Exogenous H2S Research Articles

Engineering a mitochondria-targeted fluorescent probe with long wavelength for rapid detection of H2S in beer, water and living cells

Hydrogen sulfide (H2S) is involved in the regulatory mechanisms of the human physiological system, it can also have adverse effects on the environment and food, resulting in health hazards. In this study, a novel fluorescent probe HMTI ((E)-2-(4-(diethylamino)-2-hydroxystyryl)-3-methylbenzo[d]thiazol-3-ium iodide)) for the discrimination of H2S was designed and synthesized. HMTI showed fluorescence signal quenching at 622 nm of long wavelength, fast response to H2S (30 s), low limit of detection (LOD = 99.68 nM), excellent ability of specific recognition and anti-interference. In addition, probe HMTI not only could be capable of detecting H2S in environmental water samples and beer, but also untilized for exogenous and endogenous H2S imaging in living cells and effectively targeting mitochondria. Collectively, these results laid the foundation for the successful development of a sensor that could be applicable for monitoring H2S in the environment, food and living cells.

Hydrogen sulfide suppresses H2O2-induced proliferation and migration of HepG2 cells through Wnt/β-catenin signaling pathway.

Both H2S and H2O2 affect many cellular events, such as cell differentiation, cell proliferation and cell death. However, there is some controversy about the roles of H2S and H2O2, since the detailed mechanisms they are involved remain unclear. In this study, low concentration of H2O2 (40μM) increased the viability of hepatocellular carcinoma cells HepG2, while both H2S and high concentration of H2O2 decreased the cell viability in a dose-dependent manner. Wound healing assay indicated that 40μM H2O2 promoted migration of HepG2 cells, which was suppressed by exogenous H2S. Further analysis revealed that administration of exogenous H2S and H2O2 changed the redox status of Wnt3a in HepG2 cells. Altered expression of proteins including Cyclin D1, TCF-4, and MMP7, which are downstream of the Wnt3a/β-catenin signaling pathway, were found after treatment with exogenous H2S and H2O2. Compared with H2S, low concentration of H2O2 showed opposite effects on these protein expression levels in HepG2 cells. These results suggest that H2S suppressed H2O2-induced proliferation and migration of HepG2 through regulating Wnt3a/β-catenin signaling pathway.

Attenuation of Sepsis-Induced Acute Kidney Injury by Exogenous H2S via Inhibition of Ferroptosis.

Sepsis-associated acute kidney injury (SA-AKI) results in significant morbidity and mortality, and ferroptosis may play a role in its pathogenesis. Our aim was to examine the effect of exogenous H2S (GYY4137) on ferroptosis and AKI in in vivo and in vitro models of sepsis and explore the possible mechanism involved. Sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice, which were randomly divided into the sham, CLP, and CLP + GYY4137 group. The indicators of SA-AKI were most prominent at 24 h after CLP, and analysis of the protein expression of ferroptosis indicators showed that ferroptosis was also exacerbated at 24 h after CLP. Moreover, the level of the endogenous H2S synthase CSE (Cystathionine-γ-lyase) and endogenous H2S significantly decreased after CLP. Treatment with GYY4137 reversed or attenuated all these changes. In the in vitro experiments, LPS was used to simulate SA-AKI in mouse renal glomerular endothelial cells (MRGECs). Measurement of ferroptosis-related markers and products of mitochondrial oxidative stress showed that GYY4137 could attenuate ferroptosis and regulate mitochondrial oxidative stress. These findings imply that GYY4137 alleviates SA-AKI by inhibiting ferroptosis triggered by excessive mitochondrial oxidative stress. Thus, GYY4137 may be an effective drug for the clinical treatment of SA-AKI.

Isoenzymatic Pattern of Hydrogen Sulfide (H2S)-Generating L-Cysteine Desulfhydrase (LCD) in Arabidopsis thaliana Seedlings: Effect of Nitric Oxide (NO) and H2S

In higher plants, hydrogen sulfide (H2S) is a recognized signaling molecule that performs multiple regulatory functions. The enzyme L-cysteine desulfhydrase (LCD) catalyzes the conversion of L-cysteine (L-Cys) to pyruvate and ammonium with the concomitant generation of H₂S, and it is considered one of the main sources of H2S in plants. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a specific assay for LCD activity, this study aims to identify the potential LCD isozymes in wild-type Arabidopsis thaliana seedlings of 16 days old grown under in vitro conditions, and to evaluate the potential impact of nitric oxide (NO) and H2S on these LCD isozymes. For this purpose, an Atnoa1 mutant characterized to have a low endogenous NO content as well as the exogenous application of H2S were used. Five LCD isozymes were detected, with LCD IV being the isozyme that has the highest activity. However, the LCD V activity was the only one that was positively modulated in the Atnoa1 mutants and by exogenous H2S. To our knowledge, this is the first report showing the different LCD isozymes present in Arabidopsis seedlings and how their activity is affected by NO and H2S content.

Transcriptomic analysis reveals the functions of H2S as a gasotransmitter independently of Cys in Arabidopsis.

Numerous studies have revealed the gasotransmitter functions of hydrogen sulfide (H2S) in various biological processes. However, the involvement of H2S in sulfur metabolism and/or Cys synthesis makes its role as a signaling molecule ambiguous. The generation of endogenous H2S in plants is closely related to the metabolism of Cys, which play roles in a variety of signaling pathway occurring in various cellular processes. Here, we found that exogenous H2S fumigation and Cys treatment modulated the production rate and content of endogenous H2S and Cys to various degrees. Furthermore, we provided comprehensive transcriptomic analysis to support the gasotransmitter role of H2S besides as a substrate for Cys synthesis. Comparison of the differentially expressed genes (DEGs) between H2S and Cys treated seedlings indicated that H2S fumigation and Cys treatment caused different influences on gene profiles during seedlings development. A total of 261 genes were identified to respond to H2S fumigation, among which 72 genes were co-regulated by Cys treatment. GO and KEGG enrichment analysis of the 189 genes, H2S but not Cys regulated DEGs, indicated that these genes mainly involved in plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid biosynthesis, and MAPK signaling pathway. Most of these genes encoded proteins having DNA binding and transcription factor activities that play roles in a variety of plant developmental and environmental responses. Many stress-responsive genes and some Ca2+ signal associated genes were also included. Consequently, H2S regulated gene expression through its role as a gasotransmitter, rather than just as a substrate for Cys biogenesis, and these 189 genes were far more likely to function in H2S signal transduction independently of Cys. Our data will provide insights for revealing and enriching H2S signaling networks.

Fast and slow releasing sulphide donors engender distinct transcriptomic alterations in Atlantic salmon hepatocytes

Hydrogen sulphide (H2S) is a naturally occurring compound generated either endogenously or exogenously and serves both as a gaseous signalling molecule and an environmental toxicant. Though it has been extensively investigated in mammalian systems, the biological function of H2S in teleost fish is poorly identified. Here we demonstrate how exogenous H2S regulates cellular and molecular processes in Atlantic salmon (Salmo salar) using a primary hepatocyte culture as a model. We employed two forms of sulphide donors: the fast-releasing salt form, sodium hydrosulphide (NaHS) and the slow-releasing organic analogue, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Hepatocytes were exposed to either a low (LD, 20 µg/L) or high (HD, 100 µg/L) dose of the sulphide donors for 24 hrs, and the expression of key sulphide detoxification and antioxidant defence genes were quantified by qPCR. The key sulphide detoxification genes sulfite oxidase 1 (soux) and the sulfide: quinone oxidoreductase 1 and 2 (sqor) paralogs in salmon showed pronounced expression in the liver and likewise responsive to the sulphide donors in the hepatocyte culture. These genes were ubiquitously expressed in different organs of salmon as well. HD-GYY4137 upregulated the expression of antioxidant defence genes, particularly glutathione peroxidase, glutathione reductase and catalase, in the hepatocyte culture. To explore the influence of exposure duration, hepatocytes were exposed to the sulphide donors (i.e., LD versus HD) either transient (1h) or prolonged (24h). Prolonged but not transient exposure significantly reduced hepatocyte viability, and the effects were not dependent on concentration or form. The proliferative potential of the hepatocytes was only affected by prolonged NaHS exposure, and the impact was not concentration dependent. Microarray analysis revealed that GYY4137 caused more substantial transcriptomic changes than NaHS. Moreover, transcriptomic alterations were more marked following prolonged exposure. Genes involved in mitochondrial metabolism were downregulated by the sulphide donors, primarily in NaHS-exposed cells. Both sulphide donors influenced the immune functions of hepatocytes: genes involved in lymphocyte-mediated response were affected by NaHS, whereas inflammatory response was targeted by GYY4137. In summary, the two sulphide donors impacted the cellular and molecular processes of teleost hepatocytes, offering new insights into the mechanisms underlying H2S interactions in fish.

Near-infrared fluorescent probe for visualizing hydrogen sulfide fluctuations during arthritis and its treatment

Arthritis, a common joint disease, has acquired extensive attention from researchers due to its high incidence and still difficult to treat. Hydrogen sulfide, a typical anti-oxidative stress signaling molecule, plays a key role in maintaining redox homeostasis and we therefore speculate that H2S is closely associated with arthritis. In this paper, a novel near-infrared H2S fluorescent probe, NIR-Ar-H2S, was rationally designed and synthesized for visualizing the changes of H2S content in arthritis. The probe possesses many advantages such as near-infrared emission, a large Stokes shift, good optical stability, and low detection limit (0.18 μM), which guarantees the detection of exogenous and endogenous H2S. More importantly, H2S levels were elevated in the arthritis model compared to normal joints, whereas H2S levels decreased after treatment. We expect that this finding will provide valuable guidance for the diagnosis and therapeutic evaluation of arthritis, thereby facilitating arthritis pathology research and drug development.

A selective fluorescent probe for hydrogen sulfide from a series of flavone derivatives and intracellular imaging

In this work, through the orthogonal design of two fluorophores and two recognition groups, a series of fluorescent probes were developed from the flavone derivatives for hydrogen sulfide (H2S). The probe FlaN-DN stood out from the primarily screening on the selectivity and response intensities. It could respond to H2S with both the chromogenic and fluorescent signals. Among the recent reported probes for the H2S detection, FlaN-DN indicated the most highlighted advantages including the rapid response (within 200 s) and the high response multiplication (over 100 folds). FlaN-DN was sensitive to the pH condition, thus could be applied to distinguish the cancer micro-environment. Moreover, FlaN-DN suggested practical capabilities including a wide linear range (0–400 μM), a relatively high sensitivity (limit of detection 0.13 μM), and high selectivity towards H2S. As a low cytotoxic probe, FlaN-DN achieved the imaging in living HeLa cells. FlaN-DN could detect the endogenous generation H2S and visualize the dose-dependent responses to the exogenous H2S level. This work provided a typical case of natural-sourced derivatives as functional implements, which might inspire the future investigations.

Monitoring H2S fluctuation during autophagic fusion of lysosomes and mitochondria using a lysosome-targeting fluorogenic probe

Hydrogen sulfide (H2S) plays a cytoprotective role during mitophagy by detoxifying superfluous reactive oxygen species (ROS), and its concentration fluctuates in this process. However, no work has been reported to reveal the variation in H2S levels during autophagic fusion of lysosomes and mitochondria. Herein, we present a lysosome-targeted fluorogenic probe, named NA-HS, for real-time monitoring of H2S fluctuation for the first time. The newly synthesized probe exhibits good selectivity and high sensitivity (detection limit of 23.6 nM). Fluorescence imaging results demonstrated that NA-HS could image exogenous and endogenous H2S in living cells. Interestingly, the colocalization results revealed that the level of H2S was upregulated after autophagy began because of the cytoprotective effect, and was finally gradually reduced during subsequent autophagic fusion. This work not only affords a powerful fluorescence tool to monitor the variations in H2S levels during mitophagy, but also offers new insights into targeting small molecules for elaborating the complex cellular signal pathways.

S-sulfhydration of SIRT3 combats BMSC senescence and ameliorates osteoporosis via stabilizing heterochromatic and mitochondrial homeostasis

Senescence of bone marrow mesenchymal stem cells (BMSCs) is one of the leading causes of osteoporosis. SIRT3, an essential NAD-dependent histone deacetylase, is highly correlated with BMSC senescence-mediated bone degradation and mitochondrial/heterochromatic disturbance. S-sulfhydration of cysteine residues favorably enhances SIRT3 activity by forming persulfides. Nevertheless, the underlying molecular mechanism of SIRT3 S-sulfhydration on mitochondrial/heterochromatic homeostasis involved in BMSC senescence remains unknown. Here, we demonstrated that CBS and CSE, endogenous hydrogen sulfide synthases, are downregulated with BMSC senescence. Exogenous H2S donor NaHS-mediated SIRT3 augmentation rescued the senescent phenotypes of BMSCs. Conversely, SIRT3 deletion accelerated oxidative stress-induced BMSC senescence through mitochondrial dysfunction and the detachment of the heterochromatic protein H3K9me3 from the nuclear envelope protein Lamin B1. H2S-mediated SIRT3 S-sulfhydration modification rescued the disorganized heterochromatin and fragmented mitochondria induced by the S-sulfhydration inhibitor dithiothreitol, thus leading to elevated osteogenic capacity and preventing BMSC senescence. The antisenescence effect of S-sulfhydration modification on BMSCs was abolished when the CXXC sites of the SIRT3 zinc finger motif were mutated. In vivo, aged mice-derived BMSCs pretreated with NaHS were orthotopically transplanted to the ovariectomy-induced osteoporotic mice, and we proved that SIRT3 ameliorates bone loss by inhibiting BMSC senescence. Overall, our study for the first time indicates a novel role of SIRT3 S-sulfhydration in stabilizing heterochromatin and mitochondrial homeostasis in counteracting BMSC senescence, providing a potential target for the treatment of degenerative bone diseases.

Hydrogen sulfide alleviates salt stress through auxin signaling in Arabidopsis

Hydrogen sulfide (H2S) is a third gas transmitter following nitric oxide and carbon monoxide, that regulates plant growth, photosynthesis, and responses to abiotic stress. Auxin, a crucial plant hormone, is widely involved in stress response and plant growth processes. H2S and auxin interact with each other to regulate root growth. Whether H2S functions in the auxin signaling pathway under salt stress remains unclear. In this study, we used the double mutant of the H2S-producing enzyme-encoding gene L-cysteine desulfhydrase (lcd des1), which found that H2S was involved in alleviating salt stress through auxin signaling in Arabidopsis using physiological and transcriptomic data. RNA-seq analysis identified 267 differentially expressed genes (DEGs) in response to salt stress and revealed that H2S activated salt-related genes and limited cellular activities associated with growth through the regulation of auxin signaling. Furthermore, auxin transport-related mutants (pin347, aux1–7) and auxin signal transduction related mutants (tir1–1, arf10 16) had a significantly inhibited root length under salt stress, and root growth inhibition were alleviated after exogenous H2S treatment. Exogenous H2S treatment, however, did not alleviate inhibition in pin2 with salt treatment. Taken together, these findings suggest that H2S regulates Arabidopsis salt tolerance and inhibits root growth by regulating PIN2 expression.

Modulation of Near-Infrared Mitochondria-Targetable fluorescent probe for H2S bioimaging through the modification of heavy atom iodine

H2S is correlated with mitochondrial dysfunction, which results in the death of cells. Two near-infrared fluorescent probes, Mito-HS-1 and Mito-HS-2, were designed for mitochondrial H2S imaging. Initially, the synthesis protocol of expensive IR-780-based hemicyanine (HXPI) was optimized with an appreciate yield of 80 % as compared with 14–56 % previously reported. Iodine atom was introduced to HXPI to obtain iodine-HXPI whose Stokes shift was increased to be 90 nm. On account of the rapid and fast nucleophilic attack of H2S, HXPI-based Mito-HS-1 could be applied for the real time imaging of mitochondrial H2S. Besides some similar optical properties with Mito-HS-1, iodine-HXPI-based Mito-HS-2 exhibited wider linear range (3–150 μM), more stable fluorescent imaging and more favorable specificity in vitro. Both Mito-HS-1 and Mito-HS-2 could be used to image exogenous H2S in cells, with Mito-HS-2 showing fairly better signal-to-noise. Additionally, the Pearson correlation coefficient of two probes demonstrated that they could successfully monitor mitochondrial H2S in A549 cells and Hela cells.

Differential sensitivity of mucosal organs to transient exposure to hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar)

Mortality related to hydrogen sulphide (H2S) has recently become a serious concern in Atlantic salmon (Salmo salar) farming, particularly in saline recirculating aquaculture systems (RASs), where the risk of H2S formation is high. H2S has a distinct odour of rotten eggs, and its production is associated with the anaerobic bacterial decomposition of protein and other sulphur-containing organic matter. Significant advances have been made in elucidating its formation in RAS, but the biological consequences of this toxicant in salmon remain elusive. We report the physiological consequences of transient exposure of post-smolt Atlantic salmon to H2S. The fish were exposed to one of three levels of H2S for 1 h: 0 μM (unexposed), 0.6 μM (low exposure), and 1.2 μM (high exposure). Fish were allowed to recover for 24 h and then sampled for gene expression, histology, and metabolomics analyses. Molecular profiling was performed on a subset of genes with known functions in sulphide detoxification, mucins, immunity, and stress responses, which focused on the gills, olfactory organ, skin, and distal gut. With the exception of interleukin 10, all genes studied were significantly affected in the skin, where high H2S triggered significant upregulation. Stress-related genes were mostly affected in the gills, where the high H2S level also induced significant upregulation. Downregulation of the marker genes was identified in the olfactory organ especially in the low-dose group. The distal gut was less sensitive to H2S, regardless of the dose. Histological health scoring of the four mucosal organs revealed no substantial structural alterations and only sporadic cases of mild-moderate unspecific tissue damage. High-throughput metabolomics revealed that transient H2S exposure had a substantial mucosal impact rather than a systemic impact, as shown by changes in skin mucus metabolome. Functional annotation indicated that 10 metabolomic pathways were significantly affected in the skin mucus, including tRNA charging, the superpathway of branched-chain amino acid biosynthesis, and glucosilinate biosynthesis from phenylalanine. The physiological alterations following transient exposure to H2S showed that the mucosal organs exhibited distinct response profiles, where transcriptional impacts were more pronounced in the skin and gills. The results contribute to a better understanding of the biological functions of exogenous H2S in teleost fish, as well as the development of mitigation strategies for salmon-production facilities and the risk of H2S exposure.

GYY4137, a H2S donor, ameliorates kidney injuries in diabetic mice by modifying renal ROS-associated enzymes

Diabetic nephropathy (DN) is a common microvascular complication of both type 1 and type 2 diabetes mellitus and often advances to end-stage renal disease. Oxidative stress plays an important role in the pathogenesis and progress of DN. Hydrogen sulfide (H2S) is considered as a promising candidate for the management of DN. But the antioxidant effects of H2S in DN have not been fully studied. In mouse model induced by high-fat diet and streptozotocin, GYY4137, a H2S donor, ameliorated albuminuria at weeks 6 & 8 and decreased serum creatinine at week 8, but not hyperglycemia. Renal nitrotyrosine and urinary 8-isoprostane were reduced along with the suppressed levels of renal laminin and kidney-injury-molecule 1. Renal NADPH oxidase (NOX) 2 was lower but heme oxygenase (HO) 2, paraoxonase (PON) 1, PON2 were higher in DN+GYY than DN group. NOX1, NOX4, HO1, superoxide dismutases 1–3 were similar between groups. Except for a rise at HO2, all the affected enzymes were unchanged in mRNA levels. The affected reactive-oxygen-species (ROS) enzymes were mainly located in the renal sodium-hydrogen-exchanger positive proximal tubules with similar distribution but changed immunofluorence in GYY4137 treated DN mice. Kidney morphological alterations in DN mice under light and electrical-microscopes were also improved by GYY4137. Thus, exogenous H2S administration may improve the renal oxidative damage in DN by reducing ROS production and enhancing ROS cleavage in kidney via the affected enzymes. This study may shed a light on therapeutic applications in diabetic nephropathy with H2S donors in the future.

Hydrogen sulfide alleviates mitochondrial damage and ferroptosis by regulating OPA3–NFS1 axis in doxorubicin-induced cardiotoxicity

Ferroptosis is a major cause of cardiotoxicity induced by doxorubicin (DOX). Previous studies have shown that hydrogen sulfide (H2S) inhibits ferroptosis in cardiomyocytes and myoblasts, but the underlying mechanism has not been fully elucidated. In this study, we investigated the role of H2S in protecting against DOX-induced cardiotoxicity both in vivo and in vitro, and elucidated the potential mechanisms involved. We found that DOX downregulated the expression of glutathione peroxidase 4 (GPX4) and NFS1, and upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression level, resulting in increased lipid peroxidation and ferroptosis. Additionally, DOX inhibited MFN2 expression and increased DRP1 and FIS1 expression, leading to abnormal mitochondrial structure and function. In contrast, exogenous H2S inhibited DOX-induced ferroptosis by restoring GPX4 and NFS1 expression, and reducing lipid peroxidation in H9C2 cells. This effect was similar to that of the ferroptosis antagonist ferrostatin-1 (Fer-1) in protecting against DOX-induced cardiotoxicity. We further demonstrated that the protective effect of H2S was mediated by the key mitochondrial membrane protein optic atrophy 3 (OPA3), which was downregulated by DOX and restored by exogenous H2S. Overexpression of OPA3 alleviated DOX-induced mitochondrial dysfunction and ferroptosis both in vivo and in vitro. Mechanistically, NFS1 has an inhibitory effect on ferroptosis, and NFS1 deficiency increases the susceptibility of cardiomyocytes to ferroptosis. OPA3 is involved in the regulation of ferroptosis by interacting with NFS1. Post-translationally, DOX promoted OPA3 ubiquitination, while exogenous H2S antagonized OPA3 ubiquitination by promoting OPA3 s-sulfhydration. In summary, our findings suggested that H2S protects against DOX-induced cardiotoxicity by inhibiting ferroptosis via targeting the OPA3–NFS1 axis. This provides a potential therapeutic strategy for the treatment of DOX-induced cardiotoxicity.

Exogenous hydrogen sulfide (H2S) exerts therapeutic potential in triple-negative breast cancer by affecting cell cycle and DNA replication pathway

Triple negative breast cancer (TNBC) is a highly aggressive subtype with a poor prognosis due to its high rates of proliferation and metastasis. Recently, hydrogen sulfide (H2S) has been recognized as a novel gasotransmitter that plays a significant role in various pathological processes, including cancer. Here, we show that exogenous H2S inhibited TNBC cancer cell proliferation, migration and invasion in vitro, and also decreased cancer malignances in the mouse model of TNBC. To investigate the underlying mechanisms of H2S's anti-cancer effects in TNBC, we performed transcriptome sequencing and bioinformatic analyses. 2121 differentially expressed genes (DEGs) were revealed, and mainly enriched in cell cycle and DNA replication pathways. Further analysis revealed changes in alternative splicing after exogenous H2S treatment. Protein-protein interaction (PPI) network analysis was performed, which identified 458 interactions among 276 DEGs enriched in cell cycle and DNA replication pathways.We identified seven hub genes (MCM3, MCM4, MCM5, MCM6, CDC6, CDC45, and GINS2) through PPI network analysis, which were up-regulated in clinical human breast cancer but down-regulated after H2S treatment. Based on the hub genes selected, we developed a model predicting that exogenous H2S mainly exerts its anti-TNBC role by delaying DNA replication. Our findings suggest that exogenous H2S has potential as a therapeutic agent in TNBC and may exert its therapeutic potential through DNA replication and the cell cycle pathway.

The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle.

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10-8-10-4M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2M) and exogenous H2S (NaHS; 10-3M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10-2M PAG (~ 40%) and 10-3M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-β-synthase (CBS) inhibitor, respectively. Y-27632 (10-6M) and GF 109203X (10-6M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

A highly sensitive and specific luminescent MOF determines nitric oxide production and quantifies hydrogen sulfide-mediated inhibition of nitric oxide in living cells.

The synthesis of a novel carboxylate-type organic linker-based luminescent MOF (Zn(H2L) (L1)) (named PUC2) (H2L = 2-aminoterephtalic acid, L1 = 1-(3-aminopropyl) imidazole) is reported by the solvothermal method and comprehensively characterized using single-crystal XRD, PXRD, FTIR, TGA, XPS, FESEM, HRTEM, and BET. PUC2 selectively reacts with nitric oxide (▪NO) with a detection limit of 0.08µM, and a quenching constant (0.5 × 104M-1) indicating a strong interaction with ▪NO. PUC2 sensitivity remains unaffected by cellular proteins or biologically relevant metals (Cu2+/ Fe3+/Mg2+/ Na+/K+/Zn2+), RNS/ROS, or H2S to score ▪NO in living cells. Lastly, we used PUC2 to demonstrate that H2S inhibition increases ▪NO production by ~ 14-30% in various living cells while exogenous H2S suppresses ▪NO production, indicating that the modulation of cellular ▪NO production by H2S is rather generic and not restricted to a particular cell type. In conclusion, PUC2 can successfully detect ▪NO production in living cells and environmental samples with considerable potential for its application in improving the understanding of the role of ▪NO in biological samples and study the inter-relationship between ▪NO and H2S.

Gold Nanocluster-Based Ratiometric Probe with Surface Structure Regulation-Triggered Sensing of Hydrogen Sulfide in Living Organisms.

The development of reliable probes for in vivo detection of hydrogen sulfide (H2S) with high sensitivity and selectivity is of great significance due to its key roles in many pathological and physiological processes. Herein, it was found that H2S could finely regulate surface structure of gold nanoclusters (AuNCs) through reduction of surface Au(I)-ligand motifs and further quench their fluorescence by a two-stage kinetic reaction process. Stage I showed the H2S-assisted surface Au(I)-ligand reduction and Au(0) core growth with a rapid fluorescence decrease; stage II showed the surface structure optimization and reconstruction with a relatively slow fluorescence quenching. By virtue of the excellent fluorescence response of AuNCs to H2S, a novel ratiometric fluorescence probe (RBDA) for sensing H2S was designed through electrostatic attraction-induced fluorescence resonance energy transfer (FRET) between AuNCs and rhodamine B. The probe was facilely prepared, showing a straightforward, rapid ratiometric fluorescence response to H2S with built-in self-calibration. It presented the high detection sensitivity with a detection limit (LOD) of 56 nM and an excellent sensing selectivity for H2S over various other biological species. The probe was demonstrated to possess high biostability, low cytotoxicity, good cell and issue penetrability, and favorable biocompatibility. It realizes successful monitoring of both exogenous and endogenous H2S levels in living cells and zebrafish.

Hydrogen sulfide alleviates lipopolysaccharide-induced myocardial injury through TLR4-NLRP3 pathway.

To investigate the effect of hydrogen sulfide (H2S) on myocardial injury in sepsis-induced myocardial dysfunction (SIMD), male C57BL/6 mice were intraperitoneally injected with lipopolysaccharide (LPS) (10 mg/kg, i.p.) to induce cardiac dysfunction without or with the H2S donor sodium hydrosulfide (NaHS) (50 µmol/kg, i.p.) administration 3 h after LPS injection. Six hours after the LPS injection, echocardiography, cardiac hematoxylin and eosin (HE) staining, myocardial damage and inflammatory biomarkers and Western blot results were analyzed. In mice, the administration of LPS decreased left ventricular ejection fraction (LVEF) by 30 % along with lowered H2S levels (35 % reduction). It was observed that cardiac troponin I (cTnI), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) levels were all increased (by 0.22-fold, 2000-fold and 0.66-fold respectively). HE staining revealed structural damage and inflammatory cell infiltration in the myocardial tissue after LPS administration. Moreover, after 6 h of LPS treatment, toll-like receptor 4 (TLR4) and nod-like receptor protein 3 (NLRP3) expressions were up-regulated 2.7-fold and 1.6-fold respectively. When compared to the septic mice, NaHS enhanced ventricular function (by 0.19-fold), decreased cTnI, TNF-alpha, and IL-1beta levels (by 11 %, 33 %, and 16 % respectively) and downregulated TLR4 and NLRP3 expressions (by 64 % and 31 % respectively). Furthermore, NaHS did not further improve cardiac function and inflammation in TLR4-/- mice or mice in which NLRP3 activation was inhibited by MCC950, after LPS injection. In conclusion, these findings imply that decreased endogenous H2S promotes the progression of SIMD, whereas exogenous H2S alleviates SIMD by inhibiting inflammation via the TLR4-NLRP3 pathway suppression.