Differential mechanisms underlying neuroprotection of hydrogen sulfide donors against oxidative stress

This study investigated whether slow-releasing organic hydrogen sulfide donors act through the same mechanisms as those of inorganic donors to protect neurons from oxidative stress. By inducing oxidative stress in a neuronal cell line HT22 with glutamate, we investigated the protective mechanisms of the organic donors: ADT-OH [5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione], the most widely used moiety for synthesizing slow-releasing hydrogen sulfide donors, and ADT, a methyl derivative of ADT-OH. The organic donors were more potent than the inorganic donor sodium hydrogensulfide (NaHS) in protecting HT22 cells against glutamate toxicity. Consistent with previous publications, NaHS partially restored glutamate-depleted glutathione (GSH) levels, protected HT22 from direct free radical damage induced by hydrogen peroxide (H₂O₂), and NaHS protection was abolished by a K_ATP channel blocker glibenclamide. However, neither ADT nor ADT-OH enhanced glutamate-depleted GSH levels or protected HT22 from H₂O₂-induced oxidative stress. Glibenclamide, which abolished NaHS neuroprotection against oxidative stress, did not block ADT and ADT-OH neuroprotection against glutamate-induced oxidative stress. Unexpectedly, we found that glutamate induced AMPK activation and that compound C, a well-established AMPK inhibitor, remarkably protected HT22 from glutamate-induced oxidative stress, suggesting that AMPK activation contributed to oxidative glutamate toxicity. Interestingly, all hydrogen sulfide donors, including NaHS, remarkably attenuated glutamate-induced AMPK activation. However, under oxidative glutamate toxicity, compound C only increased the viability of HT22 cells treated with NaHS, but did not further increase ADT and ADT-OH neuroprotection. Thus, suppressing AMPK activation likely contributed to ADT and ADT-OH neuroprotection. In conclusion, hydrogen sulfide donors acted through differential mechanisms to confer neuroprotection against oxidative toxicity and suppressing AMPK activation was a possible mechanism underlying neuroprotection of organic hydrogen sulfide donors against oxidative toxicity.     

Hydrogen-rich water regulates cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner

Hydrogen gas (H₂) is an endogenous gaseous molecule in plants. Although its reputation is as a “biologically inert gas”, recent results suggested that H₂ has therapeutic antioxidant properties in animals and plays fundamental roles in plant responses to environmental stresses. However, whether H₂ regulates root morphological patterns is largely unknown. In this report, hydrogen-rich water (HRW) was used to characterize H₂ physiological roles and possible signaling transduction pathways in the promotion of adventitious root (AR) formation in cucumber explants. Our results showed that a 50% concentration of HRW was able to mimic the effect of hemin, an inducer of a carbon monoxide (CO) synthetic enzyme, and heme oxygenase-1 (HO-1), in restoring AR formation in comparison with the inhibition effect conferred by auxin-depletion treatment alone. It was further shown that the inducible effect of HRW could be further blocked by the co-treatment with N-1-naphthylphtalamic acid (NPA; an auxin transport inhibitor). The HRW-induced response, at least partially, was HO-1-dependent. This conclusion was supported by the fact that the exposure of cucumber explants to HRW up-regulates cucumber HO-1 gene expression and its protein levels. HRW-mediated induction of representative target genes related to auxin signaling and AR formation, such as CsDNAJ-1, CsCDPK1/5, CsCDC6, CsAUX22B-like, and CsAUX22D-like, and thereafter AR formation (particularly in the AR length) was differentially sensitive to the HO-1 inhibitor zinc protoporphyrin IX (ZnPP). Above blocking actions were clearly reversed by CO, further confirming that the above response was HO-1/CO-specific. However, the addition of a well-known antioxidant, ascorbic acid (AsA), failed to influence AR formation triggered by HRW, thus ruling out the involvement of redox homeostasis in this process. Together, these results indicated that HRW-induced adventitious rooting is, at least partially, correlated with the HO-1/CO-mediated responses. We also suggested that exogenous HRW treatment on plants might be a good option to induce root organogenesis.     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

Hydrogen sulfide alleviates uranium-induced kidney cell apoptosis mediated by ER stress via 20S proteasome involving in Akt/GSK-3β/Fyn-Nrf2 signaling

Abstract

Hydrogen sulfide (H₂S) shows antioxidative, anti-inflammatory, antiapoptotic, and cytoprotective effects in kidneys. Recently, H₂S has been reported to alleviate uranium-induced rat nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways. Here, the protective effect and molecular mechanism of H₂S on uranium-induced apoptosis were examined in normal rat kidney proximal cells (NRK-52^E) in vitro. The results indicate that NaHS (an H₂S donor) administration in uranium-intoxicated kidney cells ameliorated uranium-induced reactive oxygen species generation, caspase-3-dependent apoptosis, and endoplasmic reticulum (ER) stress identified through several key markers including GRP78, C/EBP homologous protein (CHOP), and caspase-12. NaHS treatment in uranium-intoxicated kidney cells abolished the effects of uranium on Akt phosphorylation, GSK-3β activation, increased Fyn nuclear expression, and concomitantly decreased Nrf2 nuclear expression. NaHS administration in uranium-treated kidney cells resorted uranium-decreased the expression of two key subunit PSMA6 and PSMB7 in 20S proteasome. But, DRB (an Nrf2 inhibitor) administration abrogated the effects of NaHS on PSMA6 and PSMB7 expression in uranium-contaminated kidney cells. Bortezomib (a proteasome inhibitor) treatment in NaHS pulsing uranium cotreated kidney cells reversed the effects of NaHS on not only PSMA6 and PSMB7 but also GRP78 and CHOP. Taken together, all data suggest that H₂S can attenuate uranium-induced kidney cell apoptosis mediated by ER stress via 20S proteasome involving in Akt/GSK-3β/Fyn-Nrf2 signaling axis.     

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H₂S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H₂S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca_v3.2 T-type Ca²⁺ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca_v3.2. In the present study, we asked if endogenous and exogenous H₂S participates in regulation of the channel functions in Ca_v3.2-transfected HEK293 (Ca_v3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca_v3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca_v3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1–0.3 mM, increased T-currents in Ca_v3.2-HEK293 cells. Similarly, Na₂S, another H₂S donor, at 0.1–0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca_v3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na₂S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na₂S was higher than NaHS. These results suggest that the function of Ca_v3.2 T-channels is tonically enhanced by endogenous H₂S synthesized by CSE in Ca_v3.2-HEK293 cells, and that exogenous H₂S is capable of enhancing Ca_v3.2 function when endogenous H₂S production by CSE is inhibited. In addition, Na₂S is considered a more potent H₂S donor than NaHS in vitro as well as in vivo.     

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H₂S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H₂S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H₂S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H₂S signaling might be further investigated as a novel target for anti-osteoporosis treatment.     

Glutathione,a tripeptide which may function as a temporary storage compound of excessive reduced sulphur in H2S fumigated spinach plants

Summary Exposure of spinach plants to 250 ppb H₂S for two days resulted in a four-fold increase of the reduced glutathione (GSH)/sulphydryl (SH) concentration and in a two-fold increase of the oxidized glutathione (GSSG) concentration of the shoots. Both in the presence and the absence of H₂S, glutathione was predominantly present in the reduced form (more than 86%). When the H₂S exposure was ceased both the levels of GSH and GSSG in the shoot rapidly decreased. There was no emission of H₂S by the leaves after the fumigation was terminated. Glutathione reductase activity in the shoots was not affected by short term H₂S fumigation. It is proposed that glutathione plays the role of a temporary storage compound of excessive reduced sulphur in spinach shoots when exposed to H₂S in the ambient air.     

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

Hydrogen sulfide (H₂S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H₂S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H₂S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.     

外源H_2S对NO_3~-胁迫下番茄幼苗生理生化特性的影响

采用营养液水培方法,通过外源施加H2S供体NaHS(100μmol/L),研究了信号分子H2S对100mmol/L NO3-胁迫下番茄幼苗生理生化特性的影响。结果表明:(1)NO3-胁迫下,随着处理时间的延长,番茄幼苗的株高、根长、鲜重和干重显著降低,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著降低,而胞间CO2浓度以及丙二醛(MDA)、H2O2含量增加,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性显著降低,抗坏血酸(AsA)和还原性谷胱甘肽(GSH)含量显著降低。(2)与NO3-胁迫处理相比,外源NaHS处理1、3、5d后,番茄幼苗的株高、根长、鲜重和干重显著增加,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著升高,而胞间CO2浓度显著降低;MDA和H2O2含量降低,SOD、POD、CAT和APX活性显著增强,AsA和GSH含量显著增加,而且幼苗的硝酸还原酶、谷氨酰胺合成酶、谷氨酸合酶的活性显著增强;L-半胱氨酸脱巯基酶活性和内源H2S含量增加。研究认为,外源H2S可能通过提高抗氧化物酶的活性和增加抗氧化物质含量来缓解NO3-对番茄幼苗造成的伤害,从而增强其对NO3-胁迫耐性。     

Hydrogen sulfide protects SH-SY5Y neuronal cells against d-galactose induced cell injury by suppression of advanced glycation end products formation and oxidative stress

d-Galactose is widely used as an agent to cause aging effects in experimental animals. The present study aims to investigate the effects of hydrogen sulfide (H₂S) in human neuroblastoma SH-SY5Y cells exposed to d-galactose. Cells were pretreated with NaHS, an H₂S donor, and then exposed to d-galactose (25–400 mM for 48 h). We found that NaHS pretreatment significantly reversed the d-galactose-induced cell death and cellular senescence. MTT assay shows that NaHS significantly increased cell viability from 62.31 ± 1.29% to 72.34 ± 0.46% compared with d-galactose (200 mM) treatment group. The underlying mechanism appeared to involve a reduction by NaHS in the formation of advanced glycation end products (AGEs), which are known to contribute to the progression of age-related diseases. In addition, NaHS decreased the elevation of reactive oxygen species from 151.17 ± 2.07% to 124.8 ± 2.89% and malondialdehyde from 1.72 ± 0.07 to 1.10 ± 0.08 (nmol/mg protein) in SH-SY5Y cells after d-galactose exposure. NaHS also stimulated activities of superoxide dismutase from 0.42 ± 0.05 to 0.73 ± 0.04 (U/mg protein) and glutathione peroxidase from 3.98 ± 0.73 to 14.73 ± 0.77 (nmol/min/mg protein) and upregulated the gene expression levels of copper transport protein ATOX1, glutathione synthetase (GSS) and thioredoxin reductase 1 (TXNRD1) while down-regulated aldehyde oxidase 1 (AOX1). In summary, our data indicate that H₂S may have potentially anti-aging effects through the inhibition of AGEs formation and reduction of oxidative stress.     

Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells

Polysulfide is a bound sulfur species derived from endogenous H₂S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells.     

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells

Hydrogen sulfide (H₂S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H₂S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca²⁺ imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H₂S donor, activates cytosolic calcium (Ca_c) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 μM) evoked Ca_c signals also in “normal” human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 μM). Remarkably H₂S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5 mM, 30 min), an inhibitor of the H₂S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca_c signals induced by VEGF (20 ng/ml). We conclude that H₂S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.     

A signaling cascade mediated by ceramide,src and PDGFRβ coordinates the activation of the redox-sensitive neutral sphingomyelinase-2 and sphingosine kinase-1

Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H₂O₂. In mesenchymal cells, H₂O₂ elicits a dose-dependent biphasic effect, mitogenic at low concentration (5 μM), and anti-proliferative and toxic at high concentration (100 μM).     

Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) cells may contribute to the progression of age-related macular degeneration. Thymoquinone (TQ), an active component derived from Nigella sativa, possesses antioxidative effect. However, the role of TQ in RPE cells under oxidative stress condition remains unclear. The present study aimed to examine the protective effect of TQ against hydrogen peroxide (H₂O₂)-induced oxidative stress in human RPE cells. Our results showed that TQ improved the cell viability and apoptosis in H₂O₂-induced ARPE cells. We also found that the levels of reactive oxygen species and malondialdehyde induced by H₂O₂ were reduced after the pretreatment of TQ. In addition, the inhibitory effect of H₂O₂ on the glutathione (GSH) level and superoxide dismutase activity was markedly attenuated by TQ pretreatment. Moreover, TQ enhanced the activation of Nrf2/heme oxygenase 1 (HO-1) signaling pathway in H₂O₂-induced ARPE cells. Knockdown of Nrf2 abolished the protective effect of TQ on H₂O₂-induced oxidative damage. These results suggested that TQ protected ARPE cells from H₂O₂-induced oxidative stress and apoptosis via the Nrf2/HO-1 signaling pathway.