two injections sustained the hypothermic effect in conscious mice for up to 6 h. This PIH treatment was initiated 15, 60, or 120 min after the onset of TBI, and significantly reduced the contusion volume measured 3 days after TBI. HPI-363 attenuated caspase-3 activation, Bax expression, and TUNEL-positive cells in the pericontusion region. In blood-brain barrier assessments, HPI-363 ameliorated extravasation of Evans blue dye and immunoglobulin G, attenuated the MMP-9 expression, and decreased the number of microglia cells in the post-TBI brain. HPI-363 decreased the mRNA expression of tumor necrosis factor-α and interleukin-1β (IL-1β), but increased IL-6 and IL-10 levels. Compared with TBI control mice, HPI-363 treatments improved sensorimotor functional recovery after TBI. These findings suggest that the second generation NTR-1 agonists, such as HPI-363, are efficient hypothermic-inducing compounds that have a strong potential in the management of TBI.

Overexpression of OLC1 Promotes Tumorigenesis of Human Esophageal Squamous Cell Carcinoma

PurposeOLC1 was recently identified to be a potential oncogene. However, the role of OLC1 in human esophageal cell carcinoma (ESCC) is unknown. The aim of this study was therefore to evaluate the expression of OLC1 in human ESCC from normal, premalignant, and malignant lesions, and to clarify the mechanisms by which OLC1 contributes to the progression of ESCC. Experimental DesignTwo hundred and fourteen paired ESCC specimens, and an independent set from 28 ESCC patients, were used to analyze the correlation between OLC1 expression and the pathological characteristics of tumors using immunohistochemistry. Stable OLC1-overexpressing and OLC1-interfering esophageal cancer cells were established and a series of experimental methods were used to investigate the biological functions and mechanisms of action of OLC1.ResultsWe showed that OLC1 was overexpressed in 145 of 214 (67.8%) of human ESCC specimens, compared with in only 59 of 214 (27.57%) paired adjacent normal tissues (P<0.001). OLC1 overexpression occurred at a rate of 35% (10/28) at the stage of mild/moderate dysplasia, but was significantly upregulated to 66% (22/33) at the stages of severe dysplasia and in situ carcinoma, while 71% positive staining (22/28) was observed in invasive carcinoma tissues compared with normal tissues (P<0.05). We also provided evidence that OLC1 abnormalities significantly altered the cell proliferation and apoptosis induced by cytotoxic agents. OLC1 overexpression suppressed apoptosis, and was associated with attenuated caspase-3 activation and increased Bcl-2 stability.ConclusionOur study provides strong evidence suggesting OLC1 abnormalities may contribute to the development of human ESCC and have some important clinical significance.

An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage-gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Nav 1.1, Nav 1.2, and Nav 1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS-induced NF-κB activation, expression of TNF-α, IL-1β and inducible nitric oxide synthase, and NO production. LPS-induced p38MAPK activation followed pro-nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS-induced JNK activation was not. TTX also inhibited caspase-3 activation and cell death of primary cortical neurons in neuron/microglia co-cultures by inhibiting LPS-induced microglia activation. Furthermore, TTX attenuated caspase-3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury.

Neuroprotective Effects of Lycium chinense Miller Against Rotenone-Induced Neurotoxicity in PC12 Cells

Rotenone, an inhibitor of mitochondrial complex I, has been widely regarded as a neurotoxin because it induces a Parkinson's disease-like syndrome. The fruit and root bark of Lycium chinense Miller have been used as traditional medicines in Asia to treat neurodegenerative diseases. In this study, we examined the neuroprotective effects of Lycium chinense Miller extracts in rotenone-treated PC12 cells. Treatment with rotenone reduced PC12 cell viability and cellular ATP levels. Conversely, caspase 3/7 activity, the ratio of Bax:Bcl-2 expression levels, mitochondrial superoxide level, and intracellular calcium (Ca(2+)) concentration were elevated. Pretreatment with Lycium chinense Miller extracts significantly increased cell viability and ATP levels. Additionally, they attenuated caspase activation, mitochondrial membrane depolarization and mitochondrial superoxide production. Moreover, confocal microscopy showed that the mitochondrial staining pattern was restored from that of extracts treated cells and that the increase in intracellular Ca (2+) level was blunted by treatment with the extracts. Our results suggest that Lycium chinense Miller extracts may have the possible beneficial effects in Parkinson's disease by attenuating rotenone induced toxicity.

Hypoxia-Inducible Factor-1α Dependent Pathways Mediate the Renoprotective Role of Acetazolamide Against Renal Ischemia-Reperfusion Injury

Background/Aims: Acute kidney injury (AKI) is a major complication of kidney transplantation, resulting in early graft dysfunction. Since diuretic acetazolamide (AZA) has been shown to improve contrast induced AKI, we hypothesized that AZA also protected against ischemia-reperfusion (I/R) caused AKI. Methods: An in vivo mouse renal I/R injury model and an in vitro H₂O₂ stimulated HK-2 cell injury model were utilized to examine the renoprotective effect of AZA. Renal injury and blood flow were measured. Nitric oxide synthase (eNOS)/Nitric oxide (NO), cell apoptosis and hypoxia-inducible factor-1α (HIF-1α) changes were analyzed. Results: AZA reduced kidney injury scores and improved renal function by decreasing serum creatinine and BUN levels after I/R. Impaired renal blood flow was restored by increasing eNOS activities and NO production, as indicated by Laser Doppler imaging. TUNEL staining presented that AZA reduced apoptotic cells due to attenuated caspase activation and increased Bcl-2/Bax ratio. Furthermore, HIF-1α induction by AZA was demonstrated. AZA also enhanced in vitro NO production, reduced cell apoptosis and increased HIF-1α expression. Knockdown of HIF-1α by RNAi confirmed that AZA exerted its protective role depending on HIF-1α. AZA's effects were significantly reduced by Akt inhibitor LY294002. Conclusions: The present study demonstrated that AZA exerted a renoprotective role against I/R induced AKI through activating HIF-1α and downstream pathways.