Peter T.-H. Wong

Hydrogen sulfide attenuates lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated protein kinase in microglia

The present study attempts to investigate the effect of H2S on lipopolysaccharide (LPS)‐induced inflammation in both primary cultured microglia and immortalized murine BV‐2 microglial cells. We found that exogenous application of sodium hydrosulfide (NaHS) (a H2S donor, 10–300 μmol/L) attenuated LPS‐stimulated nitric oxide (NO) in a concentration‐dependent manner. Stimulating endogenous H2S production decreased LPS‐stimulated NO production, whereas lowering endogenous H2S level increased basal NO production. Western blot analysis showed that both exogenous and endogenous H2S significantly attenuated the stimulatory effect of LPS on inducible nitric oxide synthase expression, which is mimicked by SB 203580, a specific p38 mitogen‐activated protein kinase (MAPK) inhibitor. Exogenously applied NaHS significantly attenuated LPS‐induced p38 MAPK phosphorylation in BV‐2 microglial cells. Moreover, both NaHS (300 μmol/L) and SB 203580 (1 μmol/L) significantly attenuated LPS‐induced tumor necrosis factor‐α secretion, another inflammatory indicator. In addition, NaHS (10–300 μmol/L) dose‐dependently decreased LPS‐stimulated NO production in primary cultured astrocytes, suggesting that the anti‐neuroinflammatory effect of H2S is not specific to microglial cells alone. Taken together, H2S produced an anti‐inflammatory effect in LPS‐stimulated microglia and astrocytes, which may be due to inhibition of inducible nitric oxide synthase and p38 MAPK signaling pathways. These findings may have important implications in the treatment of neuroinflammation‐related diseases.

Hydrogen Sulfide Is a Mediator of Cerebral Ischemic Damage

Background and Purpose— We observed recently that elevated plasma cysteine levels are associated with poor clinical outcome in acute stroke patients. In a rat stroke model, cysteine administration increased the infarct volume apparently via its conversion to hydrogen sulfide (H2S). We therefore investigated the effects of H2S and the inhibition of its formation on stroke. Methods— Cerebral ischemia was studied in a rat stroke model created by permanent occlusion of the middle cerebral artery (MCAO). The resultant infarct volume was measured 24 hours after occlusion. Results— Administration of sodium hydrosulfide (NaHS, an H2S donor) significantly increased the infarct volume after MCAO. The NaHS-induced increase in infarct volume was abolished by the administration of dizolcilpine maleate (an N-methyl-d-aspartate receptor channel blocker). MCAO caused an increase in H2S level in the lesioned cortex as well as an increase in the H2S synthesizing activity. Administration of 4 different inhibitors of H2S synthesis reduced MCAO-induced infarct volume dose dependently. The potency of these inhibitors in effecting neuroprotection in vivo appeared to parallel their potency as inhibitors of H2S synthesis in vitro. It also appeared that most of the H2S synthesizing activity in the cortex results from the action of cystathionine β-synthase. Conclusions— The present results strongly suggest that H2S plays a part in cerebral ischemic damage after stroke. Inhibition of H2S synthesis should be investigated for its potential as a novel neuroprotective stroke therapy.

Hydrogen Sulfide Inhibits Rotenone-Induced Apoptosis via Preservation of Mitochondrial Function

Hydrogen sulfide (H2S) has been proposed as a novel neuromodulator, which plays critical roles in the central nervous system affecting both neurons and glial cells. However, its relationship with neurodegenerative diseases is unexplored. The present study was undertaken to investigate the effects of H2S on cell injury induced by rotenone, a commonly used toxin in establishing in vivo and in vitro Parkinsons disease (PD) models, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report here that sodium hydrosulfide (NaHS), an H2S donor, concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death. NaHS also prevented rotenone-induced p38- and c-Jun NH2-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) phosphorylation and rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (ΔΨm) dissipation, cytochrome c release, caspase-9/3 activation and poly(ADP-ribose) polymerase cleavage. Furthermore, 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-sensitive potassium (mitoKATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Thus, we demonstrated for the first time that H2S inhibited rotenone-induced cell apoptosis via regulation of mitoKATP channel/p38- and JNK-MAPK pathway. Our data suggest that H2S may have potential therapeutic value for neurodegenerative diseases, such as PD.

Hydrogen sulfide: A novel signaling molecule in the central nervous system

Hydrogen sulfide is an endogenously produced gas in the central nervous system and has been touted as the bodys third gaseous signaling molecule after nitric oxide and carbon monoxide. A review of the current understanding of hydrogen sulfide is necessary in view of the current plethora of research in this area. The aim of this review is to present the current understanding of H(2)S as a signaling molecule in the central nervous system (CNS). This objective is achieved by discussing the involvement of H(2)S in the regulation of (1) intracellular signaling molecules such as protein kinase A, receptor tyrosine kinases, mitogen kinases and oxidative stress signaling, (2) ion channels such as calcium (L-type, T-type and intracellular stores), potassium (K(ATP) and small conductance channels) and cystic fibrosis transmembrane conductance regulator chloride channels, (3) the release and function of neurotransmitters such as gamma-aminobutyric acid, N-methyl-D-aspartate, glutamate and catecholamines. The role of H(2)S as an important mediator in a myriad of neural functions inclusive of neuroprotection is also discussed throughout the review.

Neuroprotection associated with running: is it a result of increased endogenous neurotrophic factors?

The possible neuroprotective effect of physical exercise was investigated in rats after middle cerebral artery occlusion (MCAO), a focal stroke model. It was found that physical exercise in the form of a 12-week treadmill running programme reduced the volume of infarction caused by MCAO. At the molecular level, reverse transcription polymerase chain reaction revealed that the runner had increased gene expression for nerve growth factor (NGF) over the nonrunner with or without MCAO. Expression of the NGF receptors, p75, was increased only in the absence of MCAO. In addition, runners showed a significantly higher number of cholinergic neurons, which constitutively expressed p75, in the horizontal diagonal band of Broca. The present findings suggest that neuroprotection after physical exercise may be a result of an increase in an endogenous neurotrophic factor nerve growth factor and the proliferation of its receptive cholinergic neurons.

Alterations in spatial learning and memory after forced exercise

Exercise has been shown to influence learning and memory. Most studies were performed with a voluntary running paradigm (e.g. running wheel) in mice. However, such effects of exercise on learning and memory are less well demonstrated using a forced running paradigm (e.g. treadmill). The present study was designed to examine the effects of 12 weeks of forced treadmill running on learning and memory performance in rats. We have previously shown that forced running resulted in qualitative and quantitative changes in the cholinergic neurons of the horizontal diagonal band of Broca (HDB) in the septum. This study was conducted in order to determine whether or not these changes occur simultaneously with enhanced learning and memory. The one-day version of the Morris water maze (MWM) test [Frick, K.M., Stillner, E.T., Berger-Sweeney, J., 2000. Mice are not little rats: species differences in a one-day water maze task. NeuroReport 11, 3461-3465] was used to test spatial learning and memory after the exercise period. Our data showed that runners displayed better spatial learning and memory when compared to nonrunners. This was evidently shown by a reduction in the time required for spatial acquisition (p<0.05) and superior probe trial performance (p<0.05). A shorter distance swam by the runners also suggested improved learning over the nonrunners (p<0.05). In an attempt to revalidate our earlier quantitative results, we used design-based stereology (DBS) to estimate the number of cholinergic neuronal profile population in the medial septum and diagonal band (MSDB). We confirmed that forced running increased the cholinergic neuronal profile subpopulation in the HDB (Coefficient of Error<0.2). Taken together, these results indicate that forced exercise could influence learning and memory with a concomitant increase in the number of cholinergic neurons in the HDB.

Hydrogen sulphide regulates calcium homeostasis in microglial cells.

Hydrogen sulphide (H2S), which is produced endogenously from L‐cysteine in mammalian tissues, has been suggested to function as a neuromodulator in the brain. However, the role of H2S in microglial cells is unclear. In this study, the effect of exogenous and endogenous H2S on intracellular calcium homeostasis was investigated in primary cultured microglial cells. Sodium hydrosulphide (NaHS), a H2S donor, caused a concentration‐dependent (0.1–0.5 mM) increase in intracellular calcium concentration ([Ca2+]i). This effect was significantly attenuated in the presence of a calcium‐free extracellular solution, Gd3+ (100 μM), a nonselective Ca2+ channel blocker, or thapsigargin (2 μM), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase. These observations suggest that the increase in [Ca2+]i in response to H2S involves both calcium influx across the plasma membrane and calcium release from intracellular stores. The H2S‐induced calcium elevation is partly attenuated by H‐89, a selective cAMP‐dependent protein kinase (PKA) inhibitor, but not by U73122, a phospholipase C (PLC) inhibitor, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of cAMP/PKA, but not PLC/PKC/phosphoinositol‐3,4,5‐inositol (IP3) pathway. Using RT‐PCR, only cystathionine γ‐lyase (CSE), a H2S producing enzyme, was detected in primary cultures of microglia. Lowering endogenous H2S level with, D,L‐propargylglycine and β‐cyano‐L‐alanine, two CSE inhibitors, significantly decreased [Ca2+]i, suggesting that endogenous H2S may have a positive tonic influence on [Ca2+]i homeostasis. These findings support the possibility that H2S may serve as a neuromodulator to facilitate signaling between neurons and microglial cells.

Upregulation of dihydropyrimidinase-related protein 2, spectrin α II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats- : A proteomics approach

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein90 pathway

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H(2)S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen-glucose deprivation or Na(2)S(2)O(4) (an oxygen scavenger) treatment. We found that treatment with NaHS (an H(2)S donor, 10-100 microM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H(2)S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-beta-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H(2)S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K(ATP)/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H(2)S in the brain during cerebral hypoxia.

A novel analgesic toxin (hannalgesin) from the venom of king cobra (Ophiophagus hannah).

The pharmacological effects of a purified neurotoxin from king cobra (Ophiophagus hannah) venom were studied. Using the hot-plate test, it is shown that this neurotoxin increased latency time dose-dependently when administered i.p. Similar analgesic action was observed when it was administered p.o. or i.c.v. The rota-rod performance, which is a good index for neurological deficits including sedation, muscle relaxant and impairment of motor activity and coordination, was not significantly affected in the dose range of 16-32 ng/g that caused analgesia. The toxin did not increase the convulsion threshold in the dose range of 8-64 ng/g in the maximal electroshock seizure tests. These results demonstrated that this neurotoxin produced analgesia in the dose range of 16-32 ng/g (i.p.) without causing any neurological or muscular deficits. It is further shown that such analgesic action was blocked by naloxone and L-NG-nitro-arginine methyl ester, suggesting the possible involvement of the opioid and nitric oxide systems, respectively. In view of the source of this neurotoxin (O. hannah) and its potent analgesic action, it is proposed that this toxin be named hannalgesin.