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Abstract

Hydrogen sulphide (H₂S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H₂S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO^•) bioactivity were significantly reduced (p < 0.05), and maximal vasorelaxation to the NO^• donor sodium nitroprusside was impaired (p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase (p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 (p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO^• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO^• efficacy and reducing superoxide production in the mouse aorta.

Keywords

Hydrogen sulphide endothelial dysfunction oxidative stress NADPH oxidase diabetes vasoprotection

Introduction

In mammalian tissues, reactive oxygen species (ROS) are produced in both pathological and physiological conditions. The parent ROS molecule superoxide $(O_{2}^{\cdot -})$ is produced by mitochondria and several oxidases within vascular tissue including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, cyclooxygenases and endothelial nitric oxide synthase (eNOS) in its uncoupled state.¹ Normally, $(O_{2}^{\cdot -})$ is rapidly dismutated to hydrogen peroxide (H₂O₂) by superoxide dismutase (SOD), then H₂O₂ is converted to H₂O and O₂ by catalase or glutathione peroxidase (GPx). However, if the production of ROS is excessive and/or ROS metabolism is impaired, oxidative stress can develop. Oxidative stress is associated with many cardiovascular diseases, including diabetes-induced vascular disease, and is believed to be a key mediator of vascular dysfunction.^2,3 One of the best characterised mechanisms by which oxidative stress can cause vascular dysfunction is via the inactivation of endothelium-derived nitric oxide (NO^•; commonly referred to as endothelial dysfunction) by $(O_{2}^{\cdot -})$ .⁴ The reaction of $(O_{2}^{\cdot -})$ with NO^• reduces its bioactivity within the vascular wall leading to increased vascular tone and loss of anti-platelet effects.⁵ In addition, this reaction can result in the formation of peroxynitrite (ONOO^•), which in turn can cause protein nitration and lipid peroxidation.⁶

Vascular dysfunction, of both micro- and macro-vessels, is a major cause of morbidity in diabetes. Endothelial dysfunction, in both conductance and resistance arteries, is an initiating factor in the pathogenesis of vascular disease in both type 1 and type 2 diabetes^7,8 There is considerable evidence that hyperglycaemia leads to oxidative stress resulting in endothelial dysfunction.^9,10

Hydrogen sulphide (H₂S) is well known as a toxic gas with the distinct smell of rotten eggs; however, there is now an abundance of literature to indicate that H₂S is a gasotransmitter with important physiological functions.¹¹ In particular, there is mounting evidence that H₂S is a cytoprotective molecule with anti-inflammatory and anti-oxidant effects,¹² including in the vasculature.¹³ Indeed, H₂S is a potent one-electron chemical reductant and nucleophile that is reported to be capable of scavenging free radicals.¹⁴ H₂S is also important in regulating mitochondrial function^15,16 and reduces mitochondrial ROS formation.¹⁷ There is evidence that H₂S decreases vascular $(O_{2}^{\cdot -})$ generation by inhibiting NADPH oxidase activity in vitro.^18,19 H₂S donors boost glutathione levels via enhanced l-cystine transport,²⁰ and increasing the activity of GPx and glutathione reductase.²¹ Moreover, H₂S donors have been shown to increase the activity and expression of SOD and synergistically increase the activity of SOD under conditions of oxidative stress, to elicit cytoprotection.¹⁷ Finally, H₂S is reported to increase the nuclear translocation of Nrf2 leading to the increased expression of downstream anti-oxidant mechanisms.²² These data indicate that H₂S has the capacity for a multi-faceted enhancement of cellular anti-oxidant defences and thus may be a useful therapeutic agent for reducing vascular oxidative stress and improving vascular function during cardiovascular disease.

The vasoprotective effects of H₂S have largely been demonstrated in vitro or in cell-based assays, but data showing that these effects are translated in vivo are lacking. There is evidence of the vasoprotective effects of H₂S in hypertension²³ and atherosclerosis,²⁴ and sodium hydrosulfide (NaHS) treatment can attenuate endothelial dysfunction in diabetic rats.²⁵ These conditions all involve vascular oxidative stress and the potential protective effects of H₂S on the vasculature under oxidative stress need to be further investigated. Therefore, the purpose of this study is to investigate the ability of chronic NaHS treatment to protect vascular function in vivo under conditions of diabetes-induced oxidative stress.

Methods

All experimental procedures were approved by the Animal Experimentation Ethics Committee of RMIT University and conformed to the National Health and Medical Research Council of Australia code of practice for the care and use of animals for scientific purposes.

Induction of diabetes

Male 8-week-old C57BL/6J mice (Animal Resource Centre, Western Australia) were randomly divided into three groups: control, diabetic and diabetic treated with NaHS. Type 1 diabetes was induced with streptozotocin (STZ, 60 mg/kg/day, ip) for 2 weeks, whereas the control group received an equivalent volume of vehicle (0.1 M citrate buffer, pH: 4.5). Five weeks after the commencement of STZ injections, half the diabetic mice were randomly allocated to receive NaHS (100 µmol/kg/day, ip) for a further 4 weeks. Blood glucose level was measured at the beginning and at the end of the experiment and twice a week during the experiment for diabetic mice. Insulin 0.1 U, ip, 50% isophane insulin (Protaphane^®) + 50% neutral insulin (Actrapid^®) was given when the blood glucose level exceeded 30 mM to prevent excessive loss of weight. Control group blood glucose level was also measured during week 5. Blood samples were obtained from the tail vein and non-fasting blood glucose concentration was measured using a one touch glucometer ACCU-CHEK Advantage^® (Roche, NSW, Australia) and glycated haemoglobin (HbA_1C) using an in2it (II) analyser (281-0000EX) (Bio-Rad, NSW, Australia). Induction of diabetes was considered successful when the blood glucose level was >25 mM.

Tissue collection

Mice were culled humanely using CO₂ asphyxiation (95% CO₂, 5% O₂) followed by cervical dislocation and decapitation, at which point blood samples were collected for determination of HbA_1C and plasma total sulphide concentrations. The whole aorta was isolated and immediately placed in ice cold Krebs bicarbonate solution [composition (mM): NaCl 118, KCl 4.7, MgSO₄ 1.18, KH₂PO₄ 1.2, NaHCO₃ 25, d-glucose 11, CaCl₂ 1.6] and then carefully cleared of fat and connective tissues. Thoracic or abdominal aorta were cut into 2 mm long rings and used immediately for the superoxide assay and vascular reactivity studies, and some aorta samples were snap frozen for western blot analysis.

Vascular reactivity studies

Aortic rings of 2-mm long were mounted on a multi-wire myograph system 610 M (Danish Myo Technology, Aarhus, Denmark) filled with Krebs bicarbonate solution. The vessels were allowed to stabilise at zero tension for 10 min before the 15 min equilibration period at 5 mN. All experiments were performed at 37°C and continuously bubbled with carbogen (95% O₂ and 5% CO₂). Changes in tension were recorded using a Powerlab data acquisition system (ADInstruments, Australia).

Assessment of vascular endothelial and smooth muscle cell function

Vascular function was examined as previously described²⁶ with the following modifications: Aortic rings were maximally contracted with the thromboxane A₂ mimetic, U46619 (1 µM) and once the U46619-induced maximum contraction reached a plateau (F_max), the aortic rings were washed with Krebs solution to regain their basal tension. Following determination of F_max, the aortic rings were then pre-contracted to ~50% F_max using titrated concentrations of U46619 (0.1–100 nM) followed by cumulative concentration–response curves to acetylcholine (ACh, 0.1 nM–10 µM) to assess endothelial function. Endothelium-independent relaxation was examined with the nitric oxide donor sodium nitroprusside (SNP, 0.1 nM–3 µM) and the K_ATP channel opener levcromakalim (LKM, 0.1 nM–10 µM). Maximal relaxation was achieved by adding the Ca²⁺ channel blocker nifedipine (10 µM) at the end of each concentration–response curve.

Assessment of basal NO^• bioactivity

In a separate set of experiments, the effect of diabetes on basal NO^• bioactivity was examined by measuring the contractile response of endothelium intact aortic rings to the NO^• synthase inhibitor N_ω-Nitro-l-arginine methyl ester (L-NAME 100 µM). Briefly, rings were pre-contracted with U46619 (0.1–10 nM) to approximately 30% of the maximal contraction followed by the addition of L-NAME. Under these conditions, the contractile response induced by L-NAME is considered to reflect basal NO^• bioactivity.²⁷

Vascular superoxide production

NADPH oxidase–derived superoxide production in the aorta was measured using lucigenin-enhanced chemiluminescence assay. Aortic rings (2 mm) were pre-incubated for 45 min at 37°C in Krebs-HEPES buffer [composition (mM): NaCl 99.9, KCl 4.7, KH₂PO₄ 1.0, MgSO₄ 1.2, d-glucose 11.0, NaHCO₃ 25.0, CaCl₂ 2.5, Na-HEPES 20.0, pH 7.4] containing diethyldithiocarbamic acid (1 mM) to inactivate endogenous SOD, and NADPH (100 µM) as a substrate for NADPH oxidase, with and without diphenyleneiodonium (DPI; 5 µM), a flavoprotein inhibitor of NADPH oxidase. Krebs-HEPES buffer containing lucigenin (5 µM) and the appropriate treatment were placed in a 96-well Optiplate, and superoxide production was measured and quantified as previously reported.²⁸

Western blotting

Western blotting was performed as described previously.²⁹ Briefly, aortae were excised, snap frozen in liquid nitrogen and homogenised in Laemmli buffer [25% glycerin, 12.5% β-mercaptoethanol, 7.5% sodium docedyl sulphate, 25% 1 mol/L Tris–HCl (pH: 8.0) and 0.25 mg/mL bromophenol blue] over liquid nitrogen. Protein concentration was determined using the reducing agent and detergent compatible (RCDC) assay (Bio-Rad). Equal amounts of protein were loaded onto a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% skim milk for 1 h and then incubated overnight at 4°C with the appropriate primary antibody (1:1000 for eNOS and NOX2, 1:2000 for β-actin) in 5% skim milk. Membranes were then incubated with a horseradish peroxidase–conjugated anti-mouse IgG for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence, quantified using a ChemiDoc XRS molecular imager (Bio-Rad) and normalised to intensity of corresponding bands for β-actin.

Plasma sulphide assay

Mouse plasma samples were added to zinc acetate (1% w/v) followed by the addition of trichloroacetic acid (10% w/v). Subsequently, N,N-dimethyl-p-phenylenediamine sulphate (20 mM) in 7.2 M HCl and FeCl₃ (30 mM) in 1.2 M HCl were added to each of the samples. After 20-min incubation at room temperature, the sample solutions were centrifuged at nine relative centrifugal force for 1 min and 250 µL of each sample supernatant was pipetted into a 96-well plate and measured at 670 nm using a spectrophotometer (Thermo Scientific Multiskan Spectrum). The H₂S concentration of each sample was calculated against a calibration curve of NaHS (0–200 µM) and results are expressed in micromolar.

Reagents

All drugs were purchased from Sigma-Aldrich (USA), except for acetylcholine perchlorate (ACh, BDH Chemicals, UK), LKM (Tocris, Australia) and U46619 (Sapphire Bioscience, Australia). All drugs were dissolved in distilled water, except LKM and nifedipine which were dissolved in ethanol.

Statistical analyses

All results are expressed as mean ± standard error of mean (SEM), n represents the number of animals per group. Concentration–response curves from mouse aorta experiments were computer fitted to a sigmoidal curve using non-linear regression (Prism version 6; GraphPad Software, USA) to calculate the sensitivity of each agonist (pEC₅₀). Maximum relaxation (R_max) to ACh, SNP and LKM was measured as a percentage of pre-contraction to U46619, where the response to nifedipine was considered to be 100%. Group pEC₅₀ and R_max values were compared by one-way analysis of variance (ANOVA) with post hoc analysis using the Holm–Sidak test. p < 0.05 was considered statistically significant.

Results

Body weight and blood glucose levels

Body weight gained, blood glucose level, HbA_1C and plasma sulphide levels are shown in Table 1. A total of 9 weeks after commencement of treatment with STZ or citrate buffer, the body weight gain in control mice was significantly greater than in diabetic mice (p < 0.05). The blood glucose and HbA_1C levels in diabetic mice were significantly greater than control mice (p < 0.05). Treatment with NaHS had no significant effect on body weight gain, blood glucose or HbA_1C levels in diabetic animals. Total plasma sulphide levels at the end of the experiment were not different between the groups.

Table 1.

Body weight, blood glucose and plasma sulphide data.

	n	Control	n	Diabetic	n	Diabetic and NaHS
Body weight gained (g)	22	8.0 ± 0.6	22	3.3 ± 0.8*	21	3.0 ± 0.5*
Blood glucose (mM)	22	9.1 ± 0.5	22	31.7 ± 0.9*	21	31.5 ± 0.8*
HbA_1C (%)	12	5.6 ± 0.2	15	11.1 ± 0.3*	12	10.4 ± 0.4*
Total plasma sulphide (µM)	8	25 ± 3	10	20 ± 2	9	20 ± 3

NaHS: sodium hydrosulfide; HbA_1C: glycated haemoglobin; ANOVA: analysis of variance.

Values recorded at the end of the experiment.

p < 0.0001 cf control, ANOVA, post hoc Holm–Sidak test.

Effect of diabetes and NaHS treatment on endothelial function

The maximal contraction elicited by U46619 was not affected by diabetes or NaHS treatment (Table 2). Pre-contraction levels for vasorelaxant concentration–response curves were matched between the groups (Table 2). Diabetes significantly reduced the sensitivity and maximal relaxation to ACh (Figure 1(a) Table 2, p < 0.05). Treatment with NaHS in diabetic mice returned the sensitivity and maximal relaxation response to ACh to that of the control group. NO^• bioactivity, assayed by the contractile response to L-NAME, was significantly reduced in the diabetic group, but this was also restored with NaHS treatment (Figure 1(b), p < 0.05).

Table 2.

Parameters for vascular reactivity experiments.

Group	n	pEC₅₀	R _max	%PC	U4_max
ACh
Control	14	7.19 ± 0.08	87.0 ± 3.2	52.5 ± 1.2	10.7 ± 0.9
Diabetic	11	6.71 ± 0.10^φ	68.1 ± 4.6*	52.5 ± 1.7	12.0 ± 1.2
Diabetic and NaHS	9	6.98 ± 0.10	78.1 ± 4.5^ψ	51.0 ± 3.3	8.8 ± 1.1
SNP
Control	7	7.98 ± 0.11	90.3 ± 1.1	51.5 ± 4.0	8.5 ± 1.0
Diabetic	8	7.58 ± 0.09	83.0 ± 2.6*	51.5 ± 2.2	11.3 ± 1.0
Diabetic and NaHS	5	7.69 ± 0.08	96.4 ± 5.5^ψ	48.6 ± 2.5	11.7 ± 1.0
LKM
Control	6	5.43 ± 0.11	72.0 ± 4.9	55.4 ± 6.2	8.9 ± 1.0
Diabetic	7	5.69 ± 0.14	87.0 ± 5.4	45.9 ± 1.1	11.4 ± 1.1
Diabetic and NaHS	5	6.01 ± 0.20	83.2 ± 0.6	47.0 ± 3.1	11.7 ± 1.0

ACh: acetylcholine; SNP: sodium nitroprusside; LKM: levcromakalim; n: number of aortic rings, each from a different animal; pEC₅₀: −log of the concentration required to elicit a response 50% of maximum; R_max: maximum vasorelaxation response (%); %PC: pre-contraction value as a percentage of the maximum attainable contraction; U4_max: maximum attained contraction (mN) with 1 µΜ U46619; ANOVA: analysis of variance; NaHS: sodium hydrosulfide.

p < 0.05 cf control pEC₅₀; *p < 0.05 cf control; ^ψp < 0.05 cf diabetic R_max; ANOVA, post hoc Holm–Sidak test.

Figure 1.

(a) Vasorelaxation response to ACh. φp < 0.05 cf control pEC₅₀, *p < 0.05 cf control R_max, ^ψp < 0.05 cf diabetic R_max, ANOVA, post hoc Holm–Sidak test, n = 9–14. (b) Basal NO^• bioactivity in aorta from control, diabetic and diabetic treated with NaHS mice *p < 0.05 cf control, ns = p > 0.05 control cf diabetic and NaHS, ANOVA, post hoc Holm–Sidak test, n = 5–7.

Effect of diabetes and NaHS treatment on vascular smooth muscle cell function

The maximum vasorelaxation response elicited by exogenous NO^•, delivered via SNP, was significantly reduced in the diabetic group although the sensitivity was unaffected. The maximum relaxation to SNP was restored in the NaHS treated group (Figure 2(a), Table 2, p < 0.05). The response to the K_ATP channel opener LKM was neither affected by diabetes nor treatment with NaHS (Table 2).

Figure 2.

Vasorelaxation response to the NO^• donor SNP in aorta from control, diabetic and diabetic treated with NaHS mice. *p < 0.05 cf control R_max, ^ψp < 0.05 cf diabetic R_max, ANOVA, post hoc Holm–Sidak test, n = 5–8.

Effect of diabetes and NaHS treatment on vascular superoxide production

The level of superoxide production detected by lucigenin-enhanced chemiluminescence in aorta from diabetic mice was significantly increased compared to control mice (Figure 3, p < 0.05). NaHS treatment significantly reduced superoxide production from diabetic aorta (p < 0.05). In all groups, superoxide production from aorta could be virtually abolished by DPI, a flavoprotein inhibitor of NADPH oxidase (Figure 3).

Figure 3.

Superoxide production measured by lucigenin-enhanced chemiluminescence in aorta from control, diabetic and diabetic treated with NaHS mice, with (filled bars) and without (open bars) the NADPH oxidase inhibitor DPI. *p < 0.05 cf control, ^ψp < 0.05 cf diabetic, ANOVA, post hoc Holm–Sidak test, n = 5–8.

Effect of diabetes and NaHS treatment on NOX2 and eNOS protein expression

NOX2 expression was significantly enhanced in the diabetic group, but this was not affected by NaHS treatment (Figure 4, p < 0.05). Total eNOS expression was unaffected by diabetes or NaHS treatment (Figure 4).

Figure 4.

NOX2 expression, normalised to β-actin in aorta from control, diabetic and diabetic treated with NaHS mice. (a) Example western blots from each group. (b). Group data, *p < 0.05 cf control, ANOVA, post hoc Holm–Sidak test, n = 9. eNOS expression, normalised to β-actin in aorta from control, diabetic and diabetic treated with NaHS mice. (c) Example western blots from each group. (d). Group data, p > 0.05, ANOVA, post hoc Holm–Sidak test, n = 9.

Discussion

This study demonstrates the ability of H₂S to protect endothelial function under conditions of oxidative stress, in diabetes. Indeed, we have shown that treatment of diabetic animals with the fast H₂S donor NaHS reduces vascular superoxide production by inhibiting vascular NADPH oxidase activity. This is associated with an improvement in endothelial function, likely as a result of reduced inactivation of NO^• by superoxide. Consistent with this, our data also show that the efficacy of exogenous NO^• is protected by NaHS treatment. This is the first study to show the specific protective effects of NaHS on the vasculature in diabetes, where there is increased oxidative stress, and it provides further evidence of the potential role of H₂S in regulating vascular NADPH oxidase function in vivo.

Daily NaHS treatment for 4 weeks following induction of diabetes had a marked protective effect on endothelial function, by restoring ACh-mediated vasorelaxation and basal NO^• bioactivity, as assessed by the contraction response to L-NAME. This may relate to an effect on eNOS activity or may be due to a protective effect of endogenous NO^•, after production. There was no significant difference in total eNOS protein expression with diabetes or NaHS treatment. Previous studies have shown that diabetes reduces the expression of the physiological dimer form of the enzyme³⁰ which produces NO^•, and increases the presence of the monomer form, which is a superoxide generating enzyme³¹ further exacerbating oxidative stress. Indeed, H₂S has been previously shown to have a stimulatory effect on the NO^• signalling pathway since there is in vitro evidence that H₂S facilitates the phosphorylation and s-sulfhydration of eNOS³² as well as increasing the production of NO^• from endothelial cells.^33,34 This study did not examine the abundance of monomer versus dimer expression of eNOS nor changes in the phosphorylation of the enzyme, which are known to be altered in diabetes³⁵ and these would be important lines of investigation for future studies.

Consistent with previous reports, we found that diabetes induced vascular oxidative stress as evidenced by increased NADPH-oxidase dependent vascular superoxide production and increased NOX2 subunit expression. In addition, endothelial function was significantly impaired, with both reduced ACh-mediated vasorelaxation and basal NO^• bioactivity, as we and others have previously described.^10,27,30

In addition, this study also showed that NaHS treatment could act to protect the activity of exogenously derived NO^•. In diabetic aorta, the maximum relaxation response to the NO^• donor SNP was significantly reduced, but treatment with NaHS restored the efficacy of exogenous NO^• to control levels.

Previous work has shown that H₂S is a K_ATP channel opener;³⁶ however, we saw no difference in the ex vivo aortic vasorelaxation response to the K_ATP channel opener LKM. These data show that the vascular smooth muscle cell function in diabetic mice was not compromised; thus, the decrease in vasorelaxation to ACh and SNP was due to a NO^•-dependent deficit.

It is known that superoxide can readily react with and reduce the availability of NO^•,⁶ so it is possible that NaHS-induced reduction of NADPH oxidase activity and therefore superoxide production may be the mechanism behind this effect.

Recent studies have focused on the ability of H₂S to elicit cytoprotective effects^12,13 in a number of tissues by mechanisms that include potentially scavenging ROS,^14,19 activating anti-oxidant gene expression (Nrf2 and nfKb) and enhancing expression of other anti-oxidant molecules.^17,22 In addition, there is evidence that H₂S may suppress ROS production by the NADPH oxidases. This study extends these findings of H₂S-induced suppression of NADPH oxidase activity in the vasculature. There are seven isoforms of mammalian NADPH oxidase, which produce both superoxide and H₂O₂. These enzymes are essential for host defence and are important for cellular signalling; however, there is compelling evidence that overactivity of NADPH oxidase contributes to oxidative stress in cardiovascular diseases.³⁷

The effect of H₂S on the various isoforms of NADPH oxidase has not been well studied. Previous studies in vascular smooth muscle cells showed that NaHS inhibited NADPH oxidase induced superoxide production, NOX1 subunit expression and Rac activity.¹⁸ Another report showed NaHS could inhibit NADPH oxidase induced superoxide production and reduce NOX2 subunit expression in endothelial cells.³⁸ More recent in vivo studies have shown NaHS inhibits NOX4 expression in a rat model of myocardial ischaemia, resulting in reduced fibrosis³⁹ and reduced vascular NADPH oxidase activity has been shown in mouse models of angiotensin II–induced hypertension²³ and atherosclerosis.²⁴ This study has focused only on the NOX2 subunit of the enzyme as it has been previously shown to be pathologically up-regulated in cardiovascular diseases.⁴⁰ The data show that NaHS treatment could ameliorate the increase in NADPH-oxidase activity seen in diabetes, but there was no effect on NOX2 subunit expression, which is in contrast to the previous study,³⁸ the main difference being that this study was in aorta samples ex vivo rather than in cultured endothelial cells. It should be noted that in this study the superoxide signal was virtually abolished by DPI, a flavoprotein inhibitor, which is specific for NADPH oxidase at 5 µΜ, indicating that the superoxide detected in response to NADPH is derived from a NADPH oxidase. The data suggest that NaHS is reducing superoxide production by inhibiting NADPH oxidase activity, an effect that can occur in the absence of a decrease in NOX2 expression. It is possible that other subunits modulate the activity of NOX2-NADPH oxidase. Although unlikely it is not possible to exclude the possibility of NaHS inhibiting other ROS enzymatic sources in addition to NADPH oxidase. There is still a lack of information regarding H₂S donors and their potential effects on NADPH oxidase subunits and more work in this area is warranted.

It is important to note that these results are obtained from a single daily ip injection of NaHS. NaHS is an inorganic salt which when dissolved releases H₂S immediately;⁴¹ thus, it is considered a ‘fast’ donor of H₂S. The t_1/2 of NaHS is estimated in the range of seconds to minutes,⁴² then it is sequestered or metabolised to sulphate. Thus, the method of administration used in this study results in a transient high concentration of H₂S which is quickly cleared as plasma sulphide levels are not different between the groups. This suggests the NaHS is having an immediate but long-lasting effect (e.g. via protein sulfhydration or altering gene expression), or that the H₂S delivered is being sequestered or bound for later release. All these possibilities have been reported previously.^17,22,43,44

In summary, chronic treatment with the fast H₂S donor NaHS results in reduced oxidative stress in diabetes. This effect is possibly mediated via the inhibition of the NOX2-containing isoform of NADPH oxidase, which leads to a protection of the endogenous production and action of NO^• in the vasculature. These data further confirm the anti-oxidant effect of H₂S and H₂S donors may prove to be a useful therapeutic in the treatment of diabetes-related vascular complications.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

References

Miller

Budzyn

Sobey

CG.

Vascular dysfunction in cerebrovascular disease: mechanisms and therapeutic intervention. Clin Sci 2010; 119: 1–17.

Brandes

Weissmann

Schroder

NADPH oxidases in cardiovascular disease. Free Radic Biol Med 2010; 49: 687–706.

De Silva

Miller

. Cerebral small vessel disease: targeting oxidative stress as a novel therapeutic strategy? Front Pharmacol 2016; 7: 61.

Graier

Posch

Fleischhacker

. Increased superoxide anion formation in endothelial cells during hyperglycemia: an adaptive response or initial step of vascular dysfunction? Diabetes Res Clin Pract 1999; 45: 153–160.

Langenstroer

Pieper

GM.

Regulation of spontaneous EDRF release in diabetic rat aorta by oxygen free radicals. Am J Physiol 1992; 263: H257–H265.

Pacher

Beckman

Liaudet

Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007; 87: 315–424.

Rask-Madsen

King

GL.

Mechanisms of disease: endothelial dysfunction in insulin resistance and diabetes. Nat Clin Pract Endocrinol Metab 2007; 3: 46–56.

Kahlberg

Qin

Anthonisz

. Adverse vascular remodelling is more sensitive than endothelial dysfunction to hyperglycaemia in diabetic rat mesenteric arteries. Pharmacol Res 2016; 111: 325–335.

Ortega Mateo

De Artiñano

. Nitric oxide reactivity and mechanisms involved in its biological effects. Pharmacol Res 2000; 42: 421–427.

10.

Leo

Parry

LJ.

Serelaxin (recombinant human relaxin-2) prevents high glucose-induced endothelial dysfunction by ameliorating prostacyclin production in the mouse aorta. Pharmacol Res 2016; 107: 220–228.

11.

Beltowski

Hydrogen sulfide in pharmacology and medicine: an update. Pharmacol Rep 2015; 67: 647–658.

12.

Wallace

Blackler

Chan

. Anti-inflammatory and cytoprotective actions of hydrogen sulfide: translation to therapeutics. Antioxid Redox Signal 2015; 22: 398–410.

13.

Streeter

Hart

JL.

Hydrogen sulfide as a vasculoprotective factor. Med Gas Res 2013; 3: 9.

14.

Wedmann

Bertlein

Macinkovic

. Working with ‘H2S’: facts and apparent artifacts. Nitric Oxide 2014; 41: 85–96.

15.

Szabo

Ransy

Modis

. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. Br J Pharmacol 2014; 171: 2099–2122.

16.

Modis

Bos

Calzia

. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part II. Pathophysiological and therapeutic aspects. Br J Pharmacol 2014; 171: 2123–2146.

17.

Sun

Liu

Chen

. Hydrogen sulfide decreases the levels of ROS by inhibiting mitochondrial complex IV and increasing SOD activities in cardiomyocytes under ischemia/reperfusion. Biochem Biophys Res Commun 2012; 421: 164–169.

18.

Muzaffar

Shukla

Bond

. Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells. J Vasc Res 2008; 45: 521–528.

19.

Al-Magableh

Kemp-Harper

. Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro. Naunyn Schmiedebergs Arch Pharmacol 2014; 387: 67–74.

20.

Kimura

Goto

Kimura

Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria. Antioxid Redox Signal 2010; 12: 1–13.

21.

Benetti

Campos

Gurgueira

. Hydrogen sulfide inhibits oxidative stress in lungs from allergic mice in vivo. Eur J Pharmacol 2013; 698: 463–469.

22.

Calvert

Jha

Gundewar

. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res 2009; 105: 365–374.

23.

Al-Magableh

Kemp-Harper

Hart

JL.

Hydrogen sulfide treatment reduces blood pressure and oxidative stress in angiotensin II-induced hypertensive mice. Hypertens Res 2015; 38: 13–20.

24.

Ford

Al-Magableh

Gaspari

. Chronic NaHS treatment is vasoprotective in high-fat-fed ApoE(-/-) mice. Int J Vasc Med 2013; 2013: 915983.

25.

Suzuki

Olah

Modis

. Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function. Proc Natl Acad Sci U S A 2011; 108: 13829–13834.

26.

Jelinic

Parry

. Increased superoxide production and altered nitric oxide-mediated relaxation in the aorta of young but not old male relaxin-deficient mice. Am J Physiol Heart Circ Physiol 2015; 309: H285–H296.

27.

Leo

Hart

Woodman

OL.

3′,4′-Dihydroxyflavonol reduces superoxide and improves nitric oxide function in diabetic rat mesenteric arteries. PLoS ONE 2011; 6: e20813.

28.

Woodman

Malakul

3′,4′-Dihydroxyflavonol prevents diabetes-induced endothelial dysfunction in rat aorta. Life Sci 2009; 85: 54–59.

29.

Andrews

Barsby

. Ghrelin-related peptides exert protective effects in the cerebral circulation of male mice through a nonclassical ghrelin receptor(s). Endocrinology 2015; 156: 280–290.

30.

Leo

Hart

Woodman

OL.

Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes. Br J Pharmacol 2011; 162: 365–377.

31.

Stuehr

Pou

Rosen

GM.

Oxygen reduction by nitric-oxide synthases. J Biol Chem 2001; 276: 14533–14536.

32.

Altaany

Yang

. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. Sci Signal 2014; 7: ra87.

33.

Kida

Sugiyama

Yoshimoto

. Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells. Eur J Pharm Sci 2013; 48: 211–215.

34.

Chen

Wang

. Hydrogen sulfide increases nitric oxide production and subsequent S-nitrosylation in endothelial cells. ScientificWorldJournal 2014; 2014: 480387.

35.

Atochin

Kashiwagi

. Deficient eNOS phosphorylation is a mechanism for diabetic vascular dysfunction contributing to increased stroke size. Stroke 2013; 44: 3183–3188.

36.

Shen

. Mechanism underlying enhanced endothelium-dependent vasodilatation in thoracic aorta of early stage streptozotocin-induced diabetic mice. Acta Pharmacol Sin 2003; 24: 422–428.

37.

Drummond

Selemidis

Griendling

. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov 2011; 10: 453–471.

38.

Muzaffar

Jeremy

Sparatore

. H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G. Br J Pharmacol 2008; 155: 984–994.

39.

Pan

Liu

Shen

. Inhibition of NADPH oxidase 4-related signaling by sodium hydrosulfide attenuates myocardial fibrotic response. Int J Cardiol 2013; 168: 3770–3778.

40.

Drummond

Sobey

CG.

Endothelial NADPH oxidases: which NOX to target in vascular disease?

Trends Endocrin Met 2014; 25: 452–463.

41.

Papapetropoulos

Whiteman

Cirino

Pharmacological tools for hydrogen sulphide research: a brief, introductory guide for beginners. Br J Pharmacol 2015; 172: 1633–1637.

42.

Reiffenstein

Hulbert

Roth

SH.

Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 1992; 32: 109–134.

43.

Mustafa

Gadalla

Sen

. H2S signals through protein S-sulfhydration. Sci Signal 2009; 2: ra72.

44.

Shen

Peter

Bir

. Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radic Biol Med 2012; 52: 2276–2283.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice

Abstract

Keywords

Introduction

Methods

Induction of diabetes

Tissue collection

Vascular reactivity studies

Assessment of vascular endothelial and smooth muscle cell function

Assessment of basal NO• bioactivity

Vascular superoxide production

Western blotting

Plasma sulphide assay

Reagents

Statistical analyses

Results

Body weight and blood glucose levels

Effect of diabetes and NaHS treatment on endothelial function

Effect of diabetes and NaHS treatment on vascular smooth muscle cell function

Effect of diabetes and NaHS treatment on vascular superoxide production

Effect of diabetes and NaHS treatment on NOX2 and eNOS protein expression

Discussion

Footnotes

Declaration of conflicting interests

Funding

References

Assessment of basal NO^• bioactivity