Li-Fang Hu

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.

Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN). The present study was designed to examine the therapeutic effect of hydrogen sulfide (H2S, a novel biological gas) on PD. The endogenous H2S level was markedly reduced in the SN in a 6‐hydroxydopamine (6‐OHDA)‐induced PD rat model. Systemic administration of NaHS (an H2S donor) dramatically reversed the progression of movement dysfunction, loss of tyrosine‐hydroxylase positive neurons in the SN and the elevated malondialdehyde level in injured striatum in the 6‐OHDA‐induced PD model. H2S specifically inhibited 6‐OHDA evoked NADPH oxidase activation and oxygen consumption. Similarly, administration of NaHS also prevented the development of PD induced by rotenone. NaHS treatment inhibited microglial activation in the SN and accumulation of pro‐inflammatory factors (e.g. TNF‐α and nitric oxide) in the striatum via NF‐κB pathway. Moreover, significantly less neurotoxicity was found in neurons treated with the conditioned medium from microglia incubated with both NaHS and rotenone compared to that with rotenone only, suggesting that the therapeutic effect of NaHS was, at least partially, secondary to its suppression of microglial activation. In summary, we demonstrate for the first time that H2S may serve as a neuroprotectant to treat and prevent neurotoxin‐induced neurodegeneration via multiple mechanisms including anti‐oxidative stress, anti‐inflammation and metabolic inhibition and therefore has potential therapeutic value for treatment of PD.

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 protects astrocytes against H2O2-induced neural injury via enhancing glutamate uptake

Excess extracellular glutamate, the main excitatory neurotransmitter, may result in excitotoxicity and neural injury. The present study was designed to study the effect of hydrogen sulfide (H(2)S), a novel neuromodulator, on hydrogen peroxide (H(2)O(2)) -induced glutamate uptake impairment and cellular injuries in primary cultured rat cortical astrocytes. We found that NaHS (an H(2)S donor, 0.1-1000 microM) reversed H(2)O(2)-induced cellular injury in a concentration-dependent manner. This effect was attenuated by L-trans-pyrrolidine-2,4-dicarboxylic (PDC), a specific glutamate uptake inhibitor. Moreover, NaHS significantly increased [(3)H]glutamate transport in astrocytes treated with H(2)O(2), suggesting that H(2)S may protect astrocytes via enhancing glutamate uptake function. NaHS also reversed H(2)O(2)-impaired glutathione (GSH) production. Blockade of glutamate uptake with PDC attenuated this effect, indicating that the effect of H(2)S on GSH production is secondary to the stimulation of glutamate uptake. In addition, it was also found that H(2)S may promote glutamate uptake activity via decreasing ROS generation, enhancing ATP production and suppressing ERK1/2 activation. In conclusion, our findings provide direct evidence that H(2)S has potential therapeutic value for oxidative stress-induced brain damage via a mechanism involving enhancing glutamate uptake function.

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.

Hydrogen sulfide protects MC3T3-E1 osteoblastic cells against H2O2-induced oxidative damage—implications for the treatment of osteoporosis

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative damage is an important contributor to the morphological and functional changes in the development of osteoporosis. We found in this study that hydrogen sulfide (H2S), a novel endogenous gaseous mediator, protected MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative injury. NaHS, an H2S donor, increased cell viability and reduced cell apoptosis caused by H2O2. NaHS also stimulated osteoblast proliferation by enhancing both transcription and activity of alkaline phosphatase in MC3T3-E1 osteoblastic cells. Moreover, treatment with NaHS stimulated the transcriptional level of osteocalcin, the main bone matrix protein, and the protein expression of collagen, a major constituent of bone tissue. The above effects were mediated by the antioxidant effect of H2S. NaHS reversed the reduced superoxide dismutase activity, decreased reactive oxygen species production, and suppressed NADPH oxidase activity in H2O2-treated osteoblasts. In addition, NaHS treatment also produced anti-inflammatory effects via inhibition of the production of nitric oxide and TNF-α, suggesting an anti-inflammatory effect of H2S. Cell viability and Western blotting analysis demonstrated that the protective effects of H2S were mediated by p38 and ERK1/2 MAPKs. In conclusion, H2S protects osteoblastic cells against oxidative stress-induced cell injury and suppression of proliferation and differentiation via a MAPK (p38 and ERK1/2)-dependent mechanism. Our findings suggest that H2S may have a potentially therapeutic value for osteoporosis.

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.

Hydrogen sulfide interacts with nitric oxide in the heart -Possible involvement of nitroxyl

AIMS The present study aims to investigate the interaction between nitric oxide (NO) and hydrogen sulfide (H(2)S), the two important gaseous mediators in rat hearts. METHODS AND RESULTS Intracellular calcium in isolated cardiomyocytes was measured with a spectrofluorometric method using Fura-2. Myocyte contractility was measured with a video edge system. NaHS (50 µM, an H(2)S donor) had no significant effect on the resting calcium level, electrically induced (EI) calcium transients, and cell contractility in ventricular myocytes. Stimulating endogenous NO production with l-arginine or exogenous application of NO donors [sodium nitroprusside (SNP) and 2-(N,N-diethylamino)-diazenolate-2-oxide] decreased myocyte twitch amplitudes accompanied by slower velocities of both cell contraction and relaxation. Surprisingly, NaHS reversed the negative inotropic and lusitropic effects of the above three NO-increasing agents. In addition, the mixture of SNP + NaHS increased, whereas SNP alone decreased, the resting calcium level and the amplitudes of EI calcium transients. Angelis salt, a nitroxyl anion (HNO) donor, mimicked the effect of SNP + NaHS on calcium handling and myocyte contractility. Three thiols, N-acetyl-cysteine, l-cysteine, and glutathione, abolished the effects of HNO and SNP + NaHS on myocyte contraction. Neither Rp-cAMP [a protein kinase A (PKA) inhibitor] nor Rp-cGMP [a protein kinase G (PKG) inhibitor] affected the effects of SNP + NaHS, suggesting a cAMP/PKA- or cGMP/PKG-independent mechanism. CONCLUSION H(2)S may interact with NO to form a thiol sensitive molecule (probably HNO) which produces positive inotropic and lusitropic effects. Our findings may shed light on the interaction of NO and H(2)S and provide new clues to treat cardiovascular diseases.

Hydrogen Sulfide Regulates Na+/H+ Exchanger Activity via Stimulation of Phosphoinositide 3-Kinase/Akt and Protein Kinase G Pathways

Intracellular pH (pHi) is an important endogenous modulator of cardiac function. Inhibition of Na+/H+ exchanger-1 (NHE-1) protects the heart by preventing Ca2+ overload during ischemia/reperfusion. Hydrogen sulfide (H2S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H2S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 μM, produced sustained decreases in pHi in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl4 prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) significantly attenuated NaHS-suppressed NHE-1 activity and/or NaHS-induced cardioprotection. Although KT5823 failed to affect NaHS-induced Akt phosphorylation, Akt inhibitor did attenuate NaHS-stimulated PKG activity. In conclusion, this work demonstrated for the first time that H2S produced cardioprotection via the suppression of NHE-1 activity involving a PI3K/Akt/PKG-dependent mechanism.