H2S Donor GYY4137 Mitigates sFlt-1-Induced Hypertension and Vascular Dysfunction in Pregnant Rats.

BACKGROUND: Gestational hypertension, often associated with elevated soluble Fms-related receptor tyrosine kinase 1 (sFlt-1), poses significant risks to both maternal and fetal health. Hydrogen sulfide (H2S), a gasotransmitter, has demonstrated blood pressure-lowering effects in hypertensive animals and humans. However, its role in pregnancy-induced hypertension remains unclear. OBJECTIVE: This study aimed to investigate the impact of GYY4137, a slow-release H2S donor, on sFlt-1-induced hypertension in pregnant rats and examine the underlying mechanisms. METHODS: Pregnant rats were administered sFlt-1 (6 µg/kg/day, intravenously) or vehicle from gestation day (GD) 12 to 20. A subset of these groups received GYY4137 (an H2S donor, 50 mg/kg/day, subcutaneously) from GD 16 to 20. Serum H2S levels, mean arterial blood pressure (CODA tail-cuff), uterine artery blood flow (ultrasonography), vascular reactivity to vasopressors and endothelial-dependent relaxation (myography), endothelial nitric oxide synthase (eNOS) protein expression in uterine arteries (Western blotting) were assessed. In addition, maternal weight gain, as well as fetal and placental weights, were measured. RESULTS: Elevated sFlt-1 reduced both maternal weight gain and serum H2S levels. GYY4137 treatment restored both weight gain and H2S levels in sFlt-1 dams. sFlt-1 increased mean arterial pressure and decreased uterine artery blood flow in pregnant rats. However, treatment with GYY4137 normalized blood pressure and restored uterine blood flow in sFlt-1 dams. sFlt-1 dams exhibited heightened vasoconstriction to phenylephrine and GYY4137 significantly mitigated the exaggerated vascular contraction. Notably, sFlt-1 impaired endothelium-dependent relaxation, while GYY4137 attenuated this impairment by upregulating eNOS protein levels and enhancing vasorelaxation in uterine arteries. GYY4137 mitigated sFlt-1-induced fetal growth restriction. CONCLUSION: sFlt-1 mediated hypertension is associated with decreased H2S levels. Replenishing H2S with the donor GYY4137 mitigates hypertension and improves vascular function and fetal growth outcomes. This suggests modulation of H2S could offer a novel therapeutic strategy for managing gestational hypertension and adverse fetal effects.

Supplementation of sperm cryopreservation media with H2S donors enhances sperm quality, reduces oxidative stress, and improves in vitro fertilization outcomes.

Cryopreservation of sperm can cause oxidative stress and damage, leading to decreased different functional parameters and fertilization potential. In this study, we evaluated two types of H2S donors: NaHS, a fast-releasing donor, and GYY4137, a slow-releasing donor during cryopreservation of goat sperm. Initially, we determined that 1.5 and 3 µM NaHS, and 15 and 30 µM GYY4137 are optimal concentrations that improved different sperm functional parameters including motility, viability, membrane integrity, lipid peroxidation, and ROS production during incubation at 38.5 °C for 90 min. We subsequently evaluated the impact of the optimal concentration of NaHS and GYY4137 supplementation on various functional parameters following thawing during cryopreservation. Our data revealed that supplementation of extender improved different parameters including post-thaw sperm motility, viability, membrane integrity, and reduced DNA damage compared to the frozen-thawed control group. The supplementation also restored the redox state, decreased lipid peroxidation, and improved mitochondrial membrane potential in the thawed sperm. Finally, we found that supplementation of the extender with NaHS and GYY4137 enhanced IVF outcomes in terms of blastocyst rate and quality of blastocysts. Our results suggest that both donors can be applied for cryopreservation as antioxidants to improve sperm quality and IVF outcomes of frozen-thawed goat sperm.

[The Protective Action of Hsp70 and Hydrogen Sulfide Donors in THP-1 Macrophages in the Lipopolysaccharide-Induced Inflammatory Response by Modulating Endocytosis].

Hsp70 and hydrogen sulfide donors reduce inflammatory processes in human and animal cells. The biological action mediated by Hsp70 and H2S donors (GYY4137 and sodium thiosulfate) depends on their protection kinetics from cell activation by lipopolysaccharides. However, the molecular mechanisms of action of Hsp70 and H2S are not well understood. We studied the effect of human recombinant Hsp70 and H2S donors on the formation of reactive oxygen species and tumor necrosis factor-alpha induced in human cells (THP-1) by lipopolysaccharides. Transcriptomic changes occurring in these cells after LPS administration in combination with GYY4137 pretreatment were investigated. The results we obtained showed that Hsp70 and hydrogen sulfide donors reduce inflammatory processes in cells activated by the action of LPS. Hsp70 and H2S donors differed in the kinetics of the protective action, while hydrogen sulfide donors turned out to be more effective. The role of endocytosis in the mechanisms of protection of cells by H2S and Hsp70 donors from the action of LPS was studied. It has been found that GYY4137 pretreatment of LPS-exposed cells reduces the LPS-induced induction of various pro-inflammatory genes and affects the expression of genes of various intracellular signaling pathways.

Ageing-Related Alterations in Renal Epithelial Glucose Transport.

The kidney plays a crucial role in glucose homeostasis by regulating glucose transport. We aimed to investigate the impact of alterations in glucose transport on glucose metabolism during ageing. Adult male Sprague Dawley rats were divided into five groups: 3-month, 6-month, and 12-month control groups, and 6- and 12-month groups receiving the hydrogen sulfide donor molecule GYY4137. The study found that, as age increased, daily urinary uric acid and protein levels increased in the 12-month group. Blood sugar level and HOMA-IR index increased in the 12-month group, and were partially improved by GYY4137. The kidney tissue showed mild glomerulosclerosis in the 12-month group, which was diminished by GYY4137. Gene expression analysis showed decreased sirtuin and increased p21 expression in the aging groups. Increased SGLT1 and SGLT2 expression was observed in the 12-month group, which was reversed by GYY4137. Both GLUT1 and GLUT2 expression was increased in the 6- and 12-month groups, and reversed by GYY4137 in the 12-month group. The study concluded that aging was associated with increased blood sugar levels and the HOMA-IR index, and the abundance of renal glucose transporters increased as aging progressed. GYY4137 effectively reversed aging-related alterations in glucose homeostasis and renal epithelial transporters.

Exogenous hydrogen sulfide alleviates hepatic endoplasmic reticulum stress via SIRT1/FoxO1/PCSK9 pathway in NAFLD.

High-fat-induced endoplasmic reticulum (ER) stress has been the main reason for the occurrence and development of nonalcoholic fatty liver disease (NAFLD). Hydrogen sulfide (H2 S) produces a marked effect on regulating lipid metabolism and antioxidation, whose effects on ER stress of NAFLD are still unclear. Here, we studied the influence of exogenous H2 S on NAFLD and its potential mechanism. In vivo, NAFLD model was induced by high-fat diet (HFD) for 12 weeks, followed by intraperitoneal injection of exogenous H2 S intervention for 4 weeks. HepG2 cells exposure to lipid mixture (LM) were used as vitro model to explore the potential mechanism. We found exogenous H2 S significantly inhibited the hepatic ER stress and improved the liver fat deposition of HFD-fed mice. These similar results were also observed in HepG2 cells dealt with LM after exogenous H2 S treatment. Further mechanism studies showed exogenous H2 S strengthened the combination of FoxO1 with the PCSK9 promoter gene through SIRT1-mediated deacetylation, thereby inhibiting the PCSK9 expression to relieve the hepatic ER stress. However, SIRT1 knockout eliminated the effects of exogenous H2 S on FoxO1 deacetylation, PCSK9 inhibition, and remission of hepatic ER stress and steatosis. In conclusion, exogenous H2 S improved NAFLD by inhibiting hepatic ER stress through SIRT1/FoxO1/PCSK9 pathway. Exogenous H2 S and ER stress may be potential drug and target for the treatment of NAFLD, respectively.

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.

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.

GYY4137-Derived Hydrogen Sulfide Donates Electrons to the Mitochondrial Electron Transport Chain via Sulfide: Quinone Oxidoreductase in Endothelial Cells.

The protective effects of hydrogen sulphide (H2S) to limit oxidative injury and preserve mitochondrial function during sepsis, ischemia/reperfusion, and neurodegenerative diseases have prompted the development of soluble H2S-releasing compounds such as GYY4137. Yet, the effects of GYY4137 on the mitochondrial function of endothelial cells remain unclear, while this cell type comprises the first target cell after parenteral administration. Here, we specifically assessed whether human endothelial cells possess a functional sulfide:quinone oxidoreductase (SQOR), to oxidise GYY4137-released H2S within the mitochondria for electron donation to the electron transport chain. We demonstrate that H2S administration increases oxygen consumption by human umbilical vein endothelial cells (HUVECs), which does not occur in the SQOR-deficient cell line SH-SY5Y. GYY4137 releases H2S in HUVECs in a dose- and time-dependent fashion as quantified by oxygen consumption and confirmed by lead acetate assay, as well as AzMC fluorescence. Scavenging of intracellular H2S using zinc confirmed intracellular and intramitochondrial sulfur, which resulted in mitotoxic zinc sulfide (ZnS) precipitates. Together, GYY4137 increases intramitochondrial H2S and boosts oxygen consumption of endothelial cells, which is likely governed via the oxidation of H2S by SQOR. This mechanism in endothelial cells may be instrumental in regulating H2S levels in blood and organs but can also be exploited to quantify H2S release by soluble donors such as GYY4137 in living systems.

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 ß-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.

Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) can cause severe respiratory illness in infants, immunocompromised, and older adults. Despite its burden, no vaccine or specific treatment is available. RSV infection is associated with increased reactive oxygen species (ROS) production, degradation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), and decreased antioxidant enzymes (AOEs), leading to oxidative damage and lung injury. Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays a physiological role in numerous cellular processes and a protective role in multiple pathological conditions, displaying vasoactive, cytoprotective, anti-inflammatory, and antioxidant activities. H2S can promote NRF2 activation through the sulfhydration of Kelch-like ECH-associated protein 1, the cytoplasmic repressor of NRF2. Here we investigated whether increasing cellular H2S levels could rescue NRF2 and NRF2-dependent gene expression in RSV-infected primary airway epithelial cells. We found that treatment with the H2S donor GYY4137 significantly increased NRF2 levels and AOEs gene expression by decreasing KEAP1 levels, and by modulating pathways involved in RSV-induced NRF2 degradation, such as NRF2 ubiquitination, and promyelocytic leukemia (PML) protein levels. These results suggest that the administration of exogenous H2S can positively impact the altered redox balance associated with RSV infection, which represents an important determinant of RSV-induced lung disease.

Inhalative as well as Intravenous Administration of H2S Provides Neuroprotection after Ischemia and Reperfusion Injury in the Rats' Retina.

BACKGROUND: Neuronal ischemia-reperfusion injury (IRI), such as it can occur in glaucoma or strokes, is associated with neuronal cell death and irreversible loss of function of the affected tissue. Hydrogen sulfide (H2S) is considered a potentially neuroprotective substance, but the most effective route of application and the underlying mechanism remain to be determined. METHODS: Ischemia-reperfusion injury was induced in rats by a temporary increase in intraocular pressure (1 h). H2S was then applied by inhalation (80 ppm at 0, 1.5, and 3 h after reperfusion) or by intravenous administration of the slow-releasing H2S donor GYY 4137. After 24 h, the retinas were harvested for Western blotting, qPCR, and immunohistochemical staining. Retinal ganglion cell survival was evaluated 7 days after ischemia. RESULTS: Both inhalative and intravenously delivered H2S reduced retinal ganglion cell death with a better result from inhalative application. H2S inhalation for 1.5 h, as well as GYY 4137 treatment, increased p38 phosphorylation. Both forms of application enhanced the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and inhalation showed a significant increase at all three time points. H2S treatment also reduced apoptotic and inflammatory markers, such as caspase-3, intracellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS). The protective effect of H2S was partly abolished by the ERK1/2 inhibitor PD98059. Inhalative H2S also reduced the heat shock response including heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) and the expression of radical scavengers such as superoxide dismutases (SOD1, SOD2) and catalase. CONCLUSION: Hydrogen sulfide acts, at least in part, via the mitogen-activated protein kinase (MAPK) ERK1/2 to reduce apoptosis and inflammation. Both inhalative H2S and intravenous GYY 4137 administrations can improve neuronal cell survival.

Slow sulfide donor GYY4137 potentiates effect of paclitaxel on colorectal carcinoma cells.

Although paclitaxel (PTX) is potent chemotherapeutic agent commonly used in variety of cancers, in colorectal carcinoma its usage is excluded because of low effectivity. Up to now, some experimental attempts were utilized to improve sensitivity of colorectal carcinoma to PTX. We used a slow sulfide donor GYY4137 to increase sensitivity of colorectal carcinoma cells to PTX. As a model of colorectal carcinoma, we utilized three different cell lines - HCT116, SW620 and DLD1. We compared IC50 for PTX and PTX/GYY4137, cell cycle, apoptosis, ATP levels and changes in intracellular pH. We observed significant decrease in IC50 levels in PTX/GYY4137 groups compared to PTX in all three cell lines. PTX arrested cell cycle in G2/M phase. Differences in S phase were observed in HCT116 and DLD1 cells treated with 20 nM PTX/GYY4137, but not in SW620 cell. GYY4137 increased early, but not late phase of apoptosis. This increase was not detected in non-cancer EAHy926 cells. Upregulation of IP3R1 suggested involvement of these receptors in PTX and/or GYY4137 induced apoptosis. We also observed partial ATP depletion and intracellular acidification in PTX treated groups. In PTX/GYY4137 groups of all three cell lines no ATP depletion was detectable and intracellular acidification was lower than in PTX treated groups. Slight differences in all measured parameters were determined among HCT116, SW620 and DLD1 cells, which is probably due to physiological variations in these cells. Taking together, sensitivity of PTX to colorectal carcinoma cell lines could be increased by slow sulfide donor GYY4137, probably through potentiation of apoptosis.

Overexpression of CBS/H2S inhibits proliferation and metastasis of colon cancer cells through downregulation of CD44.

BACKGROUND: The role of hydrogen sulfide (H2S) in cancer biology is controversial, including colorectal cancer. The bell-shaped effect of H2S refers to pro-cancer action at lower doses and anti-cancer effect at higher concentrations. We hypothesized that overexpression of cystathionine-beta-synthase (CBS)/H2S exerts an inhibitory effect on colon cancer cell proliferation and metastasis. METHODS: Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), clone-formation and sphere formation assay. Cell migration was evaluated by transwell migration assay. Intracellular H2S was detected by H2S probe. Chromatin immunoprecipitation (ChIP) analysis was carried out to examine DNA-protein interaction. Cell experiments also included western blotting, flow cytometry, immunohistochemistry (IHC) and immunofluorescence analysis. We further conducted in vivo experiments to confirm our conclusions. RESULTS: Overexpression of CBS and exogenous H2S inhibited colon cancer cell proliferation and migration in vitro. In addition, overexpression of CBS attenuated tumor growth and liver metastasis in vivo. Furthermore, CD44 and the transcription factor SP-1 was probably involved in the inhibitory effect of CBS/H2S axis on colon cancer cells. CONCLUSIONS: Overexpression of CBS and exogenous provision of H2S inhibited colon cancer cell proliferation and migration both in vivo and in vitro. Molecular mechanisms might involve the participation of CD44 and the transcription factor SP-1.

Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis.

A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.

GYY4137 Regulates Extracellular Matrix Turnover in the Diabetic Kidney by Modulating Retinoid X Receptor Signaling.

Diabetic kidney is associated with an accumulation of extracellular matrix (ECM) leading to renal fibrosis. Dysregulation of retinoic acid metabolism involving retinoic acid receptors (RARs) and retinoid X receptors (RXRs) has been shown to play a crucial role in diabetic nephropathy (DN). Furthermore, RARs and peroxisome proliferator-activated receptor γ (PPARγ) are known to control the RXR-mediated transcriptional regulation of several target genes involved in DN. Recently, RAR and RXR have been shown to upregulate plasminogen activator inhibitor-1 (PAI-1), a major player involved in ECM accumulation and renal fibrosis during DN. Interestingly, hydrogen sulfide (H2S) has been shown to ameliorate adverse renal remodeling in DN. We investigated the role of RXR signaling in the ECM turnover in diabetic kidney, and whether H2S can mitigate ECM accumulation by modulating PPAR/RAR-mediated RXR signaling. We used wild-type (C57BL/6J), diabetic (C57BL/6-Ins2Akita/J) mice and mouse mesangial cells (MCs) as experimental models. GYY4137 was used as a H2S donor. Results showed that in diabetic kidney, the expression of PPARγ was decreased, whereas upregulations of RXRα, RXRß, and RARγ1 expression were observed. The changes were associated with elevated PAI-1, MMP-9 and MMP-13. In addition, the expressions of collagen IV, fibronectin and laminin were increased, whereas elastin expression was decreased in the diabetic kidney. Excessive collagen deposition was observed predominantly in the peri-glomerular and glomerular regions of the diabetic kidney. Immunohistochemical localization revealed elevated expression of fibronectin and laminin in the glomeruli of the diabetic kidney. GYY4137 reversed the pathological changes. Similar results were observed in in vitro experiments. In conclusion, our data suggest that RXR signaling plays a significant role in ECM turnover, and GYY4137 modulates PPAR/RAR-mediated RXR signaling to ameliorate PAI-1-dependent adverse ECM turnover in DN.

The Hypothermic Effect of Hydrogen Sulfide Is Mediated by the Transient Receptor Potential Ankyrin-1 Channel in Mice.

Hydrogen sulfide (H2S) has been shown in previous studies to cause hypothermia and hypometabolism in mice, and its thermoregulatory effects were subsequently investigated. However, the molecular target through which H2S triggers its effects on deep body temperature has remained unknown. We investigated the thermoregulatory response to fast-(Na2S) and slow-releasing (GYY4137) H2S donors in C57BL/6 mice, and then tested whether their effects depend on the transient receptor potential ankyrin-1 (TRPA1) channel in Trpa1 knockout (Trpa1-/-) and wild-type (Trpa1+/+) mice. Intracerebroventricular administration of Na2S (0.5-1 mg/kg) caused hypothermia in C57BL/6 mice, which was mediated by cutaneous vasodilation and decreased thermogenesis. In contrast, intraperitoneal administration of Na2S (5 mg/kg) did not cause any thermoregulatory effect. Central administration of GYY4137 (3 mg/kg) also caused hypothermia and hypometabolism. The hypothermic response to both H2S donors was significantly (p < 0.001) attenuated in Trpa1-/- mice compared to their Trpa1+/+ littermates. Trpa1 mRNA transcripts could be detected with RNAscope in hypothalamic and other brain neurons within the autonomic thermoeffector pathways. In conclusion, slow- and fast-releasing H2S donors induce hypothermia through hypometabolism and cutaneous vasodilation in mice that is mediated by TRPA1 channels located in the brain, presumably in hypothalamic neurons within the autonomic thermoeffector pathways.

GYY4137 attenuates functional impairment of corpus cavernosum and reduces fibrosis in rats with STZ-induced diabetes by inhibiting the TGF-ß1/Smad/CTGF pathway.

Erectile dysfunction (ED) is a common diabetic complication. Recent evidence has illuminated the role of hydrogen sulfide (H2S) as a dynamic mediator of the erection process. H2S is a potent endogenous relaxant gas. It has been shown to relax human and animal penile tissue in vitro and induce erection in animals in vivo. The reported penile expression of H2S-synthesizing enzymes also supports the potential role of the endogenous L-cysteine/H2S pathway in penile homeostasis. Several pathological changes take place in the diabetic penile tissue, including inflammation, oxidative stress, neuropathy and fibrosis of the corpus cavernosum (CC), the major erectile structure of the penis. The present study is experimental and has been performed in the diabetic rat model. The study will investigate the role of H2S as a potential protective mediator against diabetes-induced structural and functional alterations in the CC by examining if it: (1) reduces corporal contraction and/or enhances corporal relaxation following pharmacological stimulation, (2) attenuates fibromuscular changes in diabetic CC, and (3) whether there is a link with H2S plasma/urine level and CC tissue generation, as well as studying the expression of some proteins in the transforming growth factor (TGF)-ß1-associated pathway. The major findings of the study reveal that- compared to the nondiabetic controls - the diabetic animals CC showed: (1) augmented contraction and attenuated relaxation in response to phenylephrine and carbachol, respectively, (2) marked fibromuscular degeneration with a significantly lower smooth muscle/collagen ratio and upregulation of TGF-ß-1/Smad/CTGF fibrosis signaling pathway, (3) reduced H2S plasma and urinary levels and cavernosal tissue generation. Chronic GYY4137 treatment prevented most of these pathological changes in diabetic CC, thus may be considered a potential new strategy for the prevention and/or treatment of diabetes-induced ED.

GYY4137 protected the integrity of the blood-brain barrier via activation of the Nrf2/ARE pathway in mice with sepsis.

Injury to the blood-brain barrier (BBB) plays a vital role in sepsis-associated encephalopathy (SAE), which is one of the most common complications of sepsis. GYY4137, a new synthetic compound of hydrogen sulfide (H2 S), has extensive biological benefits. In this study, we focused on the protective effects of GYY4137 on the BBB in septic mice and the underlying mechanisms. The results suggested that whether administrated at the same time or 3 hours after LPS injection, GYY4137 both significantly alleviated the clinical symptoms and the long-term prognosis. Besides, GYY4137 improved the pathological abnormalities of septic mice. Moreover, the degradation of tight junctions in the BBB was considerably inhibited by GYY4137. In addition, GYY4137 significantly attenuated inflammation and apoptosis in the brain. Furthermore, GYY4137 activated the Nrf2/ARE pathway through the sulfhydrylation of Keap1 and inhibited oxidative stress. ML385, the specific inhibitor of Nrf2, significantly reversed the protective effects of GYY4137 in sepsis mice. In conclusion, this study indicated that through the sulfhydrylation of Keap1, GYY4137 activated the Nrf2/ARE pathway and exerted anti-inflammatory, anti-apoptotic and antioxidant effects in septic mice that consequently protected the integrity of the BBB and improved the clinical outcome of sepsis. Our findings suggest that GYY4137 might be a promising agent for the treatment of SAE.

Protective Effects of GYY4137 on Renal Ischaemia/Reperfusion Injury through Nrf2-Mediated Antioxidant Defence.

INTRODUCTION/AIMS: Hydrogen sulfide (H2S) is considered to be the third most important endogenous gasotransmitter in organisms. GYY4137 is a long-acting donor for H2S, a gas transmitter that has been shown to prevent multi-organ damage in animal studies. We previously reported the effect of GYY4137 on cardiac ischaemia reperfusion injury (IRI) in diabetic mice. However, the role and mechanism of GYY4137 in renal IRI are poorly understood. The aims of this study were to determine whether GYY4137 can effectively alleviate the injury induced by renal ischaemia reperfusion and to explore its possible mechanism. METHODS: Mice received right nephrectomy and clipping of the left renal pedicle for 45 min. GYY4137 was administered by intraperitoneal injection for 2 consecutive days before the operation. The model of hypoxia/reoxygenation injury was established in HK-2 cells, which were pre-treated with or without GYY4137. Renal histology, function, apoptosis, and oxidative stress were measured. Western blot was used to measure the target -protein after renal IRI. RESULTS: The results indicated that GYY4137 had a clear protective effect on renal IRI as reflected by the attenuation of renal dysfunction, renal tubule injury, and apoptosis. Moreover, GYY4137 remarkably reduced renal IRI-induced oxidative stress. GYY4137 significantly elevated the nuclear translocation of nuclear factor-erythroid-2-related factor 2 (Nrf2) and the expression of antioxidant enzymes regulated by Nrf2, including SOD, HO-1, and NQO-1. CONCLUSIONS: GYY4137 alleviates ischaemia reperfusion-induced renal injury through activating the antioxidant effect mediated by Nrf2 signalling.

GYY4137 and Sodium Hydrogen Sulfide Relaxations Are Inhibited by L-Cysteine and KV7 Channel Blockers in Rat Small Mesenteric Arteries.

Donors of H2S may be beneficial in treating cardiovascular diseases where the plasma levels of H2S are decreased. Therefore, we investigated the mechanisms involved in relaxation of small arteries induced by GYY4137 [(4-methoxyphenyl)-morpholin-4-yl-sulfanylidene-sulfido-λ5-phosphane;morpholin-4-ium], which is considered a slow-releasing H2S donor. Sulfides were measured by use of 5,5'-dithiobis-(2-nitro benzoic acid), and small rat mesenteric arteries with internal diameters of 200-250 µm were mounted in microvascular myographs for isometric tension recordings. GYY4137 produced similar low levels of sulfides in the absence and the presence of arteries. In U46619-contracted small mesenteric arteries, GYY4137 (10-6-10-3 M) induced concentration-dependent relaxations, while a synthetic, sulfur-free, GYY4137 did not change the vascular tone. L-cysteine (10-6-10-3 M) induced only small relaxations reaching 24 ± 6% at 10-3 M. Premixing L-cysteine (10-3 M) with Na2S and GYY4137 decreased Na2S relaxation and abolished GYY4137 relaxation, an effect prevented by an nitric oxide (NO) synthase inhibitor, L-NAME (Nω-nitro-L-arginine methyl ester). In arteries without endothelium or in the presence of L-NAME, relaxation curves for GYY4137 were rightward shifted. High extracellular K+ concentrations decreased Na2S and abolished GYY4137 relaxation suggesting potassium channel-independent mechanisms are also involved Na2S relaxation while potassium channel activation is pivotal for GYY4137 relaxation in small arteries. Blockers of large-conductance calcium-activated (BKCa) and voltage-gated type 7 (KV7) potassium channels also inhibited GYY4137 relaxations. The present findings suggest that L-cysteine by reaction with Na2S and GYY4137 and formation of sulfides, inhibits relaxations by these compounds. The low rate of release of H2S species from GYY4137 is reflected by the different sensitivity of these relaxations towards high K+ concentration and potassium channel blockers compared with Na2S. The perspective is that the rate of release of sulfides plays an important for the effects of H2S salt vs. donors in small arteries, and hence for a beneficial effect of GYY4137 for treatment of cardiovascular disease.