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

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

Change in the molecular properties of sarcoplasmic reticulum Ca-ATPase during modification of the membranes with cholesterol

The thiol reagent NBD-chloride (4-chloro-7-nitro-benzo-2-oxo-1,3-diazole) was used to determine the amount and reactivity of SH-groups of Ca-ATPase of rat skeletal muscle sarcoplasmic reticulum during hypercholesterolemia. Modification of membranes with cholesterol brought about a decrease in the total amount and reactivity of SH-groups at the cost of reduction of rapid SH-groups and decrease of the modification constant of these SH-groups. The masking effect of high concentrations of ATP on the reactivity of SH-groups in hypercholesterolemia was noticed. It is inferred that the reduced efficacy of Ca-pump work found under the same experimental conditions before is a consequence of the modification of sarcoplasmic reticulum membranes with cholesterol and change in the molecular conformation of Ca-ATPase.     

Investigation of sarcoplasmic reticulum SH-groups]

The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant.     

Interleukin-10 down-regulates oxLDL induced expression of scavenger receptor A and Bak-1 in macrophages derived from THP-1 cells

Here, we investigated the therapeutic potential of IL-10 by testing its effects on oxLDL-induced lipoprotein uptake and apoptosis by flow cytometry in THP-1-derived macrophages. The mRNA and protein expressions of lipid scavenger receptors (SR-A, CD36) and apoptosis-related proteins (Bcl-2, Bak-1) were also detected. Co-incubation of oxLDL with IL-10 reduced DiI-oxLDL uptake by 16.1 ± 3.8%, 35.2 ± 3.8% and 28.9 ± 1.8% at 6, 12 and 24 h of treatment, respectively. Furthermore, treatment with oxLDL for 24 h enhanced the SR-A mRNA and protein expressions by 89.3 ± 17.1% and 70.1 ± 17.6%, respectively. IL-10 abrogated the oxLDL-induced SR-A mRNA expression by 50.2 ± 3.9% and its protein by 45.6 ± 1.9%. Meanwhile IL-10 had no effect on the oxLDL-induced increase of CD36 expression. IL-10 inhibited the oxLDL-induced cell apoptosis in a time-dependent manner by 17.3 ± 3.3%, 36.4 ± 2.8% and 31.0 ± 4.3% at 6, 12 and 24 h, respectively. OxLDL increased Bak-1 mRNA and protein expressions by 38.4 ± 13.3% and 36.9 ± 12.1%, respectively. However co-stimulation of oxLDL with IL-10 for 24 h inhibited Bak-1 expression to 28.4 ± 7.2% (mRNA) and 25.7 ± 6.3% (protein). Meanwhile, IL-10 had no effect on the oxLDL-induced decrease of Bcl-2 expression. Our findings suggested that IL-10 reduced the oxLDL-induced lipoprotein uptake and apoptosis partly via down-regulating the oxLDL-induced expression of SR-A and Bak-1 in THP-1-derived macrophages.     

Binding to tubulin of an allocolchicine spin probe: interaction with the essential SH groups and other active sites

EPR titration of tubulin with an allocolchicine spin probe showed more than one binding site: one high-affinity binding site (Kd = 8 microM), consistent with the Ki found for competition with colchicine, and one or more low-affinity site(s) (Kd higher than 50 microM). No disturbance of the EPR signal of the tubulin-bound allocolchicine spin probe could be observed at room temperature in the presence of other paramagnetic probes: Mn(II) for the binding site of Mg(II), Co(II) for the Zn(II) binding site and Cr(III)GTP for the binding site of the exchangeable GTP. Labelling of tubulin with both the allocolchicine and a SH-group spin probe also showed lack of interaction. The colchicine-binding site is thus sterically isolated from the binding sites for GTP, Mg(II), Zn(II) and the two essential SH-groups. In the tubulin-colchicin complex, all SH-groups could still be labelled with an excess of the SH-reagent, N-ethylmaleimide. Furthermore, colchicine binding was only minimally influenced by the blocking of the two essential SH-groups. However, the rate constant of the reaction of two equivalents of the SH-reagent, a maleimide spin probe, with the tubulin-colchicine complex was only 50% of the rate constant found with uncomplexed tubulin. As direct steric interaction of the essential SH-groups with the colchicine-binding site can be excluded, we can now definitively decide that binding of colchicine to tubulin induces a conformational change, which affects the accessibility of the most reactive SH-groups.     

Cleavage of Bovine Brain Microtubule-Associated Protein-2 by Human Immunodeficiency Virus Proteinase

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.     

The mitochondrial carnitine carrier: characterization of SH-groups relevant for its transport function.

The transport function of the purified and reconstituted carnitine carrier from rat liver mitochondria was correlated to modification of its SH-groups by various reagents. The exchange activity and the unidirectional transport, both catalyzed by the carnitine carrier, were effectively inhibited by N-ethylmaleimide and submicromolar concentrations of mercurial reagents, e.g., mersalyl and p-(chloromercuri)benzenesulfonate. When 1 microM HgCl2 or higher concentrations of the above mentioned mercurials were added, another transport mode of the carrier was induced. After this treatment, the reconstituted carnitine carrier catalyzed unidirectional substrate-efflux and -influx with significantly reduced substrate specificity. Control experiments in liposomes without carrier or with inactivated carrier protein proved the dependence of this transport activity on the presence of active carnitine carrier. The mercurial-induced uniport correlated with inhibition of the 'physiological' functions of the carrier, i.e., exchange and substrate specific unidirectional transport. The effect of consecutive additions of various reagents including N-ethylmaleimide, mercurials, Cu(2+)-phenanthroline and diamide on the transport function revealed the presence of at least two different classes of SH-groups. N-Ethylmaleimide blocked the carrier activity by binding to SH-groups of one of these classes. At least one of these SH-groups could be oxidized by the reagents forming S-S bridges. Besides binding to the class of SH-groups to which N-ethylmaleimide binds, mercurials also reacted with SH-groups of the other class. Modification of the latter led to the induction of the efflux-type of carrier activity characterized by loss of substrate specificity.     

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain

Summary Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3±0.11 and 0.8±0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.This work was supported by grants from Statens Naturvetenskapliga. Forsknigsråd (No. B0294-020), Stiftelsen Lars Hiertas Minne, Adlerbertska Forskningsfonden, Hierta-Retzius' Stipendiefond, Anna Ahrenbergs Fond, and Wilhelm och Martina Lundgrens Vetenskapsfond. Appreciation is extended to Inger Holmqvist for her excellent technical assistance     

Tubulin microheterogeneity during mouse liver development

Liver tubulin was analyzed and characterized during the development of the mouse, from embryonic stages to adult. Separation and identification of isotubulins were achieved by isoelectric focusing, two-dimensional gel electrophoresis and peptide mapping. Liver tubulin is a heterogeneous population composed of 7 isoforms, 4 α and 3 β. By comparison with the events occurring at the tubulin level during brain development, we observed that 1) tubulin heterogeneity is less important in liver than in brain 2) no appearance of new isotubulins takes place during development 3) the only developmental change detectable in our gels is an accumulation of an α tubulin subunit which is absent from brain isotubulin population. The relationship between this α isotubulin accumulation and liver secretion during development is discussed.     

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

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

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

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

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

A kinetic study of the reactive capabilities of xanthine oxidase sulfhydryl groups with regard to n-chlormercuribenzoate]

Kinetic characteristics for reactivity of SH-groups of milk xanthine oxidase were obtained under different conditions. Two types of SH-groups with rate constant values, differing by a factor of about 50, were found in a phosphate buffer at pH 7.0. The slow stage of reaction is followed by protein precipitation. The number of fast- (12) and slowly-reacting (60) groups were calculated from the kinetic data. The blocking of the fast-reacting groups occurs without loss of the enzyme activity. The values of activation energy for the fast- and slowly-reacting groups are 15 and 48 kcal/mol respectively. The formation of the enzyme-substrate complex stabilizes the enzyme molecule; the number of fast-reacting SH-groups and the rate constant values for both types of groups remain unchanged, whereas the number of slowly-reacting SH-groups markedly decreases (37). The values of activation energy for both types of SH-groups show no changes in the presence of substrate. Conformations of the enzyme in different denaturating solvents were characterized by a number of SH-groups, reacting with p-chloromercurybenzoate. 54 groups are exposed in solutions of groups exposed in 7.0-8.5 M urea solutions is 35-38. In all solvents studied the protein molecule is probably not completely unfolded, since the number of exposed SH-groups is less than the full number of SH-groups determined by the amino acid analysis. Only 42 SH-groups reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) under the same conditions.     

Heat stress pretreatment mitigates postischemic arachidonic acid accumulation in rat heart

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Diabetes-induced increase of renal medullary hydrogen peroxide and urinary angiotensinogen is similar in normotensive and hypertensive rats

Aims

Activation of renal renin–angiotensin system (RAS) and reactive oxygen species (ROS) are the main pathophysiological mechanisms associated with kidney injury both in diabetes and hypertension. However, the contribution to medullary damage when the two pathologies coexist has seldom been explored.

Main methods

Diabetes was induced with streptozotocin in twelve week-old male Wistar and spontaneously hypertensive rats (SHR) rats; controls received vehicle. Three weeks later, systolic blood pressure (SBP), plasma and urinary angiotensinogen (AGT), renal oxidative stress and metabolic status were evaluated.

Key findings

SBP was higher in SHR-controls than in Wistar-controls (200 ± 6 and 127 ± 3 mmHg, respectively) and decreased in SHR-diabetics but not in Wistar-diabetics (143 ± 8 and 122 ± 6 mmHg, respectively). Renal medullary hydrogen peroxide (H₂O₂) production was similarly increased in diabetics (Wistar: 0.32 ± 0.04 and 1.11 ± 0.10 nmol/mg protein, respectively; SHR: 0.40 ± 0.05 and 0.90 ± 0.14 nmol/mg protein, respectively) and positively correlated with glycemia (Wistar: r = 0.7166, SHR: r = 0.7899, p < 0.05) and urinary AGT excretion (Wistar: r = 0.8333; SHR: r = 0.8326, p < 0.05). Cortical H₂O₂ production was higher in SHR-controls than in Wistar-controls (1.10 ± 0.09 and 0.26 ± 0.04 nmol/mg protein, respectively) and diabetes induction decreased it in SHR (0.70 ± 0.09 nmol/mg protein). Diabetes increased urinary AGT excretion by more than 7-fold and decreased plasma AGT concentration by more than 1.5-fold in both strains.

Significance

Our results show that STZ-induced diabetes increases medullary H₂O₂ production and urinary AGT excretion with similar magnitude in normotensive and hypertensive animals. Reducing blood pressure attenuates hypertension-associated cortical damage but does not prevent medullary dysfunction.     

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

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

Autoregulation of tubulin synthesis in hepatocytes and fibroblasts

Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.     

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

Background

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

Methods

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

Results

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

Conclusions

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

     

GYY4137 ameliorates sepsis-induced cardiomyopathy via NLRP3 pathway

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