Involvement of HIF-1 in the migration-promoting effects of hydrogen sulfide in vascular endothelial cells under normoxic conditions

The purpose of this study was to investigate the molecular mechanisms whereby hydrogen sulfide (H2S) exerts the promoting effect on vascular endothelial cells migration. We used wound healing assay to study the effect of NaHS (H2S donor) on the migration ability of rhesus retinal pigment epithelial cell line, RF/6A cells, under normoxic conditions. Real-time PCR was used to measure hypoxia-inducible factor 1α (HIF-1α) mRNA level. Western blot was used to measure the expression of HIF-1α protein. The probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to measure intracellular reactive oxygen species (ROS) level. The results showed that NaHS (10-100 μmol/L) could significantly promote RF/6A cells migration under normoxic conditions, and this effect could be inhibited by 50 μmol/L HIF-1 inhibitor, CdCl2. NaHS increased the protein level of HIF-1α in a dose- and time-dependent manner, and up-regulated the mRNA level of HIF-1α quickly and continuously. Moreover, NaHS could significantly decrease ROS levels in RF/6A cells under normoxic conditions. These results suggest HIF-1 may mediate the promoting effect of H2S on vascular endothelial cells migration under normoxic conditions. ROS, as an upstream regulator of HIF-1α, may be involved in the migration-promoting effect of H2S.     

Hydrogen sulfide regulates cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells

The present study aims to investigate the regulatory effect of hydrogen sulfide (H(2)S) on cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells. It was found in the present study that NaHS at 0.1-10 μM significantly decreased cAMP production in As4.1 cells treated with isoproterenol (a β-adrenoceptor agonist), forskolin (an adenylyl cyclase activator), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). NaHS at 10 μM suppressed adenylate cyclase activity but stimulated phosphodiesterase activity. We continued to study whether H(2)S may mediate cAMP-dependent renin degranulaion in As4.1 cells. It was found that NaHS at 0.1-10 μM significantly increased intracellular renin protein level. Moreover, NaHS reversed the declined renin content within As4.1 cells and normalized the upregulated renin activity in the culture medium of As4.1 cells treated with the above three stimuli. RT-PCR showed that cystathionine-γ-lyase is the main enzyme to produce endogenous H(2)S in As4.1 cells. Overexpression of cystathionine-γ-lyase increased endogenous H(2)S production and suppressed isoproterenol-induced renin release, suggesting that endogenous H(2)S may also inhibit renin release from As4.1 cells. We also tested whether H(2)S has a similar effect in renin-rich kidney cells. It was found that isoproterenol elevated intracellular cAMP level and extracellular renin activity but decreased renin protein level in the renin-rich kidney cells. Pretreatment with NaHS abolished these effects. In conclusion, H(2)S regulates cAMP homeostasis via inhibition of adenylate cyclase and stimulation of phosphodiesterase. Our findings suggest that H(2)S plays a critical role in regulation of renin degranulation in As4.1 and rat renin-rich kidney cells.     

Nitric oxide synthase inhibition abrogates hydrogen sulfide-induced cardioprotection in mice

The cardioprotective property of hydrogen sulfide (H(2)S) is recently reported. However, cellular signaling cascades mediated by H(2)S are largely unclear. This study was undertaken to explore the molecular mechanism of H(2)S-induced cardioprotection in mouse heart by utilizing in vivo model of cardiac injury. We report here that intraperitoneal administration of sodium hydrogen sulfide (NaHS, 50 μmol kg(-1 )day(-1) for 2 days), a H(2)S donor, significantly (P ≤ 0.05) increased nitric oxide levels in serum as well as myocardium without any sign of myocardial injury. Typical characteristics of myocardial injury induced by isoproterenol (ISO) administration was significantly (P ≤ 0.05) abrogated by NaHS administration as evidenced from reduction in elevated thiobarbituric acid reactive substances (TBARS) and normalization of glutathione (GSH), glutathione peroxidase, superoxide dismutase (SOD), and catalase activity. Further, decrease in TNF-α expression and improvement in myocardial architecture was also observed. However, co-administration of N-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, and Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor along with NaHS and ISO abrogated the beneficial effect of H(2)S differentially. Inhibition of NOS significantly (P ≤ 0.05) increased serum creatine kinase, lactate dehydrogenase, serum glutamic oxaloacetic transaminase activity and myocardial TBARS, along with significant (P ≤ 0.05) reduction of myocardial GSH, SOD, and catalase. This was followed by increase in TNF-α expression and histopathological changes. Our results revealed that H(2)S provides myocardial protection through interaction with NOS and COX-2 pathway and inhibition of NOS completely abrogates the hydrogen sulfide-induced cardioprotection in mice.     

S-diclofenac protects against doxorubicin-induced cardiomyopathy in mice via ameliorating cardiac gap junction remodeling

Hydrogen sulfide (H(2)S), as a novel gaseous mediator, plays important roles in mammalian cardiovascular tissues. In the present study, we investigated the cardioprotective effect of S-diclofenac (2-[(2,6-dichlorophenyl)amino] benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-yl)phenyl ester), a novel H(2)S-releasing derivative of diclofenac, in a murine model of doxorubicin-induced cardiomyopathy. After a single dose injection of doxorubicin (15 mg/kg, i.p.), male C57BL/6J mice were given daily treatment of S-diclofenac (25 and 50 μmol/kg, i.p.), diclofenac (25 and 50 μmol/kg, i.p.), NaHS (50 μmol/kg, i.p.), or same volume of vehicle. The cardioprotective effect of S-diclofenac was observed after 14 days. It showed that S-diclofenac, but not diclofenac, dose-dependently inhibited the doxorubicin-induced downregulation of cardiac gap junction proteins (connexin 43 and connexin 45) and thus reversed the remodeling of gap junctions in hearts. It also dose-dependently suppressed doxorubicin-induced activation of JNK in hearts. Furthermore, S-diclofenac produced a dose-dependent anti-inflammatory and anti-oxidative effect in this model. As a result, S-diclofenac significantly attenuated doxorubicin-related cardiac injury and cardiac dysfunction, and improved the survival rate of mice with doxorubicin-induced cardiomyopathy. These effects of S-diclofenac were mimicked in large part by NaHS. Therefore, we propose that H(2)S released from S-diclofenac in vivo contributes to the protective effect in doxorubicin-induced cardiomyopathy. These data also provide evidence for a critical role of H(2)S in the pathogenesis of doxorubicin-induced cardiomyopathy.     

Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases, including Parkinson's disease. The present study attempted to investigate the effect of hydrogen sulfide (H(2)S) on 6-hydroxydopamine (6-OHDA)-induced ER stress in SH-SY5Y cells. We found in the present study that exogenous application of sodium hydrosulfide (NaHS; an H(2)S donor, 100 μM) significantly attenuated 6-OHDA (50 μM)-induced cell death. NaHS also reversed the upregulation of cleaved poly(ADP-ribose) polymerase and caspase 9 in 6-OHDA-treated cells. Consistent with its cytoprotective effects, NaHS markedly reduced 6-OHDA induced-ER stress responses, including the upregulated levels of eukaryotic initiation factor-2α phosphorylation, glucose-regulated protein 78, and C/EBP homologous protein expression. The protective effect of H(2)S on ER stress was attenuated by blockade of Akt activity with an Akt inhibitor or inhibition of heat shock protein (Hsp)90 with geldanamycin but not by suppression of ERK1/2 with PD-98059. Blockade of Akt also significantly decreased the protein abundance of Hsp90 in SH-SY5Y cells. Moreover, overexpression of cystathionine β-synthase (a main H(2)S-synthesizing enzyme in the brain) elevated the Hsp90 protein level and suppressed 6-OHDA-induced ER stress. In conclusion, the protective effect of H(2)S against 6-OHDA-induced ER stress injury in SH-SY5Y cells involves the Akt-Hsp90 pathway.     

Exogenous hydrogen sulfide delays the senescence of human umbilical vein endothelial cells by lessening oxidative stress

The present study was aimed to investigate the effect of pretreatment with hydrogen sulfide (H2S) on human umbilical vein endothelial cells (HUVECs) senescence and the underlying mechanism. Cultured HUVECs at twelfth and fourth passages were taken as old and young groups, respectively. Sodium hydrosulfide (NaHS, donor of H2S) group was treated with NaHS from fourth to twelfth passage. The cell senescence was determined by senescence-associated β-galactosidase (SA β-gal) staining. DAPI fluorescent dye was used to detect cellular apoptosis. Western blot was used to analyze the expression levels of xanthine oxidase (XOD), manganese-superoxide dismutase (Mn-SOD) and the subunits p67(phox) of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the HUVECs. Colorimetric method was used to detect SOD activity and cellular hydrogen peroxide (H2O2) level. The results showed that, compared with young group, the old group exhibited higher SA β-gal positive rate and cellular apoptosis, while NaHS pretreatment decreased SA β-gal positive rate and cellular apoptosis. Compared with the young group, the old group showed increased expression levels of XOD and p67(phox), as well as lower Mn-SOD expression level. With the pretreatment of NaHS, the up-regulations of XOD and p67(phox) levels and down-regulation of Mn-SOD level were inhibited. Compared with the young group, the old group showed lower SOD activity and higher H2O2 level, whereas NaHS pretreatment reversed the changes of SOD activity and H2O2 level. These results suggest that H2S delays senescence of HUVECs through lessening oxidative stress.     

Hydrogen sulfide inhibits the development of atherosclerosis with suppressing CX3CR1 and CX3CL1 expression

Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H(2)S affects atherosclerosis remain unclear. Therefore, the present study aimed to investigate the potential role of H(2)S in atherosclerosis and the underlying mechanism with respect to chemokines (CCL2, CCL5 and CX3CL1) and chemokine receptors (CCR2, CCR5, and CX3CR1) in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS (50 μM, 100 μM, 200 μM), an H(2)S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H(2)S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H(2)S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE(-/-) mice were fed a high-fat diet and then randomly given NaHS (1 mg/kg, i.p., daily) or DL-propargylglycine (PAG, 10 mg/kg, i.p., daily). NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H(2)S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H(2)S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H(2)S hampers the progression of atherosclerosis in fat-fed apoE(-/-) mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.     

硫化氢对离体豚鼠乳头状肌的电生理效应

应用细胞内微电极技术,观察硫化氢（hydrogen sulfide,H2S）对离体豚鼠乳头状肌细胞的电生理效应。结果表明：（1）NaHS （H,S的供体,50、100、200μmol/L）可浓度依赖地缩短正常乳头状肌的动作电位时程。（2）对部分去极化乳头状肌,NaHS （100μmol／L）除缩短动作电位时程外,还降低动作电位幅值和超射值,减慢零相最大上升速度。（3）预先应用ATP敏感性钾（ATP- sensitive K＋,KATP）通道阻断剂格列苯脲（glibenclamide,Gli,20μmol／L）,可部分阻断NaHS（100μmol／L）的电生理效应。（4）预先应用L型钙通道开放剂Bay K8644（0．5μmol／L）,可部分阻断NaHS（100μmol／L）的电生理效应。（5）预先应用含Gli（20μmol／L）的无钙Krebs-Henseleit液灌流标本,可完全阻断NaHS（100μmol／L）的电生理效应。（6）DL-propargylglycine（PPG,一种胱硫醚-γ-裂解酶的不可逆抑制剂,200μmol／L）可延长正常乳头状肌的动作电位时程。以上结果提示,H,S可能通过兴奋KATP通道促进K＋外流,同时抑制Ca2＋内流,进而影响豚鼠乳头状肌电生理效应。乳头状肌中内源性H2S可能发挥重要的电生理作用。     

A method for controlling hydrogen sulfide in water by adding solid phase oxygen

This work evaluates the addition of solid phase oxygen, a magnesium peroxide (MgO(2)) formulation manufactured by Regenesis (oxygen-releasing compounds, ORC), to inhibit the production of hydrogen sulfide (H(2)S) in an SRB-enriched environment. The initial rate of release of oxygen by the ORC was determined over a short period by adding sodium sulfite (Na(2)SO(3)), which was a novel approach developed for this study. The ability of ORCs to control H(2)S by releasing oxygen was evaluated in a bench-scale column containing cultured sulfate reducing bacteria (SRB). After a series of batch tests, 0.4% ORC was found to be able to inhibit the formation of H(2)S for more than 40 days. In comparison, the concentration of H(2)S dropped from 20 mg S/L to 0.05 mg S/L immediately after 0.1% hydrogen peroxide (H(2)O(2)) was added, but began to recover just four days later. Thus, H(2)O(2) does not seem to be able to inhibit the production of sulfide for an extended period of time. By providing long-term inhibition of the SRB population, ORC provides a good alternative means of controlling the production of H(2)S in water.     

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.     

Hydrogen sulfide protects astrocytes against H(2)O(2)-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.     

The role of nitric oxide signaling in renoprotective effects of hydrogen sulfide against chronic kidney disease in rats: Involvement of oxidative stress,autophagy and apoptosis

The interplay between H₂S and nitric oxide (NO) is thought to contribute to renal functions. The current study was designed to assess the role of NO in mediating the renoprotective effects of hydrogen sulfide in the 5/6 nephrectomy (5/6 Nx) animal model. Forty rats were randomly assigned to 5 experimental groups: (a) Sham; (b) 5/6 Nx; (c) 5/6Nx+sodium hydrosulfide-a donor of H ₂S, (5/6Nx+sodium hydrosulfide [NaHS]); (d) 5/6Nx+NaHS+ L -NAME (a nonspecific nitric oxide synthase [NOS] inhibitor); (e) 5/6Nx+NaHS+aminoguanidine (a selective inhibitor of inducible NOS [iNOS]). Twelve weeks after 5/6 Nx, we assessed the expressions of iNOS and endothelial NOS (eNOS), oxidative/antioxidant status, renal fibrosis, urine N-acetyl-b-glucosaminidase (NAG) activity as the markers of kidney injury and various markers of apoptosis, inflammation, remodeling, and autophagy. NaHS treatment protected the animals against chronic kidney injury as depicted by improved oxidative/antioxidant status, reduced apoptosis, and autophagy and attenuated messenger RNA (mRNA) expression of genes associated with inflammation, remodeling, and NAG activity. Eight weeks Nω-nitro-l-arginine methyl ester ( L -NAME) administration reduced the protective effects of hydrogen sulfide. In contrast, aminoguanidine augmented the beneficial effects of hydrogen sulfide. Our finding revealed some fascinating interactions between NO and H ₂S in the kidney. Moreover, the study suggests that NO, in an isoform-dependent manner, can exert renoprotective effects in 5/6 Nx model of CKD.     

Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat Seedlings

Protective role of hydrogen sulfide (H2S) on seed germination and seedling growth was studied in wheat (Triticum) seeds subjected to aluminum (Al3+) stress. We show that germination and seedling growth of wheat is inhibited by high concentrations of AICI3. At 30 mmol/L AICI3 germination is reduced by about 50% and seedling growth is more dramatically inhibited by this treatment. Pre-incubation of wheat seeds in the H2S donor NaHS alleviates AICI3-induced stress in a dose-dependant manner at an optimal concentration of 0.3 mmol/L. We verified that the role of NaHS in alleviating Al3+ stress could be attributed to H2S/HS- by showing that the level of endogenous H2S increased following NaHS treatment. Furthermore, other sodium salts containing sulfur were ineffective in alleviating Al3+ stress. NaHS pretreatment significantly increased the activities of amylases and esterases and sustained much lower levels of MDA and H2O2 in germinating seeds under Al3+ stress. Moreover, NaHS pretreatment increased the activities of guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase and catalase and decreased that of lipoxygenase. NaHS pretreatment also decreased the uptake of Al3+ in AICI3-treated seed. Taken together these results suggest that H2S could increase antioxidant capability in wheat seeds leading to the alleviation of Al3+ stress.     

NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid is a potent inhibitor of colon cancer cell growth in vitro and in a xenograft mouse model

Nonsteroidal anti-inflammatory drugs (NSAIDs) are prototypical anti-cancer agents. However, their long-term use is associated with adverse gastrointestinal effects. Recognition that endogenous gaseous mediators, nitric oxide (NO) and hydrogen sulfide (H(2)S) can increase mucosal defense mechanisms has led to the development of NO- and H(2)S-releasing NSAIDs with increased safety profiles. Here we report on a new hybrid, NOSH-aspirin, which is an NO- and H(2)S-releasing agent. NOSH-aspirin inhibited HT-29 colon cancer growth with IC(50)s of 45.5 ± 2.5, 19.7 ± 3.3, and 7.7 ± 2.2 nM at 24, 48, and 72 h, respectively. This is the first NSAID based agent with such high degree of potency. NOSH-aspirin inhibited cell proliferation, induced apoptosis, and caused G(0)/G(1) cell cycle block. Reconstitution and structure-activity studies representing a fairly close approximation to the intact molecule showed that NOSH-aspirin was 9000-fold more potent than the sum of its parts towards growth inhibition. NOSH-aspirin inhibited ovine COX-1 more than ovine COX-2. NOSH-ASA treatment of mice bearing a human colon cancer xenograft caused a reduction in volume of 85%. Taken together, these results demonstrate that NOSH-aspirin has strong anti-cancer potential and merits further evaluation.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

Effects of hydrogen sulfide on hypoxic pulmonary vascular structural remodeling

To study the role of hydrogen sulfide (H2S) in hypoxic pulmonary vascular structural remodeling (HPVSR), a total of 24 Wistar rats were randomly divided into three groups: control group (n = 8), hypoxia group (n = 8) and hypoxia with sodium hydrosulfide (hy + NaHS) group (n = 8). The mean pulmonary artery pressure (mPAP), plasma H2S and the percentage of muscularized arteries (MA), partially muscularized arteries (PMA) and nonmuscularized arteries (NMA) in small pulmonary vessels were measured. Collagen I and III, elastin, transforming growth factor-beta3 (TGF-beta3), proliferative cell nuclear antigen (PCNA) and human urotensin II(U-II) expressions were detected by immunohistochemical assay. The mRNA expressions of procollagen I and III, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinease-1 (TIMP-1) were detected by in situ hybridization. The results showed that NaHS significantly increased plasma H2S, decreased mPAP and the percentage of MA and PMA of small pulmonary vessels in rats under hypoxia. Meanwhile, NaHS inhibited the proliferation of pulmonary artery smooth muscle cells (PASMCs) represented by a decrease in the expressions of PCNA and human U-II in pulmonary artery wall. NaHS reduced the expression of collagen I and III, elastin and TGF-beta3 protein and decreased the expressions of procollagen I and III mRNA in pulmonary arteries of rats under hypoxia, but it did not impact the ratio of TIMP-1 mRNA to MMP-1mRNA in pulmonary arteries of rats under hypoxia. These data suggested that H2S played an important role in the development of HPVSR.     

A Novel Hydrogen Sulfide-releasing N-Methyl-D-Aspartate Receptor Antagonist Prevents Ischemic Neuronal Death

Physiological levels of H(2)S exert neuroprotective effects, whereas high concentrations of H(2)S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H(2)S and NMDAR antagonism, we synthesized a novel H(2)S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H(2)S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na(2)S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na(2)S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H(2)S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.     

硫化氢对大鼠心脏成纤维细胞增殖的抑制作用

本文旨在研究硫化氢(hydrogen sulfide,H2S)对大鼠心脏成纤维细胞增殖的抑制作用。以原代培养新生大鼠心脏成纤维细胞(neonatal ratcardiac fibroblasts,NRCFs)为研究对象,用不同浓度血管紧张素II(angiotensin Ⅱ,Ang Ⅱ)或胎牛血清(fetalbovineserum,FBS)刺激NRCFs,建立NRCFs增殖模型。不同浓度硫氢化钠(NaHS,H2S的供体)处理该NRCFs增殖模型后,采用5’-溴-2’-脱氧尿嘧啶(5’-bromo-2’-deoxyuridine,BrdU)掺入法检测NRCFs增殖情况,用2’,7’-二氯荧光素乙酰乙酸盐(2’,7’-di-chlorofluorescein diacetate,DCFH-DA)荧光探针法检测细胞活性氧类(reactive oxygen species,ROS)水平。结果显示,较低浓度的NaHS(1×105mol/L)能促进FBS(2%、10%)对NRCFs的诱导增殖作用,但对Ang Ⅱ(1×107mol/L)所引起的NRCFs增殖的作用不明显,而较高浓度NaHS(5×105、1×104mol/L)...     

Design,synthesis and biological evaluation of novel hydrogen sulfide releasing capsaicin derivatives

Capsaicin (CAP), the prototypical TRPV1 agonist, is the major active component in chili peppers with health-promoting benefits. However, its use is limited by the low bioavailability and irritating quality. In this study, for improving the activity of CAP and alleviating its irritating effects, a series of H₂S-releasing CAPs were designed and synthesized by combining capsaicin and dihydro capsaicin with various hydrogen sulfide donors. The resulting compounds were evaluated their TRPV1 agonist activity, analgesic activity, anticancer activities, H₂S-releasing ability, and gastric mucosa irritation. Biological evaluation indicated that the most active compound B₉, containing 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione moiety as H₂S donor, had better analgesic activity and displayed more potent cytotoxic effects on the test cell lines than the lead compound CAP. Furthermore, the preferred compound, B₉ reduced rat gastric mucosa irritation caused by CAP. Notably, the improved properties of this derivative are associated with its H₂S-releasing capability.