Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Administration of metformin alleviates atherosclerosis by promoting H2S production via regulating CSE expression.

The therapeutic effect of metformin (Met) on atherosclerosis was studied here. Effects of methionine and Met on the induction of inflammatory response and H2 S expression in peritoneal macrophages were evaluated. Enzyme-linked immunosorbent assay, immunohistochemistry assay, western blot, and quantitative reverse transcription polymerase chain reaction were conducted to observe the levels of cystathionine γ-lyase (CSE), DNA methyltransferases 1 (DNMT1), DNMT3a, DNMT3b, tumor necrosis factor (TNF- α), interleukin 1b (IL-1ß), and hydrogen sulfide (H 2 S). Luciferase and bisulfite sequencing assays were also utilized to evaluate the CSE promoter activity as well as the methylation status of CSE in transfected cells. Methionine significantly elevated Hcy, TNF-a, H 2 S, and IL-1ß expression while decreasing the level of CSE in C57BL/6 mice. In contrary, co-treatment with Methionine and Met reduced the detrimental effect of Methionine. Homocysteine (Hcy) decreased H 2 S expression while promoting the synthesis of IL-1ß and TNF-α in THP-1 and raw264.7 cells. Treatment of THP-1 and raw264.7 cells with methionine and Met reduced the activity of methionine in dose dependently. Moreover, Hcy increased the expression of DNMT and elevated the level of methylation in the CSE promoter, whereas the co-treatment with methionine and Met attenuated the effects of Hcy. Methionine significantly decreased plasma level of CSE while increasing the severity of inflammatory responses and plasma level of Hcy, which in turn suppressed H 2 S synthesis and enhanced DNA hypermethylation of CSE promoter to promote the pathogenesis of atherosclerosis. In contrary, co-treatment with methionine and Met reduced the detrimental effect of methionine.

Gasotransmitters in pregnancy: from conception to uterine involution.

Gasotransmitters are endogenous small gaseous messengers exemplified by nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S or sulfide). Gasotransmitters are implicated in myriad physiologic functions including many aspects of reproduction. Our objective was to comprehensively review basic mechanisms and functions of gasotransmitters during pregnancy from conception to uterine involution and highlight future research opportunities. We searched PubMed and Web of Science databases using combinations of keywords nitric oxide, carbon monoxide, sulfide, placenta, uterus, labor, and pregnancy. We included English language publications on human and animal studies from any date through August 2018 and retained basic and translational articles with relevant original findings. All gasotransmitters activate cGMP signaling. NO and sulfide also covalently modify target protein cysteines. Protein kinases and ion channels transduce gasotransmitter signals, and co-expressed gasotransmitters can be synergistic or antagonistic depending on cell type. Gasotransmitters influence tubal transit, placentation, cervical remodeling, and myometrial contractility. NO, CO, and sulfide dilate resistance vessels, suppress inflammation, and relax myometrium to promote uterine quiescence and normal placentation. Cervical remodeling and rupture of fetal membranes coincide with enhanced oxidation and altered gasotransmitter metabolism. Mechanisms mediating cellular and organismal changes in pregnancy due to gasotransmitters are largely unknown. Altered gasotransmitter signaling has been reported for preeclampsia, intrauterine growth restriction, premature rupture of membranes, and preterm labor. However, in most cases specific molecular changes are not yet characterized. Nonclassical signaling pathways and the crosstalk among gasotransmitters are emerging investigation topics.

Comparative localization of cystathionine beta synthases and cystathionine gamma lyase in canine, non-human primate and human retina.

Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H2S) and glutathione (GSH) may play an important role in ameliorating disease progression. H2S production is driven by cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H2S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primate (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and Mϋller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser capture microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H2S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.

Distinctive expression pattern of cystathionine-ß-synthase and cystathionine-γ-lyase identifies mesenchymal stromal cells transition to mineralizing osteoblasts.

Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up-regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up-regulated while CSE expression was not affected. Ex-vivo findings confirmed that mature h-osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation.

Korean red ginseng ameliorated experimental pancreatitis through the inhibition of hydrogen sulfide in mice.

AIM: Effective therapy to treat acute pancreatitis (AP) or to prevent its recurrence/complication is still not available. Based on previous results that suggest that: i) hydrogen sulfide (H2S) levels were significantly increased in pancreatitis and gastritis and ii) Korean red ginseng (KRG) efficiently attenuated Helicobacter pylori-associated gastritis through the suppressive actions of H2S, we hypothesized that KRG can ameliorate experimental pancreatitis through suppression of H2S generation. METHODS: C57BL/6 mice were pre-administered KRG and then subjected to cerulein injection or pancreatic duct ligation (PDL) to induce pancreatitis. Blood and pancreas tissues were collected and processed to measure serum levels of amylase, lipase and myeloperoxidase and the concentration of H2S and the levels of various inflammatory cytokine in pancreatic tissues of mice with induced AP. RESULTS: KRG significantly inhibited NaHS-induced COX-2 and TNF-α mRNA in pancreatic cells, but dl-propargylglycine did not. KRG ameliorated cerulein-induced edematous pancreatitis accompanied with significant inactivation of NF-κB and JNK in pancreatic tissues of C57BL/6 mice (p < 0.001) and also significantly ameliorated PDL-induced necrotizing pancreatitis (p<0.01); in both conditions, the significant suppression of H2S resulting from KRG pretreatment afforded rescuing outcomes. Along with suppressed levels of H2S consequent to depressed expressions of CBS and CSE mRNA, KRG administration efficiently decreased the serum level of amylase, lipase, and myeloperoxidase and the expression of inflammatory cytokines in animal models of mild or severe AP. CONCLUSIONS: These results provide evidence for the preventive and therapeutic roles of KRG against AP mediated by H2S suppression.

Urotensin II Protects Cardiomyocytes from Apoptosis Induced by Oxidative Stress through the CSE/H2S Pathway.

Plasma urotensin II (UII) has been observed to be raised in patients with acute myocardial infarction; suggesting a possible cardiac protective role for this peptide. However, the molecular mechanism is unclear. Here, we treated cultured cardiomyocytes with H2O2 to induce oxidative stress; observed the effect of UII on H2O2-induced apoptosis and explored potential mechanisms. UII pretreatment significantly reduced the number of apoptotic cardiomyocytes induced by H2O2; and it partly abolished the increase of pro-apoptotic protein Bax and the decrease of anti-apoptotic protein Bcl-2 in cardiomyocytes induced by H2O2. SiRNA targeted to the urotensin II receptor (UT) greatly inhibited these effects. Further analysis revealed that UII increased the production of hydrogen sulfide (H2S) and the level of cystathionine-γ-lyase (CSE) by activating the ERK signaling in H2O2-treated-cardiomyocytes. Si-CSE or ERK inhibitor not only greatly inhibited the increase in CSE level or the phosphorylation of ERK induced by UII but also reversed anti-apoptosis of UII in H2O2-treated-cadiomyocytes. In conclusion, UII rapidly promoted the phosphorylation of ERK and upregulated CSE level and H2S production, which in turn activated ERK signaling to protect cardiomyocytes from apoptosis under oxidative stress. These results suggest that increased plasma UII level may protect cardiomyocytes at the early-phase of acute myocardial infarction in patients.

Pharmacological Inhibition of Endogenous Hydrogen Sulfide Production Slows Bladder Cancer Progression in an Intravesical Murine Model.

Present bladder cancer therapies have relatively limited therapeutic impact and account for one of the highest lifetime treatment costs per patient. Therefore, there is an urgent need to explore novel and optimized treatment strategies. The present study investigated the effects of inhibiting endogenous hydrogen sulfide (H2S) production on bladder cell viability and in vivo tumor progression. We targeted the H2S-producing enzyme, cystathionine γ-lyase, in 5637 cells using propargylglycine (H2S inhibitor) and performed cytofluorimetric analysis to evaluate cell viability. We then tested the efficacy of propargylglycine alone or in combination with gemcitabine (conventional chemotherapy) in an intravesical murine model of bladder cancer. Magnetic resonance imaging and immunohistochemical staining for cell proliferation, apoptosis, immune-cell infiltration, and neovascularization were performed to evaluate tumor response. Compared to control conditions or cohorts, propargylglycine administration significantly attenuated bladder cancer cell viability in vitro (p < 0.0001) and tumor growth (p < 0.002) and invasion in vivo. Furthermore, propargylglycine enhanced the anti-cancer effects of gemcitabine, resulting in tumor regression (p < 0.0001). Moreover, propargylglycine induced cleaved PARP-1-activated apoptosis (p < 0.05), as well as intratumoral CD8+ T cell (p < 0.05) and F4/80+ macrophage (p < 0.002) infiltration. Propargylglycine also reduced intratumoral neovascularization (p < 0.0001) and cell proliferation (p < 0.0002). Importantly, the pro-apoptotic and anti-neovascularization effects of gemcitabine were enhanced by propargylglycine co-administration. Our findings suggest that inhibition of endogenous H2S production can be protective against bladder cancer by enhancing the chemotherapeutic action of gemcitabine and may be a novel pharmacological target and approach for improved bladder cancer diagnosis and treatments in the future.

H2S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine.

Lung cancer is a lethal disease with no truly efficient therapeutic management despite the progresses, and metabolic profiling can be a way of stratifying patients who may benefit from new therapies. The present study is dedicated to profiling cysteine metabolic pathways in NSCLC cell lines and tumor samples. This was carried out by analyzing hydrogen sulfide (H2S) and ATP levels, examining mRNA and protein expression patterns of cysteine catabolic enzymes and transporters, and conducting metabolomics analysis using nuclear magnetic resonance (NMR) spectroscopy. Selenium-chrysin (SeChry) was tested as a therapeutic alternative with the aim of having an effect on cysteine catabolism and showed promising results. NSCLC cell lines presented different cysteine metabolic patterns, with A549 and H292 presenting a higher reliance on cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) to maintain H2S levels, while the PC-9 cell line presented an adaptive behavior based on the use of mercaptopyruvate sulfurtransferase (MST) and cysteine dioxygenase (CDO1), both contributing to the role of cysteine as a pyruvate source. The analyses of human lung tumor samples corroborated this variability in profiles, meaning that the expression of certain genes may be informative in defining prognosis and new targets. Heterogeneity points out individual profiles, and the identification of new targets among metabolic players is a step forward in cancer management toward personalized medicine.

Differential effects of subchronic acrylonitrile exposure on hydrogen sulfide levels in rat blood, brain, and liver.

Background: Hydrogen sulfide (H2S), as the third gasotransmitter participates in both cellular physiological and pathological processes, including chemical-induced injuries. We recently reported acute acrylonitrile (AN) treatment inhibited endogenous H2S biosynthesis pathway in rat and astrocyte models. However, there is still no evidence to address the correlation between endogenous H2S and sub-chronic AN exposure. Objectives: This study aims to explore the modulatory effects of prolonged AN exposure on endogenous H2S levels and its biosynthetic enzymes in rat blood, brain and liver. Methods: A total of 50 male Sprague-Dawley rats were randomly divided into 5 groups, including the control group and AN-treated groups at dosages of 6.25, 12.5, 25 or 50 mg/kg. Rats received one exposure/day, 5 days/week, for 4 consecutive weeks. The rat bodyweight and brain/liver organ coefficient were detected, along with liver cytochrome P450 2E1(CYP2E1) expression. In addition, the H2S contents in rat serum and plasma, and in cerebral cortex and liver tissues were measured by methylene blue method. The expression of H2S-generating enzymes, including cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MPST) was also measured with Western blot both in rat cerebral cortex and liver. Results: Subchronic exposure to AN significantly inhibited bodyweight-gain and increased the liver CYP2E1 expression compared with the control. In addition, AN significantly increased H2S levels in rat plasma and serum, but not in liver. The endogenous H2S level in rat cerebral cortex was also significantly increased upon AN treatment, when expression of the major H2S-generating enzymes, CBS and 3-MPST were significantly enhanced. However, hepatic protein levels of CBS and CSE were significantly increased, whereas hepatic levels of 3-MPST were significantly decreased. Conclusion: This study showed that sub-chronic AN exposure increased endogenous H2S contents in rat blood and brain tissues, but not liver, which may be resulted from the distinct expression profile of H2S-producing enzymes in response to AN. The blood H2S contents may be applied as a potential novel biomarker for surveillance of chronically AN-exposed populations. Highlights: Subchronic intraperitoneal exposure to acrylonitrile increased H2S content in rat blood and cerebral cortex, but not in liver.Distinct tissue expression profiles of H2S-producing enzymes contribute to the acrylonitrile-induced differential effects on the H2S level.Blood H2S level may be a biomarker for subchronic exposure to acrylonitrile.

Activation of renal CSE/H2S pathway by alpha-lipoic acid protects against histological and functional changes in the diabetic kidney.

INTRODUCTION: We previously reported that alpha-lipoic acid (ALA) supplementation protects against progression of diabetic kidney disease (DKD). In this study, we aim to investigate whether the mechanism of renal protection by ALA involves renal cystathionine γ-lyase/hydrogen sulfide (CSE/H2S) system in type 2 diabetes mellitus (T2DM). METHODS: Thirty-seven male Sprague-Dawley rats underwent 12 h of overnight fasting. To induce T2DM, 30 of these rats received intraperitoneal administration of nicotinamide (110 mg/kg) and streptozotocin (55 mg/kg). T2DM rats then received either oral administration of ALA (60 mg/kg/day) or intraperitoneal administration of 40 mg/kg/day DL-propargylglycine (PAG, a CSE inhibitor) or both for 6 weeks after which rats were sacrificed and samples collected for analysis. Untreated diabetic and non-diabetic rats served as diabetic and healthy controls respectively. RESULTS: T2DM was characterized by reduced pancreatic ß-cell function and hyperglycemia. Histologically, untreated diabetic rats showed significantly damaged pancreatic islets, glomerular and tubular injury, with elevated levels of renal function markers compared to healthy control rats (p < 0.001). These pathological changes worsened significantly following PAG administration (p < 0.05). While some renal protection was observed in ALA+PAG rats, ALA administration in untreated diabetic rats provided superior protection comparable to healthy control rats, with improved antioxidant status, lipid profile and reduced inflammation. Mechanistically, ALA significantly activated renal CSE/H2S system in diabetic rats, which was markedly suppressed in PAG-treated rats (p < 0.001). CONCLUSION: Our data suggest that ALA protects against DKD development and progression by activating renal CSE/H2S pathway. Hence, CSE/H2S pathway may represent a therapeutic target in the treatment or prevention of DKD in diabetic patients.

Reducing Inflammation and Vascular Invasion in Intervertebral Disc Degeneration via Cystathionine-γ-Lyase Inhibitory Effect on E-Selectin.

The incidence of degenerative spinal diseases, such as cervical spondylosis and thoracic and lumbar disc herniation, is increasing. These health problems have adversely affected human life and work. Surgical intervention is effective when intervertebral disc degeneration (IDD) causes nerve compression and/or severely limits daily activity. Early IDD patients generally do not require surgery. However, there is no effective method of impeding IDD progression. Thus, novel approaches to alleviating IDD deterioration are urgently required. Cystathionine-γ-lyase (CSE) and E-selectin (CD62E) are vital factors regulating vascular function and inflammation. However, their effects on IDD and vascular invasion in intervertebral discs (IVDs) are pending further exploration. Here, bioinformatics and human nucleus pulposus (NP) tissues analyses revealed that CSE was significantly downregulated and CD62E was upregulated in the NP tissues of IDD patients. We demonstrated that CSE overexpression, CD62E downregulation, and NF-κB (P65) inhibition mitigate inflammation and recover metabolic function in NP cells. Similarly, CSE attenuated vascular invasion induced by inflammatory irritation. Using a rat IDD model, we showed that CSE improved degeneration, inflammation, and microvascular invasion in NP tissue, whereas CD62E had the opposite effect. Taken together, our results indicated that the CSE/CD62E pathway could effectively improve the inflammatory environment and vascular invasion in IVD. Hence, the findings of this study propose a promising and valuable strategy for the treatment of patients with early IDD as well as postoperative adjuvant therapy in patients with severe IDD.

Cystathionine γ-lyase promotes estrogen-stimulated uterine artery blood flow via glutathione homeostasis.

During pregnancy, estrogen (E2) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H2S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E2 stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E2 stimulation. We quantified serum and uterine artery NO metabolites (NOx), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E2 stimulation of UBF. Renal blood flow was E2-insensitive for both genotypes. While serum and uterine artery NOx were similar between genotypes at baseline, E2 decreased NOx in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E2 and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E2 or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E2 stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds.

The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells.

BACKGROUND: Physiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H2S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs. METHODS: Human EPC-derived CSE activity was detected by colorimetric assay, and H2S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot. RESULTS: Human EPCs were treated with shear stress levels of 5-25 dyn/cm2 for up to 3 h, and 25 dyn/cm2 for up to 24 h. H2S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro. CONCLUSIONS: This study gives new insight into the regulatory effect of physiological shear stress on the CSE/H2S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

Hydrogen sulfide increases glutathione biosynthesis, and glucose uptake and utilisation in C2C12 mouse myotubes.

Diabetic patients have lower blood concentrations of hydrogen sulfide (H2S), L-cysteine (LC), and glutathione (GSH). Using C2C12 mouse myotubes as a model, this study investigates the hypothesis that the beneficial effects of LC supplementation are mediated by upregulation of the H2S status under diabetic conditions. Results show that exogenous administration of sodium hydrosulfide (NaHS, 10 or 20 µM; 6 hours), a H2S donor, significantly (p < .05) upregulates the gene expression of cystathionine-γ-lyase (CSE), LC transporter (Slc7a11/xCT), and the genes involved in GSH biosynthesis. Additionally, it reduces homocysteine (HCys), reactive oxygen species (ROS) production, and enhances cellular LC, H2S, and glucose uptake and utilisation in myoblasts. The use of CSE siRNA to induce deficient endogenous H2S production causes an increase in H2O2, ROS, HCys levels, and downregulation of GSH biosynthesis pathway enzymes. In additional, CSE knockdown downregulates glucose transporter type 4 (GLUT4) and gene expression of its key transcription factors, and reduces glucose uptake in C2C12 myotubes. CSE knockdown cells showed specific increases in the protein S-glutathionylation of LC transporter and GLUT4 along with increased total protein S-glutathionylation. Taken together, evidence from this study provides molecular insights into the importance of the CSE/H2S system in maintaining the cellular glutathione and glucose homeostasis in C2C12 myotubes.

Effects of CCK-8 and Cystathionine γ-Lyase/Hydrogen Sulfide System on Acute Lung Injury in Rats.

Acute lung injury (ALI) is mainly characterized by diffusive injuries to lung epithelium and increased permeability of alveolar-capillary membranes caused by various factors, which leads to pulmonary edema and pulmonary closure. Lipopolysaccharide (LPS), which is the main component of the cell wall of gram-negative bacteria, is one of the most important factors causing pulmonary infection and ALI. More and more reports have indicated that hydrogen sulfide (H2S) is closely correlated with ALI and has anti-inflammation function, while the specific mechanism needs further investigation. Cholecystokinin-octapeptide (CCK-8), which is an important endogenous functional fragment belonging to CCK family, participates in anti-inflammatory and anti-endotoxic shock (ES). Whether CCK-8 plays important roles in curing ALI also needs further investigation. Herein, we concluded that CCK-8 alleviated the ALI induced by LPS via regulating the catalytic activity of cystathionine γ-lyase (CSE) and the formation of H2S. This work provides new medicine-designed target for clinical doctor to prevent and cure ALI.

The immunomodulation of inducible hydrogen sulfide in Pacific oyster Crassostrea gigas.

Hydrogen sulfide (H2S) is an important gasotransmitter, which plays indispensable roles in cardiovascular, nervous and immune systems of vertebrates. However, the information about the immunomodulation of H2S in invertebrates is still very limited. In the present study, the temporal expression profile of cystathionine γ lyase in oyster Crassostrea gigas (CgCSE) was investigated after the oysters were stimulated by lipopolysaccharide. The expression levels of CgCSE mRNA transcripts in hemocytes increased significantly at 12h (1.31-fold of the PBS group, P<0.05) after LPS stimulation. The immunomodulation of inducible H2S in oyster was examined by monitoring the alterations of both cellular and humoral immune parameters in response to the stimulations of LPS, LPS+Na2S and LPS+propargylglycine (PAG). The total hemocyte counts (THC) and hemolymph PO activity increased significantly after LPS stimulation, and the increase could be further enhanced by adding PAG, while inhibited by appending Na2S. The phagocytosis activity of hemocytes was also increased firstly after LPS treatment, and the increase was enhanced by adding Na2S but inhibited after appending PAG. The anti-bacterial activity in hemolymph increased at 3h post LPS treatment, and then decreased after adding PAG. The total SOD activity of hemolymph was also elevated at 6h post LPS treatment, and the elevated activity was depressed by adding Na2S. These results collectively indicated that H2S might play crucial roles in the immune response of oyster via modulating the turnover and phagocytosis of hemocytes, and regulating the anti-bacterial activity and proPO activation in the hemolymph.

Correlation between the cystathionine-γ-lyase (CES) and the severity of peptic ulcer disease.

BACKGROUND: The infection of Helicobacter pylori (H. pylori) is one of the most important causes of gastric ulcer disease. The role of hydrogen sulfide (H2S) production in H. pylori-induced gastric ulcer disease. AIM: The expression of cystathionine-γ-lyase (CSE) was determined, and correlated with the severity of gastric ulcer disease. METHODS: One hundred and eight patients were selected based on the determination of gastric ulcer and the infection of Helicobacter pylori (H. pylori), including 36 normal control, 36 patients with H. Pylori-negative gastric ulcer, and 36 patients with H. Pylori-positive gastric ulcer. RT-PCR determination was performed to determine the expression of CSE, NF-κB and IL-8. RESULTS: The expression of CSE, NF-κB and IL-8 was higher in the gastric ulcer group than control group (p<0.05). Compared with the H. pylori-negative gastric ulcer, the expression of CSE, NF-κB and IL-8 was higher than H. pylori-positive gastric ulcer group (p<0.05). For H. pylori-negative gastric ulcer group, the expression of CSE positively correlated with the expression of NF-κB (r=0.98, p<0.05) and IL-8 (r=0.95, p<0.05). For H. pylori-positive gastric ulcer group, the expression of CSE also positively correlated with the expression of NF-κB (r=0.99, p<0.05) and IL-8 (r=0.85, p<0.05). CONCLUSION: The expression of CSE was positively correlated with the severity of gastric ulcer.