The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H₂S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H₂S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund''s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO₂⁻), PGE₂, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.     

The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo

The slow-releasing hydrogen sulfide (H₂S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H₂S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H₂S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H₂S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G₂/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H₂S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H₂S donors should be investigated further as potential anti-cancer agents.     

GYY4137 ameliorates sepsis-induced cardiomyopathy via NLRP3 pathway

Sepsis-induced cardiomyopathy (SICM) has a poor prognosis, with no effective therapeutic strategy currently. This study aimed to explore the mechanism underlying SICM and investigate the protective role of the hydrogen sulfide (H₂S) donor GYY4137. This study included patients with SICM and animal models of SICM with wild-type and Nlrp3^?/? mice, which were treated with or without GYY4137. Echocardiography, ELISA, TUNEL staining, and immunofluorescence were used to investigate phenotypic alterations. Serum levels of H₂S and cytokines were measured. Inflammatory cell infiltration in the myocardial tissue was identified using immunohistochemistry and immunofluorescence. RNA expression profiles were identified using RNA sequencing. The protective mechanism of GYY4137 was further validated in the crosstalk between macrophages and cardiomyocytes using immunoblotting, real-time polymerase chain reaction (RT-PCR), and immunofluorescence when conditional medium of macrophages boosted by LPS were co-cultured with cardiomyocytes. Patients and animal models of SICM presented with lower serum H₂S levels and heart dysfunction. GYY4137 reduced macrophage infiltration in septic heart tissue. GO analysis suggested that GYY4137 was involved in the inflammatory process. GYY4137 inhibited NLRP3 inflammasome activity in macrophages, reduced the secretion of inflammatory factors, and decreased the production of reactive oxygen species (ROS) in cardiomyocytes, thus exerting protective effects against SICM. We further found that the protective effects of GYY4137 were absent in Nlrp3-knockout models. GYY4137 ameliorates myocardial injury in SICM via the NLRP3 pathway by inhibiting the inflammatory response and reducing the production of myocardial ROS.     

A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation

Effects of hydrogen sulfide (H₂S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H₂S to tissues. Now new compounds which give a less severe H₂S shock and a more prolonged exposure to H₂S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H₂S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H₂S and to determine how H₂S may fit into plant cell signalling pathways.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

新型硫化氢供体GYY4137对脂多糖诱导的小鼠急性肺损伤的保护作用

目的:急性肺损伤是临床上常见的危重病,发病急,死亡率高,目前仍缺乏有效的治疗手段,新型的外源性硫化氢供体GYY4137具有抗炎、抗休克、抗癌及抗血栓等作用,本研究探讨其对脂多糖诱导的小鼠急性肺损伤的保护作用及其机制。方法:将BALB/c小鼠(18-20 g)随机分为3组:正常对照组(20只),脂多糖组(20只),治疗组(20只),然后复制小鼠脂多糖诱导的急性肺损伤模型:给予小鼠腹腔注射脂多糖(10 mg/kg)复制小鼠急性肺损伤模型模型,治疗组注射脂多糖1小时后给予腹腔注射GYY4137(50 mg/kg),在给予脂多糖8小时后将小鼠处死,留取血清与组织标本。检测小鼠血清中的炎症因子肿瘤坏死因子α、白介素6及白介素10的表达,检测小鼠血清中H2S的含量,测得肺脏湿/干比,检测肺组织中的髓过氧化物酶活性,并测得肺组织中与氧化应激相关的H2O2、·OH与SOD因子的含量。结果:脂多糖引起了严重的肺损伤,GYY4137对脂多糖导致的肺水肿、炎症反应及氧化应激损伤有不同程度的改善,保护了脂多糖造成的肺损伤,降低了脂多糖诱导的小鼠肺脏氧化应激损伤。其保护作用于抗炎、抗氧化有关。结论:GYY4137可能通过抗炎、抗氧化作用途径保护了脂多糖造成的急性肺损伤,可能在炎症疾病模型中也发挥相同作用,并且为未来临床使用缓释硫化氢供体提供了基础资料。     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Hydrogen sulphide may be a novel downstream signal molecule in nitric oxide‐induced heat tolerance of maize (Zea mays L.) seedlings

Nitric oxide (NO) is a second messenger with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. In recent years, hydrogen sulphide (H₂S) has been found to have similar functions, but crosstalk between NO and H₂S in the acquisition of heat tolerance is not clear. In this study, pretreatment with the NO donor sodium nitroprusside (SNP) improved the survival percentage of maize seedlings and alleviated an increase in electrolyte leakage and a decrease in tissue vitality as well as accumulation of malondialdehyde, indicating that pretreatment with SNP improved the heat tolerance of maize seedlings. In addition, pretreatment with SNP enhanced the activity of L‐cystine desulfhydrase, which, in turn, induced accumulation of endogenous H₂S, while application of H₂S donors, NaHS and GYY4137, increased endogenous H₂S content, followed by mitigating increase in electrolyte leakage and enhanced survival percentage of seedlings under heat stress. Interestingly, SNP‐induced heat tolerance was enhanced by application of NaHS and GYY4137, but was eliminated by inhibitors of H₂S synthesis DL‐propargylglycine, aminooxyacetic acid, potassium pyruvate and hydroxylamine, and the H₂S scavenger hypotaurine. All of the above‐mentioned results suggest that SNP pretreatment could improve heat tolerance, and H₂S may be a downstream signal molecule in NO‐induced heat tolerance of maize seedlings.     

GYY4137, a novel hydrogen sulfide-releasing molecule,likely protects against high glucose-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells

Diabetic cardiomyopathy (DCM) has become a major cause of diabetes-related morbidity and mortality. Increasing evidences have proved that hydrogen sulfide (H₂S) fulfills a positive role in regulating diabetic myocardial injury. The present study was designed to determine whether GYY4137, a novel H₂S-releasing molecule, protected H9c2 cells against high glucose (HG)-induced cytotoxicity by activation of the AMPK/mTOR signal pathway. H9c2 cells were incubated in normal glucose (5.5 mM), 22, 33, and 44 mM glucose for 24 h to mimic the hyperglycemia in DCM in vitro. Then we added 50, 100, and 200 μM GYY4137, and measured the cell viability, lactate dehydrogenase (LDH) enzyme activity, and mitochondrial membrane potential (MMP). 0.5 mM 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) and 1 mM adenine 9-β-d-arabinofuranoside (Ara-A, an AMPK inhibitor) were used to identity whether the AMPK/mTOR signal pathway was involved in GYY4137-mediated cardioprotection. We demonstrated that HG decreased cell viability and increased LDH enzyme activity in a concentration-dependent manner. 33 mM HG treatment for 24 h was chosen as our model group for further study. Both 100 and 200 μM GYY4137 treatments significantly attenuated HG-induced cell viability decrement, LDH enzyme activity increase, and MMP collapse. AICAR had similar effects to GYY4137 treatment while Ara-A attenuated GYY4137-mediated cardioprotection. Importantly, both GYY4137 and AICAR increased AMPK phosphorylation and decreased mTOR phosphorylation compared with the HG model group while Ara-A attenuated GYY4137-mediated AMPK phosphorylation increase and mTOR phosphorylation decrement. In conclusion, we propose that GYY4137 likely protects against HG-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells.     

A New Slow Releasing,H2S Generating Compound,GYY4137 Relaxes Spontaneous and Oxytocin-Stimulated Contractions of Human and Rat Pregnant Myometrium

Better tocolytics are required to help prevent preterm labour. The gaseotransmitter Hydrogen sulphide (H₂S) has been shown to reduce myometrial contractility and thus is of potential interest. However previous studies used NaHS, which is toxic and releases H₂S as a non-physiological bolus and thus alternative H₂S donors are sought. GYY4137 has been developed to slowly release H₂S and hence better reflect endogenous physiological release. We have examined its effects on spontaneous and oxytocin-stimulated contractility and compared them to NaHS, in human and rat myometrium, throughout gestation. The effects on contractility in response to GYY4137 (1 nM–1 mM) and NaHS (1 mM) were examined on myometrial strips from, biopsies of women undergoing elective caesarean section or hysterectomy, and from non-pregnant, 14, 18, 22 day (term) gestation or labouring rats. In pregnant rat and human myometrium dose-dependent and significant decreases in spontaneous contractions were seen with increasing concentrations of GYY4137, which also reduced underlying Ca transients. GYY4137 and NaHS significantly reduced oxytocin-stimulated and high-K depolarised contractions as well as spontaneous activity. Their inhibitory effects increased as gestation advanced, but were abruptly reversed in labour. Glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of GYY4137. These data suggest (i) H₂S contributes to uterine quiescence from mid-gestation until labor, (ii) that H₂S affects L-type calcium channels and K_ATP channels reducing Ca entry and thereby myometrial contractions, (iii) add to the evidence that H₂S plays a physiological role in relaxing myometrium, and thus (iv) H₂S is an attractive target for therapeutic manipulation of human myometrial contractility.     

H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal KATP channel

Here we explored the impact of hydrogen sulfide (H₂S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H₂S in the range of 10–275 μM, and H₂S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24 h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H₂S synthesizing enzyme) and ATP-sensitive potassium (K_ATP) channels. The K_ATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the K_ATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H₂S causes the relaxation of human ASM and implicate as well the role for sarcolemmal K_ATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H₂S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.     

Experimental research into the potential therapeutic effect of GYY4137 on Ovariectomy-induced osteoporosis

Background

Evidence has shown that endogenous H₂S plays an important role in the physiological and pathophysiological processes of many organs. The study aimed to explore whether exogenous H₂S has a potential therapeutic effect on a rat ovariectomy-induced model of osteoporosis.

Methods

The OVX osteoporosis model was established in female Sprague-Dawley rats by full bilateral ovariectomy. The rats were randomly divided into four groups, with the two experimental groups receiving an intraperitoneal injection of GYY4137 or sodium alendronate. The level of H₂S in the plasma was determined and common laboratory indicators to diagnose osteoporosis, such as alkaline phosphatase (ALP) activity and the levels of osteocalcin (OCN), calcitonin, parathyroid hormone and leptin were measured. The bone mineral density (BMD) of the 4th and 5th lumbar vertebrae was measured using dual-energy X-ray absorptiometry. The maximum stress of femoral fracture was obtained through a three-point bending test of the femur.

Results

The OVX osteoporosis model was successfully established. GYY4137 was injected to increase the level of H₂S in the plasma in one group, designated OVX-GYY during the observation period (p?<?0.05). At 12 weeks, the BMD value of the fourth lumbar vertebra in the OVX-GYY group had increased (p?<?0.05). The BMD femur value in the OVX-vehicle group had decreased (p?<?0.05). Bilateral ovariectomy leads to biochemical disorders related to bone metabolism and hormone levels in rat plasma (all p?<?0.05). Ovariectomy also reduced blood calcium, blood phosphate and calcitonin, and increased parathyroid hormone and leptin. The opposite results were obtained for the groups with alendronate sodium or GYY4137 treatment (all p?<?0.05).

Conclusions

Through the slow release of H₂S, GYY4137 did an excellent job of simulating endogenous neuroendocrine gaseous signaling molecules. Exogenous H₂S had a regulatory effect on osteoporosis in ovariectomized rats, showing potential value for the treatment of human postmenopausal osteoporosis.

     

Discovery of medium ring thiophosphorus based heterocycles as antiproliferative agents

Hydrogen sulfide (H₂S) has been investigated for its potential in therapy. Recently, we reported novel H₂S donor molecules based on a thiophosphorus core, which slowly release H₂S and have improved anti-proliferative activity in cancer cell lines compared to the most widely studied H₂S donor GYY4137 (1). Herein, we have prepared new thiophosphorus organic H₂S donors with different ring sizes and evaluated them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17, was found to be the most potent with sub-micromolar IC_50s in breast (0.76 μM) and ovarian (0.76 μM) cancer cell lines. No significant H₂S release was detected in aqueous solution for this compound. However, confocal imaging showed that H₂S was released from 17 inside cells at a similar level to the widely studied H₂S donor GYY4137, which was shown to release 10 μM H₂S after 12 h at a concentration of 400 μM. Comparison of 17 with its non-sulfur oxygen analogue, 26, provided evidence that the sulfur atom is important for its potency. However, the significant potency observed for 26 (5.94–11.0 μM) indicates that the high potency of 17 is not entirely due to release of H₂S. Additional mechanism(s) appear to be responsible for the observed activity, hence more detailed studies are required to better understand the role of H₂S in cancer with potent thiophosphorus agents.     

Hydrogen Sulfide Plays a Key Role in the Inhibitory Neurotransmission to the Pig Intravesical Ureter

According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H₂S) role in the neurogenic relaxation of the pig intravesical ureter. We have performed western blot and immunohistochemistry to study the expression of the H₂S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), measurement of enzymatic production of H₂S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A₂ analogue U46619, electrical field stimulation (EFS) and the H₂S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and N^G-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H₂S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K⁺ (K_ATP) channels, transient receptor potential A1 (TRPA₁), transient receptor potential vanilloid 1 (TRPV₁), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031, AMG9810, PACAP_6–38 and CGRP_8–37, respectively. These results suggest that H₂S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via K_ATP channel activation. H₂S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA₁, TRPV₁ and related ion channel activation.     

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea,In Vitro

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H₂S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H₂S-donating moiety; l-cysteine, a substrate for endogenous production of H₂S and GYY 4137, a slow donor of H₂S. We also examined the contribution of prostaglandins to the pharmacological actions of the H₂S donors on release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation. ACS67, l-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [³H]NE release from isolated bovine iris-ciliary bodies without affecting basal [³H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and l-cysteine on stimulated [³H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-β-synthase and glibenclamide, a K_ATP channel blocker reversed the inhibition of evoked NE release induced by the H₂S donors. We conclude that H₂S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H₂S and prostanoids, and is mediated by an action on K_ATP channels.     

Hydrogen Sulfide in Paraventricular Nucleus Enhances Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats

Background

Intracerebroventricular infusion of NaHS, a hydrogen sulfide (H₂S) donor, increased mean arterial pressure (MAP). This study was designed to determine the roles of H₂S in the paraventricular nucleus (PVN) in modulating sympathetic activity and cardiac sympathetic afferent reflex (CSAR) in chronic heart failure (CHF).

Methodology/Principal Findings

CHF was induced by left descending coronary artery ligation in rats. Renal sympathetic nerve activity (RSNA) and MAP were recorded under anesthesia. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of low doses of a H₂S donor, GYY4137 (0.01 and 0.1 nmol), had no significant effects on RSNA, MAP and CSAR. High doses of GYY4137 (1, 2 and 4 nmol) increased baseline RSNA, MAP and heart rate (HR), and enhanced CSAR. The effects were greater in CHF rats than sham-operated rats. A cystathionine-β-synthase (CBS) inhibitor, hydroxylamine (HA) in PVN had no significant effect on the RSNA, MAP and CSAR. CBS activity and H₂S level in the PVN were decreased in CHF rats. No significant difference in CBS level in PVN was found between sham-operated rats and CHF rats. Stimulation of cardiac sympathetic afferents with capsaicin decreased CBS activity and H₂S level in the PVN in both sham-operated rats and CHF rats.

Conclusions

Exogenous H₂S in PVN increases RSNA, MAP and HR, and enhances CSAR. The effects are greater in CHF rats than those in sham-operated rats. Endogenous H₂S in PVN is not responsible for the sympathetic activation and enhanced CSAR in CHF rats.     

Cinnamomum cassia Bark in Two Herbal Formulas Increases Life Span in Caenorhabditis elegans via Insulin Signaling and Stress Response Pathways

Background

Proving the efficacy and corresponding mode of action of herbal supplements is a difficult challenge for evidence-based herbal therapy. A major hurdle is the complexity of herbal preparations, many of which combine multiple herbs, particularly when the combination is assumed to be vitally important to the effectiveness of the herbal therapy. This issue may be addressed through the use of contemporary methodology and validated animal models.

Methods and Principal Findings

In this study, two commonly used traditional herbal formulas, Shi Quan Da Bu Tang (SQDB) and Huo Luo Xiao Ling Dan (HLXL) were evaluated using a survival assay and oxidative stress biomarkers in a well-established C. elegans model of aging. HLXL is an eleven herb formula modified from a top-selling traditional herbal formula for the treatment of arthritic joint pain. SQDB consists of ten herbs often used for fatigue and energy, particularly in the aged. We demonstrate here that SQDB significantly extend life span in a C. elegans model of aging. Among all individual herbs tested, two herbs Cinnamomum cassia bark (Chinese pharmaceutical name: Cinnamomi Cortex, CIN) and Panax ginseng root (Chinese pharmaceutical name: Ginseng Radix, GS) significantly extended life span in C. elegans. CIN in both SQDB and HLXL formula extended life span via modulation of multiple longevity assurance genes, including genes involved in insulin signaling and stress response pathways. All the life-span-extending herbs (SQDB, CIN and GS) also attenuated levels of H₂O₂ and enhanced small heat shock protein expression. Furthermore, the life span-extending herbs significantly delayed human amyloid beta (Aβ)-induced toxicity in transgenic C. elegans expressing human Aβ.

Conclusion/Significance

These results validate an invertebrate model for rapid, systematic evaluation of commonly used Chinese herbal formulations and may provide insight for designing future evidence-based herbal therapy(s).     

Selenium Inhibits Root Elongation by Repressing the Generation of Endogenous Hydrogen Sulfide in Brassica rapa

Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H₂S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H₂S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H₂S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H₂S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H₂S by the application of H₂S donor NaHS could significantly alleviate Se(IV)-induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H₂S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H₂S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H₂S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H₂S in modulating Se(IV)-induced phytotoxicity in roots.     

Hydrogen sulphide reduces hyperhomocysteinaemia-induced endothelial ER stress by sulfhydrating protein disulphide isomerase to attenuate atherosclerosis

Hyperhomocysteinaemia (HHcy)-impaired endothelial dysfunction including endoplasmic reticulum (ER) stress plays a crucial role in atherogenesis. Hydrogen sulphide (H₂S), a metabolic production of Hcy and gasotransmitter, exhibits preventing cardiovascular damages induced by HHcy by reducing ER stress, but the underlying mechanism is unclear. Here, we made an atherosclerosis with HHcy mice model by ApoE knockout mice and feeding Pagien diet and drinking L-methionine water. H₂S donors NaHS and GYY4137 treatment lowered plaque area and ER stress in this model. Protein disulphide isomerase (PDI), a modulation protein folding key enzyme, was up-regulated in plaque and reduced by H₂S treatment. In cultured human aortic endothelial cells, Hcy dose and time dependently elevated PDI expression, but inhibited its activity, and which were rescued by H₂S. H₂S and its endogenous generation key enzyme-cystathionine γ lyase induced a new post-translational modification-sulfhydration of PDI. Sulfhydrated PDI enhanced its activity, and two cysteine-terminal CXXC domain of PDI was identified by site mutation. HHcy lowered PDI sulfhydration association ER stress, and H₂S rescued it but this effect was blocked by cysteine site mutation. Conclusively, we demonstrated that H₂S sulfhydrated PDI and enhanced its activity, reducing HHcy-induced endothelial ER stress to attenuate atherosclerosis development.