Cystathionine Γ-lyase Research Articles

Characterization of human cystathionine γ-lyase enzyme activities toward d-amino acids.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine β-lyase possesses amino acid racemase activity in addition to β-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine β-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Regulation of CyR61 expression and release by 3-mercaptopyruvate sulfurtransferase in colon cancer cells

Cysteine-rich angiogenic inducer 61 (CYR61, also termed CCN family member 1 or CCN1), is a matricellular protein encoded by the CYR61 gene. This protein has been implicated in the regulation of various cancer-associated processes including tumor growth, angiogenesis, tumor cell adhesion, migration, and invasion as well as the regulation of anticancer drug resistance. Hydrogen sulfide (H2S) is a gaseous endogenous biological mediator, involved in the regulation of cellular bioenergetics, angiogenesis, invasion, and chemotherapeutic resistance in several types of cancer. H2S is produced by three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). The current studies were set up to investigate if CBS or 3-MST regulates CyR61 in colon cancer cells in the context of the regulation of proliferation, migration, and survival. The study mainly utilized HCT116 cells, in which two of the principal H2S-producing enzymes, CBS and 3-MST, are highly expressed. The H2S donor GYY4137 and the polysulfide donor Na2S3 activated the CyR61 promoter in a concentration-dependent fashion. Aminooxyacetic acid (AOAA), a pharmacological inhibitor of CBS as well as HMPSNE: 2-[(4-hydroxy-6- methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one, a pharmacological inhibitor of 3-MST inhibited CyR61 mRNA expression. This effect was more pronounced in response to HMPSNE than to AOAA and occurred through the modulation of S1PR via ATF1 and CREB. CyR61 was found to play an active, but relatively minor role in maintaining colon cell proliferation. HMPSNE markedly suppressed the secretion/release of CyR61 from the colon cancer cells. Moreover, HMPSNE promoted colon cancer cell apoptosis; endogenously produced CyR61 was found to counteract this effect, at least in part via RhoA activation. Taken together, we conclude that the upregulation of 3-MST in cancer cells exerts cytoprotective effects and confers the cancer cells a more aggressive phenotype – at least in part via the modulation of CyR61 expression and release.

Transcriptome Analysis of Dauer Moulting of a Plant Parasitic Nematode, Bursaphelenchus xylophilus Promoted by Pine Volatile β-Pinene

Pinewood nematode, Bursaphelenchus xylophilus, a pine-parasitic nematode, poses a serious threat to pine trees globally, causing pine wilt disease. When dispersal-stage juvenile 4 (dauer, JIV, a durable stage) of B. xylophilus enters the new pine, it transforms into a propagative adult (dauer moulting) and reproduces quickly. Our previous studies have found that pine-volatile β-pinene promotes dauer moulting of B. xylophilus; however, this mechanism is not clear. Here, this study is attempting to unravel the molecular process underlying dauer moulting of B. xylophilus through signal chemical tests and transcriptome analysis. The results showed that β-pinene could promote dauer moulting of B. xylophilus, while other common dauer moulting signals, such as dafachronic acid (DA), part of the TGF/insulin signal pathway, were inoperative. Moreover, the JIV soaked in 1% β-pinene for only 6 h could transform into adults at a significant rate. Therefore, the transcriptomes of JIV soaked in 1% β-pinene for 6 h were sequenced. It was found that 15,556 genes were expressed; however, only 156 genes were expressed differentially and enriched in the metabolism of xenobiotics, peroxisome, fatty acid metabolism, and carbon metabolism, indicating that energy metabolism was active at the early stage of dauer moulting. With a stricter parameter, the number of differential genes fell to 19, including 4 sterol hydroxylase, 5 dehydrogenase, 2 glucuronosyltransferase, 5 nuclear-related factor, 1 calcium-binding protein, 1 nitrogen metabolic regulation protein, and 1 cystathionine gamma-lyase. These results indicated that dauer moulting of B. xylophilus into adults might not be regulated by the TGF-β/insulin signal pathway but by another new signal pathway related to the 19 differential genes which need more exploration. Our results contribute to the understanding of the molecular mechanisms behind dauer moulting and may be useful in reducing pine wilt disease by suppressing this moulting to cut the life cycle of B. xylophilus.

Hydrogen Sulfide Ameliorates Myocardial Injury Caused by Sepsis Through Suppressing ROS-Mediated Endoplasmic Reticulum Stress

To investigate the effect of hydrogen sulfide (H 2S) on reactive oxygen species (ROS)-mediated endoplasmic reticulum stress in myocardial injury caused by sepsis. A sepsis model was induced in Sprague-Dawley (SD) rats by cecal ligation and puncture (CLP). The rats were randomly divided into sham operation (sham) group, sepsis (CLP) group, and sepsis+sodium hydrosulfide (NaHS) (CLP+NaHS) group. The left ventricular function of the rats was observed with echocardiography and their plasma H 2S levels were measured. Lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH) levels were measured and HE staining was done to evaluate the level of myocardial oxidative stress in rats. HE staining was done to observe the morphological changes of rat myocardium, and transmission electron microscope was used to observe the ultrastructure of myocardial mitochondria. Western blot was done to examine changes in the expression of two endogenous hydrogen sulfide synthases, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), and changes in the expression of endoplasmic reticulum stress (ERS) marker proteins, including phosphorylated (p) protein kinase R-like endoplasmic reticulum kinase (p-PERK), p-eukaryotic translation initiation factor 2α (p-eIF2α), p-inositol requires enzyme 1α (IRE1α), recombinant activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). TUNEL staining was performed to observe the changes of cardiomyocyte apoptosis in rats. Left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS) and plasma H 2S decreased in septic rats ( P<0.05). Plasma H 2S exhibited linear correlation with LVEF and LVFS ( r 2=0.62 and r 2=0.64, all P<0.05). The ROS levels were significantly elevated in rats of the CLP group. In addition, these rats showed increased level of LDH ( P<0.05), increased expression of MDA ( P<0.05), and decreased expression of GSH ( P<0.05). Inflammatory cell infiltration and cardiomyocyte edema were observed in HE staining. Transmission electron microscopic observation revealed significant mitochondrial damage, observable mitochondrial edema, and cristae structure dissolution. The Western blot results showed that the expression levels of CSE and 3-MST decreased ( P<0.05), while the ERS marker proteins, including p-PERK, p-eIF2, IRE1α, ATF4, and CHOP, were expressed at increased levels ( P<0.05). TUNEL staining showed significant increase of apoptosis in cardiomyocytes ( P<0.05). After NaHS treatment, LVEF and LVFS increased ( P<0.05) and plasma H 2S increased in septic rats ( P<0.05). Myocardial oxidative stress levels decreased. HE staining and transmission electron microscopy showed improved myocardial morphology. Mitochondrial damage was reduced and CSE and 3-MST levels were significantly increased ( P<0.05). The expression of p-PERK, p-eIF2α, p-IRE1α, and CHOP proteins decreased ( P<0.05). A decrease in cardiomyocyte apoptosis levels was observed by TUNEL staining ( P<0.05). H 2S reduces septic cardiomyocyte apoptosis by inhibiting ROS-mediated ERS, thereby improving myocardial dysfunction in sepsis.

P07-04 Contribution of Nrf2 and cystathionine gamma-lyase to environmental electrophile-mediated toxicity in mice

P07-04 Contribution of Nrf2 and cystathionine gamma-lyase to environmental electrophile-mediated toxicity in mice

Esculentoside A Alleviates Intestinal Dysmotility in Ulcerative Colitis by Regulating H2S/CSE and NO/nNOS Systems.

Background Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that commonly affects the health of many individuals. Esculentoside A (EsA), a saponin extracted from the roots of Phytolacca esculenta, has antioxidative and anti-inflammatory effects against various diseases. Nonetheless, its role in UC is undetermined. Hence, in this study, we examined the therapeutic effects of EsA in UC. Methods Primary intestinal neuronal cells (in vitro) were treated with lipopolysaccharide (LPS) to induce inflammatory injury. An in vivo UC rat model was created by the administration of dextran sulfate sodium (DSS) to rats, which were subsequently treated with different doses of EsA. The effects of EsA on intestinal motility, histological score, inflammatory response, hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) system, NO/neuronal nitric oxide synthase (nNOS) system, and LPS-induced primary intestinal neuronal cell viability loss, proliferation inhibition, and apoptosis were detected. Results In vitro, EsA treatment increased the number of DSS-inhibited bowel movements and body weight, improved the histological score of colitis, and inhibited the inflammatory response by reducing IL-6 and TNF-α levels in rats. More importantly, EsA reduced the NO and H2S levels in serum and CSE, CBS, and nNOS expressions in the colon tissue. In vivo, EsA treatment eased the viability loss, proliferation inhibition, and apoptosis of LPS-stimulated primary intestinal neuronal cells, as well as inhibited the expressions of IL-6, TNF-α, CSE, CBS, and nNOS in cells. Conclusion EsA improved intestinal motility and suppressed inflammatory response in DSS-induced UC, which may be mediated by H2S/CSE and NO/nNOS systems.

Activation of DDX58/RIG‑I suppresses the growth of tumor cells by inhibiting STAT3/CSE signaling in colon cancer.

Some patients with colon cancer eventually develop metastasis during treatment, and the 5-year survival rate of patients with metastatic colon cancer remains relatively low, which is most likely due to the ineffectiveness of the current standard treatment. Systemic treatment for patients with colon cancer has expanded from chemotherapy to targeted therapy and immunotherapy. Immunotherapy holds promise in the treatment of colon cancer. The present study revealed the role of innate immune receptor helicase DExD/H-box helicase 58 (DDX58), which encodes retinoic acid-inducible gene-I (RIG-I), in colon cancer. It was demonstrated that colon cancer patients with a low protein expression of DDX58/RIG-I had a worse 5-year survival rate of patients compared with patients with a high expression of DDX58/RIG-I. The activation of DDX58/RIG-I inhibited the proliferation, migration and invasion of colon cancer cells, as well as tumor growth in a nude mouse xenograft model of colon cancer. To investigate the mechanisms of action of DDX58/RIG-I in colon cancer, the role of signal transducer and activator of transcription 3 (STAT3)/cystathionine-γ-lyase (CSE) signaling in the upor downregulation of DDX58 was examined. The data demonstrated that DDX58 regulated the STAT3/CSE signaling pathway by interacting with STAT3 and consequently affecting the proliferation of tumor cells in colon cancer. In addition, the RIG-I agonist, SB9200, induced proliferation, migration and invasion of human colon cancer. On the whole, the present study demonstrates that DDX58/RIG-I affects the proliferation of tumor cells by regulating STAT3/CSE signaling in colon cancer. The findings presented herein suggest that DDX58/RIG-I activation may be an effective treatment strategy, and DDX58/RIG-I agonists may be potential therapeutic candidates for colon cancer.

Cystathionine Gamma-Lyase Regulate Psilocybin Biosynthesis in Gymnopilus dilepis Mushroom via Amino Acid Metabolism Pathways.

As a potential medicine for the treatment of depression, psilocybin has gradually attracted attention. To elucidate the molecular mechanism regulating psilocybin synthesis in Gymnopilus dilepis, ultra-performance liquid chromatography (UPLC) was used to detect the changes in psilocybin content after S-adenosyl-l-homocysteine (SAH) treatment and the changes of psilocybin content in different parts (stipe and pileus), and RNA-Seq was used to explore the mechanism of psilocybin content changes. In this study, the psilocybin content in G. dilepis mycelia treated with SAH was significantly lower than that in the control group, and the content of psilocybin in the stipe was significantly higher than that in the pileus. Transcriptome analysis revealed that differential expression genes (DEGs) were associated with cysteine and methionine metabolism. In particular, the transcription levels of genes encoding Cystathionine gamma-lyase (CTH) in different treatments and different parts were positively correlated with psilocybin content. In addition, we found that the exogenous addition of CTH activity inhibitor (DL-propargylglycine, PAG) could reduce the content of psilocybin and L-serine, and the content of psilocybin and L-serine returned to normal levels after L-cysteine supplementation, suggesting that psilocybin synthesis may be positively correlated with L-cysteine or CTH, and L-cysteine regulates the synthesis of psilocybin by affecting L-serine and 4-hydroxy-L-tryptophan. In conclusion, this study revealed a new molecular mechanism that affects psilocybin biosynthesis, which can provide a theoretical basis for improving psilocybin synthesis and the possibility for the development of biomedicine.

Vasoactive Effects of Chronic Treatment with Fructose and Slow-Releasing H2S Donor GYY-4137 in Spontaneously Hypertensive Rats: The Role of Nitroso and Sulfide Signalization.

Increased fructose consumption induces metabolic-syndrome-like pathologies and modulates vasoactivity and the participation of nitric oxide (NO) and hydrogen sulfide (H2S). We investigated whether a slow-releasing H2S donor, GYY-4137, could exert beneficial activity in these conditions. We examined the effect of eight weeks of fructose intake on the blood pressure, biometric parameters, vasoactive responses, and NO and H2S pathways in fructose-fed spontaneously hypertensive rats with or without three weeks of GYY-4137 i.p. application. GYY-4137 reduced triacylglycerol levels and blood pressure, but not adiposity, and all were increased by fructose intake. Fructose intake generally enhanced endothelium-dependent vasorelaxation, decreased adrenergic contraction, and increased protein expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and concentration of conjugated dienes in the left ventricle (LV). Although GYY-4137 administration did not affect vasorelaxant responses, it restored disturbed contractility, LV oxidative damage and decreased protein expression of TNFα in fructose-fed rats. While the participation of endogenous H2S in vasoactive responses was not affected by fructose treatment, the expression of H2S-producing enzyme cystathionine β-synthase in the LV was increased, and the stimulation of the NO signaling pathway improved endothelial function in the mesenteric artery. On the other hand, chronic treatment with GYY-4137 increased the expression of H2S-producing enzyme cystathionine γ-lyase in the LV and stimulated the beneficial pro-relaxant and anti-contractile activity of endogenous H2S in thoracic aorta. Our results suggest that sulfide and nitroso signaling pathways could trigger compensatory vasoactive responses in hypertensive rats with metabolic disorder. A slow H2S-releasing donor could partially amend metabolic-related changes and trigger beneficial activity of endogenous H2S.

NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Cystathionine-y-lyase (CSE) is a critical enzyme for hydrogen sulfide (H2S) biosynthesis and plays a key role in respiratory syncytial virus (RSV) pathogenesis. The transcription factor NRF2 is the master regulator of cytoprotective and antioxidant gene expression, and is degraded during RSV infection. While some evidence supports the role of NRF2 in CSE gene transcription, its role in CSE expression in airway epithelial cells is not known. Here, we show that RSV infection decreased CSE expression and activity in primary small airway epithelial (SAE) cells, while treatment with tert-butylhydroquinone (tBHQ), an NRF2 inducer, led to an increase of both. Using reporter gene assays, we identified an NRF2 response element required for the NRF2 inducible expression of the CSE promoter. Electrophoretic mobility shift assays demonstrated inducible specific NRF2 binding to the DNA probe corresponding to the putative CSE promoter NRF2 binding sequence. Using chromatin immunoprecipitation assays, we found a 50% reduction in NRF2 binding to the endogenous CSE proximal promoter in SAE cells infected with RSV, and increased binding in cells stimulated with tBHQ. Our results support the hypothesis that NRF2 regulates CSE gene transcription in airway epithelial cells, and that RSV-induced NRF2 degradation likely accounts for the observed reduced CSE expression and activity.

Sulfide regulation of cardiovascular function in health and disease.

Hydrogen sulfide (H2S) has emerged as a gaseous signalling molecule with crucial implications for cardiovascular health. H2S is involved in many biological functions, including interactions with nitric oxide, activation of molecular signalling cascades, post-translational modifications and redox regulation. Various preclinical and clinical studies have shown that H2S and its synthesizing enzymes — cystathionine γ-lyase, cystathionine β-synthase and 3-mercaptosulfotransferase — can protect against cardiovascular pathologies, including arrhythmias, atherosclerosis, heart failure, myocardial infarction and ischaemia–reperfusion injury. The bioavailability of H2S and its metabolites, such as hydropersulfides and polysulfides, is substantially reduced in cardiovascular disease and has been associated with single-nucleotide polymorphisms in H2S synthesis enzymes. In this Review, we highlight the role of H2S, its synthesizing enzymes and metabolites, their roles in the cardiovascular system, and their involvement in cardiovascular disease and associated pathologies. We also discuss the latest clinical findings from the field and outline areas for future study.

β3 Relaxant Effect in Human Bladder Involves Cystathionine γ-Lyase-Derived Urothelial Hydrogen Sulfide.

It is now well established that the urothelium does not act as a passive barrier but contributes to bladder homeostasis by releasing several signaling molecules in response to physiological and chemical stimuli. Here, we investigated the potential contribution of the hydrogen sulfide (H2S) pathway in regulating human urothelium function in β3 adrenoceptor-mediated relaxation. The relaxant effect of BRL 37344 (0.1–300 µM), a selective β3 adrenoceptor agonist, was evaluated in isolated human bladder strips in the presence or absence of the urothelium. The relaxant effect of BRL 37344 was significantly reduced by urothelium removal. The inhibition of cystathionine-γ-lyase (CSE), but not cystathionine-β-synthase (CBS), significantly reduced the BRL 37344 relaxing effect to the same extent as that given by urothelium removal, suggesting a role for CSE-derived H2S. β3 adrenoceptor stimulation in the human urothelium or in T24 urothelial cells markedly increased H2S and cAMP levels that were reverted by a blockade of CSE and β3 adrenoceptor antagonism. These findings demonstrate a key role for urothelium CSE-derived H2S in the β3 effect on the human bladder through the modulation of cAMP levels. Therefore, the study establishes the relevance of urothelial β3 adrenoceptors in the regulation of bladder tone, supporting the use of β3 agonists in patients affected by an overactive bladder.

Hydrogen Sulfide Ameliorated High Choline-Induced Cardiac Dysfunction by Inhibiting cGAS-STING-NLRP3 Inflammasome Pathway.

Although it is an essential nutrient, high choline intake directly or indirectly via its metabolite is associated with increased risk of cardiovascular disease, the mechanism of which remains to be elucidated. The present study was performed to investigate whether hydrogen sulfide (H2S) was involved in high choline-induced cardiac dysfunction and explore the potential mechanisms. We found that ejection fraction (EF) and fractional shortening (FS), the indicators of cardiac function measured by echocardiography, were significantly decreased in mice fed a diet containing 1.3% choline for 4 months as compared to the control, while applying 3,3-dimethyl-1-butanol (DMB) to suppress trimethylamine N-oxide (TMAO, a metabolite of choline) generation ameliorated the cardiac function. Subsequently, we found that feeding choline or TMAO significantly increased the protein levels of cyclic GMP-AMP (cGAMP) synthase (cGAS), stimulator of interferon genes (STING), NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) as compared to the control, which indicated the activation of cGAS-STING-NLRP3 inflammasome axis. Moreover, the protein expression of cystathionine γ-lyase (CSE), the main enzyme for H2S production in the cardiovascular system, was significantly increased after dietary supplementation with choline, but the plasma H2S levels were significantly decreased. To observe the effect of endogenous H2S, CSE knockout (KO) mice were used, and we found that the EF, FS, and plasma H2S levels in WT mice were significantly decreased after dietary supplementation with choline, while there was no difference between CSE KO + control and CSE KO + choline group. To observe the effect of exogenous H2S, mice were intraperitoneally injected with sodium hydrosulfide (NaHS, a H2S donor) for 4 months, and we found that NaHS improved the cardiac function and reduced the protein levels of cGAS, STING, NLRP3, caspase-1, and IL-1β in mice receiving dietary choline. In conclusion, our studies revealed that high choline diet decreased plasma H2S levels and induced cardiac dysfunction via cGAS-STING-NLRP3 inflammasome axis while H2S treatment could restore the cardiac function by inhibiting cGAS-STING-NLRP3 inflammasome axis.

Deletion of cystathionine-γ-lyase in bone marrow-derived cells promotes colitis-associated carcinogenesis

Ulcerative colitis (UC) is characterized by widespread relapsing inflammation of the colonic mucosa. Colitis-associated cancer (CAC) is one of the most serious complications of a prolonged history of UC. Hydrogen sulfide (H2S) has emerged as an important physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to injury. Inhibition of cystathionine-γ-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. It is unknown whether CSE plays a role in CAC, or the downregulation of CSE expression and/or activity promotes CAC development.In humans, we observed a significant decrease in CSE expression in colonic biopsies from patients with UC. Using the dextran sodium sulfate (DSS) model of epithelium injury-induced colitis and global CSE KO mouse strain, we demonstrated that CSE is critical in limiting mucosal inflammation and stimulating epithelial cell proliferation in response to injury. In vitro studies showed that CSE activity stimulates epithelial cell proliferation, basal and cytokine-stimulated cell migration, as well as cytokine regulation of transepithelial permeability. In the azoxymethane (AOM)/DSS model of CAC, the loss of CSE expression accelerated both the development and progression of CAC. The increased tumor multiplicity and severity of CAC observed in CSE-KO mice were associated with reduced levels of mucosal IL-10 expression and increased levels of IL-6. Restoring CSE expression in bone marrow (BM) cells of CSE-KO mice through reciprocal BM transplantation raised mucosal IL-10 expression, decreased IL-6 level, and reduced the number of aberrant crypt foci and tumors in AOM/DSS-treated mice.These studies demonstrate that CSE expression in BM cells plays a critical role in suppressing CAC in mice. Furthermore, the data suggest that the inhibitory effects of CSE on the development of CAC are due, in part, to the modulation of mucosal pro-and anti-inflammatory cytokine expression.

Deficiency of cystathionine gamma-lyase promotes aortic elastolysis and medial degeneration in aged mice

Enzymatic degradation of elastin by matrix metalloproteinases (MMPs) leads to the permanent dilation of aortic wall and constitutes the most prominent characters of aortic aneurysm and aging-related medial degeneration. Hydrogen sulfide (H2S) as a gasotransmitter exhibits a wide variety of cardio-protective functions through its anti-inflammatory and anti-oxidative actions. Cystathionine gamma-lyase (CSE) is a main H2S-generating enzyme in cardiovascular system. The regulatory roles of CSE/H2S system on elastin homeostasis and blood vessel degeneration have not yet been explored. Here we found that aged CSE knockout mice had severe aortic dilation and elastic degradation in abdominal aorta and were more sensitive to angiotensin II-induced aortic elastolysis and medial degeneration. Administration of NaHS would protect the mice from angiotensin II-induced inflammation, gelatinolytic activity, elastin fragmentation, and aortic dilation. In addition, human aortic aneurysm samples had higher inflammatory infiltration and lower expression of CSE. In cultured smooth muscle cells (SMCs), TNFα-induced MMP2/9 hyperactivity and elastolysis could be attenuated by exogenously applied NaHS or CSE overexpression while further deteriorated by complete knockout of CSE. It was further found that H2S inhibited MMP2 transcription by posttranslational modification of Sp1 via S-sulfhydration. H2S also directly suppressed MMP hyperactivity by S-sulfhydrating the cysteine switch motif. Taken together, this study revealed the involvement of CSE/H2S system in the pathogenesis of aortic elastolysis and medial degeneration by maintaining the inactive form of MMPs, suggesting that CSE/H2S system can be a target for the prevention of age-related medial degeneration and treatment of aortic aneurysm.

Hydrogen Sulfide Regulates Irisin and Glucose Metabolism in Myotubes and Muscle of HFD-Fed Diabetic Mice.

Irisin, a novel myokine, is secreted by the muscle following proteolytic cleavage of fibronectin type III domain containing 5 (FNDC5) and is considered a novel regulator of glucose homeostasis. Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H2S) and is involved in glucose homeostasis. We examined the hypothesis that H2S deficiency leads to decreased FNDC5 and irisin secretion, and thereby alters glucose metabolism. High-fat diet-fed mice exhibited elevated blood glucose and significantly reduced levels of CSE, H2S, and PGC-1α, with decreased FNDC5/irisin levels and increased oxidative stress in the muscle compared with those of normal diet-fed mice (control). High glucose or palmitate decreases CSE/PGC-1α/FNDC5 levels and glucose uptake in myotubes. Inhibitors (propargylglycine and aminooxyacetate) of H2S producing enzymes or CSE siRNA significantly decreased levels of H2S and FNDC5 along with PGC-1α; similar H2S-deficient conditions also resulted in decreased GLUT4 and glucose uptake. The levels of H2S, PGC-1α, and FNDC5 and glucose uptake were significantly upregulated after treatment with l-cysteine or an H2S donor. Myoblast differentiation showed upregulation of PGC-1α and FNDC5, which was consistent with the increased expression of CSE/H2S. These findings suggest that the upregulation of H2S levels can have beneficial effects on glucose homeostasis via activation of the PGC-1α/FNDC5/irisin signaling pathway.

Exercise restores endogenous H2 S synthesis and mitochondrial function in the heart of old rats.

Ageing is accompanied by a decrease in endogenous hydrogen sulphide (H2 S) synthesis and the development of mitochondrial dysfunction. The aim of our work was to study the possible participation of exercise training-induced regulation of endogenous H2 S production in the restoration of mitochondrial function in old rats. Male rats were divided into three groups: adult, old and exercise-trained old. Exercise training of old rats was performed for 4 weeks. The mRNA expression cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) were determined using reverse transcription and real-time polymerase chain reaction analysis. Mitochondrial dysfunction was determined by mPTP opening, which was investigated by spectrophotometric registration of the swelling of mitochondria isolated from the rat heart. We also studied the effect of exercise on H2 S content, oxidative stress and mtNOS activity. Exercise training in old animals significantly increased the expression of H2 S-synthesizing enzymes CSE and 3-MST and restored endogenous H2 S production in cardiac tissue and cardiac mitochondria to levels of adult animals. In addition, the training significantly reduced oxidative stress in old rats, in particular the rate of formation of •O2 - and H2 O2 , diene conjugates and malondialdehyde levels in the mitochondria of the heart. Simultaneously, in the hearts of these animals, resistance of mPTP to the inducer of its opening of calcium ions was increased. Thus, exercise training restores endogenous H2 S production, and significantly reduces oxidative stress in cardiac mitochondria of old rats that are associated with the inhibition of calcium-induced mPTP opening as an indicator of mitochondrial dysfunction.

The expression of hydrogen sulfide-producing enzymes in primary and lung metastatic osteosarcoma.

Hydrogen sulfide (H₂S) is a novel gas transmitter signaling molecule. H₂S is synthesized by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST). There have been no reports about the roles of these enzymes in osteosarcoma and its metastases. We detected H₂S synthase expression levels in human primary osteosarcoma and lung metastatic osteosarcoma. Immunohistochemistry was performed in primary osteosarcoma (n=19), lung metastatic osteosarcoma (n=11), osteoblastoma (n=10) and bony callus (n=2). The expression of CBS, CSE, and MST was defined as negative, moderately positive and strongly positive. MST staining was moderately to strongly positive in all cases. CSE staining was negative in 94.7% (18/19) of primary osteosarcoma cases and 90.9% (10/11) of lung metastatic osteosarcoma cases. CBS staining was strongly positive in 68.4% (13/19) of primary osteosarcoma cases, moderately positive in 15.8% (3/19) of cases, and negative in 15.8% (3/19) of cases. In lung metastatic osteosarcoma, the proportions of negative, moderately positive and strongly positive cases were 63.6% (7/11), 18.2% (2/11) and 18.2% (2/11), respectively. CBS and CSE expression, especially CSE expression, decreased in both primary osteosarcoma and lung metastatic osteosarcoma, which may suggest that CBS and CSE play roles in osteoblast cell malignant transformation and osteosarcoma progression. These enzymes could be used as new prognostic assessment factors and may represent new therapeutic targets for osteosarcoma and metastasis prevention.

Hepatocellular cystathionine γ lyase/hydrogen sulfide attenuates nonalcoholic fatty liver disease by activating farnesoid X receptor.

Hydrogen sulfide (H2 S) plays a protective role in NAFLD. However, whether cystathionine γ lyase (CSE), a dominant H2 S generating enzyme in hepatocytes, has a role in the pathogenesis of NAFLD is currently unclear. We showed that CSE protein expression is dramatically downregulated, especially in fibrotic areas, in livers from patients with NAFLD. In high-fat diet (HFD)-induced NAFLD mice or an oleic acid-induced hepatocyte model, the CSE/H2 S pathway is also downregulated. To illustrate a regulatory role for CSE in NAFLD, we generated a hepatocyte-specific CSE knockout mouse (CSELKO ). Feeding an HFD to CSELKO mice, they showed more hepatic lipid deposition with increased activity of the fatty acid de novo synthesis pathway, increased hepatic insulin resistance, and higher hepatic gluconeogenic ability compared to CSELoxp control mice. By contrast, H2 S donor treatment attenuated these phenotypes. Furthermore, the protection conferred by H2 S was blocked by farnesoid X receptor (FXR) knockdown. Consistently, serum deoxycholic acid and lithocholic acid (FXR antagonists) were increased, and tauro-β-muricholic acid (FXR activation elevated) was reduced in CSELKO . CSE/H2 S promoted a post-translation modification (sulfhydration) of FXR at Cys138/141 sites, thereby enhancing its activity to modulate expression of target genes related to lipid and glucose metabolism, inflammation, and fibrosis. Sulfhydration proteomics in patients' livers supported the CSE/H2 S modulation noted in the CSELKO mice. FXR sulfhydration is a post-translational modification affected by hepatic endogenous CSE/H2 S that may promote FXR activity and attenuate NAFLD. Hepatic CSE deficiency promotes development of nonalcoholic steatohepatitis. The interaction between H2 S and FXR may be amenable to therapeutic drug treatment in NAFLD.

Brain Histology and Immunohistochemistry After Resuscitation From Hemorrhagic Shock in Swine With Pre-Existing Atherosclerosis and Sodium Thiosulfate (Na2S2O3) Treatment.

BackgroundThe hydrogen sulfide (H2S) and the oxytocin/oxytocin receptor (OT/OTR) systems interact in the central nervous and cardiovascular system. As a consequence of osmotic balance stress, H2S stimulates OT release from the paraventricular nuclei (PVN) in the hypothalamic regulation of blood volume and pressure. Hemorrhagic shock (HS) represents one of the most pronounced acute changes in blood volume, which, moreover, may cause at least transient brain tissue hypoxia. Atherosclerosis is associated with reduced vascular expression of the main endogenous H2S producing enzyme cystathionine-γ-lyase (CSE), and, hence, exogenous H2S administration could be beneficial in these patients, in particular after HS. However, so far cerebral effects of systemic H2S administration are poorly understood. Having previously shown lung-protective effects of therapeutic Na2S2O3 administration in a clinically relevant, long-term, porcine model of HS and resuscitation we evaluated if these protective effects were extended to the brain.MethodsIn this study, available unanalyzed paraffin embedded brain sections (Na2S2O3 N = 8 or vehicle N = 5) of our recently published HS study were analyzed via neuro-histopathology and immunohistochemistry for the endogenous H2S producing enzymes, OT, OTR, and markers for brain injury and oxidative stress (glial fibrillary acidic protein (GFAP) and nitrotyrosine).ResultsNeuro-histopathological analysis revealed uninjured brain tissue with minor white matter edema. Protein quantification in the hypothalamic PVN showed no significant inter-group differences between vehicle or Na2S2O3 treatment.ConclusionsThe endogenous H2S enzymes, OT/OTR co-localized in magnocellular neurons in the hypothalamus, which may reflect their interaction in response to HS-induced hypovolemia. The preserved blood brain barrier (BBB) may have resulted in impermeability for Na2S2O3 and no inter-group differences in the PVN. Nonetheless, our results do not preclude that Na2S2O3 could have a therapeutic benefit in the brain in an injury that disrupts the BBB, e.g., traumatic brain injury (TBI) or acute subdural hematoma (ASDH).