EXOGENOUS HYDROGEN SULFIDE ATTENUATES OXIDATIVE STRESS IN SPONTANEOUSLY HYPERTENSIVE RATS

EXOGENOUS HYDROGEN SULFIDE ATTENUATES OXIDATIVE STRESS IN SPONTANEOUSLY HYPERTENSIVE RATS

Fiaz ud din Ahmad*¹, Munavvar A. Sattar ¹, Hassaan A. Rathore ¹, Abdullah Ijaz Hussain ¹, Tan Yong Chia ¹, Oh Hui Jin ¹, Yen Pei Pei ¹, Ishfaq Ahmad ¹, Nor A. Abdullah ² and Edward J. Johns ³

School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800, Penang, Malaysia

Department of Pharmacology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia

Department of Physiology, Western Gateway Building, University College Cork, Cork, Ireland

ABSTRACT:Vascular oxidative stress occurs in hypertensive states and the potential role of antihypertensive drugs as antioxidants is currently under review. Hydrogen sulfide (H₂S) is a recently recognized member of gasotransmitters, which may act to decrease blood pressure in experimental hypertension. The present study evaluated the antioxidant potential of hydrogen sulfide in SHR. NaHS, donor of H₂S, was subjected to a series of in vitro tests to evaluate its antioxidant capacity. For in vivo study Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were divided in 4 groups namely WKY control (I), WKY-NaHS treated (II), SHR control (III) and SHR-NaHS treated (IV). Groups II and IV received NaHS, 56 µM/kg i.p. daily for 4 weeks. Blood pressure, renal cortical blood perfusion and pulse wave velocity were measured in acute studies. Oxidative and antioxidant markers from plasma were measured at the end of 4 weeks. In vitro, NaHS was found to be a free radical scavenger, reductant and inhibitor of lipid peroxidation. In vivo, the SHR control rats had higher blood pressure, lower renal cortical blood perfusion, lower H₂S and nitric oxide (NO) in plasma and oxidative stress than compared to the WKY control as evidenced by decreased superoxide dismutase, glutathione and total antioxidant, and increased malondialdehyde plasma levels. NaHS treatment reduced blood pressure, increased renal cortical blood perfusion and increased H₂S and NO plasma levels, and up-regulated the antioxidant defences in SHR-NaHS treated rats. The findings of this study suggest that the administration of NaHS not only reduces the blood pressure but also attenuates the oxidative stress in SHR.

SHR, H₂S, Oxidative stress, Antioxidant, SOD, MDA, T-AOC, Glutathione

INTRODUCTION:Oxidative stress implies an increased steady state level of molecular oxygen or reactive oxygen species (ROS) due to an imbalance between their production and elimination ¹.

The generation of reactive oxygen species (ROS) is an inevitable consequence of aerobic metabolism. Formation of superoxide (O₂¯), a primary ROS, occurs during the normal metabolic processes or oxygen “activation” by physical irradiation ².

Hypertension is a chronic medical condition usually associated with increased cardiac and vascular ROS ³. Increased vascular oxidative stress has been observed in different models of experimental hypertension, for example, angiotensin II-induced hypertension, Dahl salt-sensitive hypertension, obesity-associated hypertension and spontaneously hypertensive rats (SHR) ⁴. A large number of studies have proposed NADPH as responsible for the increased formation of O₂¯ in resistance and conductance vessels of SHR ^5-7. Increased activation of vascular NADPH oxidase, xanthine oxidase and uncoupling of endothelial nitric oxide synthase (eNOS) have been implicated in enhanced superoxide generation in experimental hypertension ⁸.

The renin-angiotensin system mediated over-activity of NADPH oxidase has been incriminated as a major contributor for the increased ROS production in hypertension ⁹. Furthermore, increased NADPH oxidase-derived vascular superoxide generation and the resulting diminished NO bioavailability may also contribute to oxidative stress in SHR ^{10, 11}.

The potential role of antihypertensive drugs as antioxidants is currently under review. H₂S, generated endogenously from cysteine by the action of cystathionine beta synthase (CBS) and cystathionine gama lyase (CSE) ¹², has been found a potential antihypertensive agent in several forms of experimental hypertension ^{13, 14}. It has been shown to be a vasodilator molecule ^{15, 16} acting on vascular smooth muscle cells through the opening of K_ATP channels ¹⁷. H₂S in vivo is metabolized by oxidation in the mitochondria or by methylation in the cytosol or scavenged by methemoglobin ¹⁸.

Despite these biochemical means for H₂S catabolism, because H₂S is a reducing agent it is likely to be consumed by endogenous oxidant species in the vasculature, such as peroxynitrite, superoxide, and hydrogen peroxide ¹⁹. Recent studies have focused on the antioxidant potential of H₂S. It has been proposed that exogenous hydrogen sulfide prevents formation of superoxide anions (O₂^-) through the inhibition of Rac₁ and the down regulation of NOX ²⁰. Similarly, H₂S replacement therapy protects the catabolism of endogenous H₂S, reduces the production of ROS and maintains normal endothelial function by preserving mitochondrial function ²¹. Hydrogen sulfide has been proposed as a scavenger of endogenous peroxynitrite ²². Indeed, hydrogen sulfide supplementation has been shown to potentiate the effects of apocyanin, glutathione, catalase and superoxide dismutase in brain endothelial cells ²³.

Moreover, it can scavenge lipid peroxides ²⁴ and can up-regulate the natural antioxidant defences by increasing the production of glutathione in neurons ^{25, 26}.

Together, these reports have strengthened the view that increased production of ROS and oxidative stress is inevitable element in hypertension, and hydrogen sulfide can act as a reducing agent with antihypertensive actions.  This was examined both in vitro and in vivo in the current report. We hypothesized that hydrogen sulfide being therapeutic molecule can reduce blood pressure and ameliorate oxidative stress contributing to its vasodilator action in spontaneously hypertensive rats.

MATERIAL AND METHODS:

Experimental Protocol: The present study was divided into in vitro and in vivo portions. For in vitro study, DPPH radical scavenging assay,Inhibition of linoleic acid peroxidation and extent of reducing power of NaHS, a donor exogenous hydrogen sulfide ^{14, 27}, was carried out. For in vivo study, WKY and Spontaneously hypertensive rats (SHR) were used and divided into 4 groups (n=7 in each group), namely WKY control (I), WKY-NaHS treated (II), SHR control (III) and SHR-NaHS treated (IV). Rats of group II and IV received sodium hydrosulfide (NaHS) (Sigma Aldrich, Selangor, Malaysia), a donor of exogenous H₂S, at a dose of 56 µM/kg intraperitoneally at the same time each day for 4 weeks ¹³. Groups I and III served as control and did not receive treatment.

Experimental animals of in vivo study: Twenty eight male rats, 14spontaneously hypertensive rats (SHR) and 14 Wistar-Kyoto (WKY), weighing 235±15g were obtained from the Animal Research Unit and Service Centre (ARASC) of Universiti Sains Malaysia. Rats were allowed to acclimatize for one week before the start of any experimental procedure. The conscious blood pressure was measured in SHR by the non-invasive blood pressure system (tail cuff method) using the CODA (Kent Scientific Corporation, USA). Only SHR exhibiting systolic blood pressure 150mmHg or above were used. All the experimental procedures were carried out according to the guidelines of Universiti Sains Malaysia Animal Ethics Committee.

Measurement of in vitro antioxidant activity of NaHS: NaHS, a donor of exogenous hydrogen sulfide, was subjected to a battery of in vitro tests to assess the antioxidant potential.

Reagents and standards for in vitro study: 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH^.) (Sigma, 90.0 %), linoleic acid, food grade synthetic antioxidant butylated hydroxytoluene (BHT) (99.0 %) was purchased from Sigma Chemicals Co (St, Louis, MO, USA). All other chemicals (analytical grade) used in this study were purchased from Merck (Darmstadt, Germany), unless stated otherwise.

DPPH Radical Scavenging Assay: 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was carried out to measure the free radical scavenging activity using published protocols ²⁸ with modification. Different concentrations (3.125-100µM) of NaHS were mixed with 2mL of methanol solution containing DPPH (90µM). After 30 min incubation at room temperature, the absorbance was read at 517 nm using a 96-well plate reader (BioTek Instruments, Inc., VT, USA). Synthetic antioxidants: butylated hydroxytoluene (BHT) was used as positive control. The percent scavenging was calculated as follows:

Scavenging (%) = 100 x (A_blank - A_sample/A_blank)

Where A_blank is the absorbance of the DPPH solution and A_sample is the absorbance of the NaHS solution. IC₅₀ values, which correspond to the concentration of NaHS that caused a 50% neutralization of DPPH radicals, were calculated from the plot of percentage against concentration inhibition.

Inhibition of linoleic acid peroxidation: The antioxidant activity of NaHS was determined by measuring the percent inhibition of linoleic acid peroxidation following a published method ²⁸. Briefly, different concentrations (3.125-100µM) of NaHS were added to a mixture of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 10 ml of 0.2 M sodium phosphate buffer (pH 7). The mixture was made up to 25 mL with distilled water and incubated at 40°C. The degree of oxidation was measured following the thiocyanate method using10 mL of ethanol (75%), 0.2 mL of an aqueous solution of ammonium thiocyanate (30%), 0.2 mL of sample solution and 0.2 mL of ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl) which were added sequentially. After 3 min of stirring, the absorption values of the mixtures were measured at 500 nm. A control was performed with linoleic acid but without NaHS. Synthetic antioxidants: butylated hydroxytoluene (BHT) was used as positive control. Inhibition of linoleic acid oxidation, expressed as percent, was calculated as follows:

Percent inhibition =

100 - [(Abs. increase of sample at 175 h/Abs. increase of control at 175 h) ×100]

Determination of reducing power: The reducing power of NaHS was determined according to a previously described procedure ²⁸ with modification. Different concentrations of NaHS (3.125-100µM) were mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50⁰C for 20 min. Then 5 mL of 10% trichloroacetic acid was added and centrifuged at 980 x g (3500 rpm approx.) for 10 min. The upper layer of the solution (5.0 mL) was diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance was read at 700 nm. Synthetic antioxidants: butylated hydroxytoluene (BHT) was used as positive control. The measurements were run in triplicate and results were averaged.

Acute experimental study: At the end of treatment period all rats were subjected to the acute experimental study for the measurement of blood pressure, renal cortical blood perfusion and pulse wave velocity under anesthesia as follows: All the rats were fasted overnight and anesthetized with pentobarbital sodium (Dorminal 20% - Alfasan, Woerden, Holland), 60 mg/kg intraperitoneally, and were supplemented intravenously with pentobarbital sodium at a dose of 50 mg/kg (diluted) if required. Tracheotomy was performed to maintain a clear airwayby using the endotracheal cannula (PP 240, Portex Ltd., Kent, UK). The right jugular vein was catheterized with PP 25 (Portex Ltd.) tubing to permit the infusion of supplementary anesthesia and drugs as necessary. The left carotid artery was cannulated with PP 25 (Portex Ltd.) tubing and the cannula was advanced up to the aortic arch.

Thereafter, the left femoral artery was cannulated with PP 25 (Portex Ltd.) tubing and the cannula was pushed upto iliac artery. Both the cannulas were connected to pressure transducers (P23 ID Gould, Statham Instruments, London, UK) and linked to a data acquisition system (PowerLab®, AD Instruments) through a Quad Amp (AD Instruments) using chart Pro (V.5.5) software (AD Instruments) on a Hewlett Packard Centrino Core2 duo Computer with Windows XP operating system. The right kidney was exposed by a small flank incision.

A laser Doppler flow probe (OxyFlow, ADInstruments) was positioned on the dorsal surface of the posterior end of the exposed kidney in order to measure renal cortical blood perfusion. The probe was connected to a laser Doppler flowmeter (ADInstruments) which was directly link to data acquisition system (PowerLab®, ADInstruments). The animals were allowed to stabilize for 1 h upon completion of the surgical procedures. After the stabilization period, renal cortical blood perfusion, mean arterial pressure, systolic blood pressure, pulse pressure, and heart rate were recorded continuously for 30 minutes and averaged.

Measurement of Pulse Wave Velocity (PWV): PWV was measured as reported previously ²⁹. Proximal and distal pressure waves were recorded at the same time and displayed on the data acquisition system. PWV was calculated by dividing the propagation distance (d) by the propagation time (t) and measured in meters per second. After euthanization of the animal, the full length of aorta upto the iliac artery was exposed and the tips of the two cannulas in the carotid and iliac arteries were identified.

The distance between these two points was determined by using a wet cotton thread laid straight along the vessels. The time for the propagation of pulse wave from the aortic arch to the iliac artery was measured by the time delay between the upstrokes (foot) of each pressure wave front (foot to foot technique). The average of 10 normal consecutive cardiac cycles was used to calculate the propagation time.

Measurement of in vivo Oxidative stress and Antioxidant markers: Before the termination of the acute experiment 5 mL of arterial blood was obtained, centrifuged and plasma was removed and stored at -30^oC till further analysis of antioxidant markers and H₂S. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione (GSH) and nitric oxide (NO) were measured using the spectrophotometric detection kits (Institute of Biological Engineering of Nanjing Jianchen, Nanjing, China). All the measurements were made according to the kit manufacturer’s instructions.

Measurement of Plasma H₂S levels: The plasma H₂S levels were measured as reported earlier ³⁰. Briefly, 100µl of aliquots of the samples were mixed with 50µl of distilled water in micro-centrifuge tubes containing 300µl of zinc acetate (1% w/v) to trap H₂S. The reaction was stopped after 5 minutes by adding 200µl of N,N-2dimethyl-p-phenylenediamine sulfate (20mM in 7.2M HCl), immediately followed by the addition of 200µl of FeCl₃ (30mM in 1.2M HCl). The mixture was kept in the dark for 20 minutes. In order to precipitate protein from the samples (even a trace amount), 150µl of trichloroacetic acid (10% w/v) was added.

The mixture was centrifuged at 10,000 rpm for 10 minutes and the absorbance of the resulting supernatant was measured at 670nm in a 96-well plate using a spectrophotometer (BioTek Instruments, Inc., VT, USA). All samples were assayed in duplicate. Finally, H₂S concentration in the plasma was calculated against the calibration curve of standard H₂S solutions (NaHS 3.125-100µM). All chemicals used were obtained in pure form from Sigma Aldrich, unless stated otherwise.

Statistical analysis: Data was analyzed using GraphPad Prisim® version 5.00 for Windows (GraphPad Software, San Diego California U.S.A). All the data were expressed as means ± SEM (wherever applicable). Pearson correlation was used to construct the relationship between concentrations of NaHS and scavenging activity, inhibition of linoleic acid peroxidationand reducing power.

In vivo experimental data was analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The difference between the means was considered significant at the 5% level.

RESULTS:

Figure 1(A) representsthe correlation between percent scavenging and concentration of NaHS. A statistically significant correlation was observed between % scavenging and concentration of NaHS (r =0.989, p < 0.001). The IC₅₀, the concentration of NaHS that causes the 50% neutralization of DPPH radical, was found to be 32.51µM/L. The reducing activity of NaHS, Figure 1(B), in terms of increase in absorbance after the addition of ferric chloride, was directly proportional to the concentration of NaHS in the test medium. Statistical significant correlation was observed between the concentrations of NaHS and increase in absorbance (r =0.975, p< 0.001)

A

B

FIGURE 1: PERCENT SCAVENGING (A) AND REDUCING POWER (B) OF NaHS. Statistical analysis was done by Pearson’s correlation. (r =0.989, p < 0.001) and (r =0.975, p< 0.001) for NaHS respectively in A and B. Butylatedhydroxytoluene (BHT) was used as positive control.

The degree of linoleic acid oxidation inhibition caused by the different concentrations of NaHS is shown in Table 1. A significant positive correlation was observed as the concentration of reducing agent in the medium increased the % inhibition of linoleic acid oxidation also increased (r =0.955, p =0.002).

TABLE 1: PERCENT INHIBITION OF LINOLEIC ACID OXIDATION OF NaHS AND BHT

The systolic blood pressure, diastolic blood pressure, means arterial blood pressure and heart rate of the SHR control and SHR-NaHS treated groups (Table 2) were significantly higher compared to WKY control (all p< 0.05). It was evident that with NaHS treatment the systolic blood pressure, diastolic blood pressure, means arterial blood pressure and heart rate reduced in SHR-NaHS treated group as compared with the SHR control group (all p< 0.05).

Moreover, SHR control group exhibited a lower renal cortical blood perfusion as compared to WKY control (p< 0.05). On the other hand, the SHR control group of rats had higher pulse wave velocity as compared with WKY control (p< 0.05).

Following the NaHS treatment, the renal cortical blood perfusion increased and pulse wave velocity decreased in SHR-NaHS treated group as compared to SHR control (all p< 0.05). The NaHS treatment had no impact on systolic blood pressure, diastolic blood pressure, means arterial blood pressure and heart rate in WKY-NaHS treated group as compared to untreated WKY control group (all p> 0.05).

The final body weight obtained at the end of treatment period was not significantly different among all the groups of study (all p> 0.05) (Table 2).

 TABLE 2: EFFECT OF EXOGENOUS H₂S ON SYSTOLIC BLOOD PRESSURE, DIASTOLIC BLOOD PRESSURE, MEAN ARTERIAL BLOOD PRESSURE, HEART RATE, RENAL CORTICAL BLOOD PERFUSION, PULSE WAVE VELOCITY AND BODY WEIGHT

Notes: SBP= systolic blood pressure, DBP= diastolic blood pressure, MAP= mean arterial blood pressure, HR= heart rate, RCBP= Renal Cortical Blood Perfusion, PWV= Pulse Wave Velocity, BPM= Beats Per Minute, BPU= Blood Perfusion Unit, m/s= meter per second. The values are mean ± SEM (n = 7). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Note:*p <0.05 versus WKY control; † <0.05 versus SHR control.

It was observed that the SHR control group exhibited significantly lower concentrations of plasma hydrogen sulfide (H₂S) (Figure 2A) and nitric oxide (NOx) (Figure 2B) as compared to the WKY control group (both p <0.05). The NaHS treatment increased the plasma H₂S and NOx levels in SHR-NaHS treated group as compared to the SHR control group (i.e., 56.2 ± 2.3 vs. 22.0 ± 1.5 µM and 25.4 ± 1.0 vs. 18.5 ± 0.9 µM respectively; both p <0.05). Conversely, the NaHS treatment did not alter the plasma H₂S and NO levels in WKY-NaHS treated group as compared to their untreated counterpart i.e. WKY control rats (all p>0.05).

FIGURE 2:  PLASMA H₂S (A) AND PLASMA NO (B) OF WKY, WKY + NaHS, SHR AND SHR + NaHS GROUPS OF RATS. The values are mean ± SEM (n = 7). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Note:*p <0.05 versus WKY control; ** <0.05 versus SHR control.

The plasma superoxide dismutase (SOD) level (Figure 3, A) was significantly lower in SHR control as compared with WKY control (i.e., 116.7 ± 3.8 vs. 150.2 ± 2.0 U/mL; p <0.05). With NaHS treatment, the SOD levels were higher in the SHR-NaHS treated group compared to the SHR control group (i.e., 133.1 ± 2.1 vs. 116.7 ± 3.8 U/mL; p <0.05).

However, the plasma malondialdehyde (MDA) level (Figure 3, B) of the SHR control group was significantly higher compared to the WKY control (p <0.05). It was observed that in the NaHS treated SHR group, the plasma MDA concentration was significantly reduced as compared to the SHR control group (i.e., 5.9 ± 0.5 vs. 8.0 ± 0.4 nmol/mL; p <0.05).

FIGURE 3: PLASMA SOD (A) AND PLASMA MDA (B) OF WKY, WKY + NaHS, SHR AND SHR + NaHS GROUPS OF RATS. The values are mean ± SEM (n = 7). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Note:*p <0.05 versus WKY control; **<0.05 versus SHR control.

The total antioxidant capacity (T-AOC) (Figure 4, A) and plasma glutathione levels (Figure 4, B) in the control SHR group were significantly lower compared to the WKY control group (both p <0.05). Moreover the treatment with NaHS resulted in increased levels of T-AOC and glutathione in the SHR-NaHS treated group as compared with SHR control group (i.e., 7.2 ± 0.2 vs. 4.9 ± 0.3 U/mL and 14.2 ± 0.3 vs. 10.4 ± 0.5 µM/L respectively; both p <0.05).

FIGURE 4: PLASMA T-AOC (A) AND PLASMA GLUTATHIONE (B) OF WKY, WKY + NaHS, SHR AND SHR + NaHS GROUPS OF RATS.The values are mean ± SEM (n = 7). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Note:*p <0.05 versus WKY control; **<0.05 versus SHR control.

DISCUSSION: This studyinvestigated the antioxidant activity of hydrogen sulfide in genetic hypertension. This was performed by studying the effects of supplementation H₂S in SHR. The key findings of this study were that the exogenously administered H₂S in the form of NaHS increased the superoxide dismutase,glutathione,total antioxidant capacity and nitric oxide plasma levels in SHR. Moreover, the supplementation of H₂S reduced blood pressure, increased plasma H₂S and significantly reversed the pulse wave velocity in SHR.

The determination of radical scavenging power, inhibition of linoleic acidperoxidation and measurement of reducing potential has been widely used to evaluate the in vitro antioxidant potential of phenolic compounds in ethno-pharmacology ^{31, 32}. The radical scavenging activity of NaHS, a donor hydrogen sulfide, was measured by the DPPH assay. DPPH is a stable free radical and a trap (scavenger) for other free radicals and possesses a deep violet color in oxidized state. Upon neutralization by receiving the electron from any donor it will become yellow.

As the concentration of reducing compounds increases, the DPPH radical scavenging activity increases, and with it the antioxidant activity ³³. It was previously reported that NaHS in solution dissociates into Na⁺ and HS^- and then HS^- combine with H⁺ to form H₂S ¹³.  It was observed that with increase in concentration of NaHS in the test medium the DPPH scavenging activity increases. These findings suggest the antioxidant activity of NaHS, as in H₂S the sulfur exists as S^-2 and can easily loose electron and behaves as a reducing agent.

Moreover, the IC₅₀ of NaHS is not significantly different from IC₅₀ of BHT, a synthetic antioxidant used as positive control. Measurement of reducing potential can reveal the some features of antioxidant capacity of NaHS. A concentration dependent increased in reducing activity has been observed in present study suggestive of reducing potential of NaHS. Furthermore, considerable inhibition of lipid per-oxidation was also observed by in vitro use of NaHS in the present study.

Up to this point little data are available in the literature regarding the in vitro antioxidant activity of NaHS by with which to compare the results of present study. However we compare our results with BHT, a synthetic antioxidant, used as a ppositive control in present study. Interestingly, it was observed that NaHS had more or less equivalent potential regarding the radical scavenging,lipid peroxidation inhibition and reducing activity as compared with BHT. This would suggest that NaHS, a donor of H₂S, has a substantial antioxidant activity in vitro.

A large number of studies provide compelling evidence that reactive oxygen species (ROS) play a key role in pathophysiology of hypertension. This is largely due to increased production of superoxide, decreased availability of endothelial derived nitric oxide (NO) and ROS induced morphological alteration in cardiovascular system ^{3, 9, 34}. Measuring the MDA and SOD is usually combined together in order to evaluate the extent of anti- and oxidative stress level of living organism. A higher concentration of plasma MDA along with low concentration of SOD generally accepted as markers of oxidative stress ³⁵.

In the present study, the SHR control group had higher levels of MDA and lower levels of SOD as compared with WKY control. These findings are in accord with previous studies ^{36, 37} and suggestive of oxidative stress in SHR. Moreover the plasma total antioxidant capacity (T-AOC), and levels of reduced glutathione were also decreased in the SHR compared to the WKY controls. The treatment with NaHS resulted in increased activity of SOD and decreased MDA concentration in SHR treated group suggesting that the level of oxidative stress had been reduced by this treatment regime. These findings are in accord with the previously reported experiments demonstrating the antioxidant potential of NaHS ³⁸.

However, the authors reported the antioxidant activity of H₂S in traumatic hemorrhagic shock and examined the acute effects of intraperitoneal NaHS. By contrast the current study examined the effects of chronic NaHS treatment in the SHR. Moreover, it was observed that the SHR treated group exhibited greater total antioxidant capacity and glutathione levels after four weeks of treatment. Glutathione acts as one of the major and potent intracellular antioxidant enzymes ³⁹. A growing body of evidence suggested that H₂S may act as an antioxidant by promoting glutathione levels ^{26, 27, 40} which supports our present findings.

The reactive oxygen species, such as superoxide, hydrogen peroxide, perioxinitrite and lipid perhydroxides have been linked to vascular pathology ⁴¹ while over activity of the vascular NADPH oxidase (NOX) has been linked to increased production of superoxide ¹¹. The in vitro results of the present study suggest that H₂S can act as a free radical scavenger and reductant molecule. Therefore, on the basis of these results it can be proposed that H₂S may directly scavenge the superoxide and hydrogen peroxide free radicals resulting in reduced oxidative stress in SHR. As we did not measure O₂¯production by vascular tissue in the present study, this suggestion needs further verification. There is considerable evidence that in hypertension either the production or availability of nitric oxide is reduced. This is due to impair activation of nitric oxide synthase (NOS) or due to oxidative inactivation of nitric oxide by the excessive production of superoxide anions in vascular wall ⁴¹.

In the present study the SHR control rats exhibited low plasma levels of nitric oxide compared to the WKY control group. The results can be best explained on the basis of an interaction between superoxide and nitric oxide to form a major product peroxynitrite, as both are highly reactive and unstable radicals ⁴². The reaction between superoxide and nitric oxide is three times faster than the dismutation of superoxide by superoxide dismutase. Hence it is possible that the increased production of superoxide in vascular wall results in a reduced bioavailability of nitric oxide causing the low levels of nitric oxide in the present study. It has been reported that the resultant peroxynitrite is more stable than either nitric oxide or superoxide and is a strong oxidant.

Recently hydrogen sulfide has been proposed as an endogenous peroxynitrite scavenger ²². Furthermore, we examined the possibility that exogenous H₂S might increase the level of nitric oxide in SHR. Indeed, it was found that after 4 weeks NaHS treatment, the NO level were elevated in the SHR-NaHS treated group compared to the non-treated counterpart and were not distinguishable from the WKY group. This was probably due to the inhibition of superoxide formation by blocking the over activity of NADPH oxidase ²⁰.

Alternatively, the increased nitric oxide level in the SHR treated group may also have resulted from increased activity of nitric oxide synthase (NOS) following the administration of exogenous H₂S in the form of NaHS ⁴³. Moreover, the low level of nitric oxide in the SHR of the present study can also be linked to endothelial dysfunction due to the over production ROS. This possibility was evaluated by examining the arterial stiffness marker, pulse wave velocity, in experimental groups. It was observed that untreated SHR exhibited increased pulse wave velocity (PWV) as compared with WKY control suggesting the impaired endothelial function.

Following NaHS treatment, PWV was lower in the SHR treated group as compared with SHR control possibly due to the increased SOD and glutathione levels. As far as the concentration of plasma H₂S concern, we have already reported a low level of plasma H₂S in SHR ³⁰.

Treating the SHR with NaHS resulted in increased level of plasma H₂S and may be due to the up-regulation of CSE enzyme activity, which is concerned with H₂S production in vascular smooth muscle cells ⁴⁴.

Hydrogen sulfide is a vasodilator molecule that can reduce the blood pressure ¹⁶. It was evident in the current study that NaHS treatment significantly reduced the blood pressure and increased the renal cortical blood perfusion in SHR-NaHS treated group as compared with SHR control. The resultant decrease in blood pressure and increase in renal cortical blood perfusion can be explained on the basis of induced vasodilatation ¹⁷ and in part due to decreased oxidative stress by administered exogenous donor of H₂S.

 CONCLUSION: In summary, our findings demonstrated that NaHS, a donor of hydrogen sulfide, exhibited an antioxidant potential in experimental in vitro evaluations and exogenously administered hydrogen sulfide significantly attenuated the oxidative stress in spontaneously hypertensive rats. A variety of mechanisms are involved in reducing the oxidative stress as the antioxidant activity of H₂S in SHR includes direct scavenging of reactive oxygen species, up-regulation of SOD, glutathione, nitric oxide and improvement in endothelial function.

The dose of NaHS i.e. 56µM/kg/day, used in present study does significantly decreased the systolic blood pressure, diastolic blood pressure, mean arterial blood pressure and heart rate in SHR treated group. These findings suggest that H₂S is potential therapeutic antihypertensive and antioxidant agent in genetic hypertension.

ACKNOWLEDGMENT: The authors fully acknowledge the Fundamental Research Grant No. 203/PFARMASI/6711217 provided by the Ministry of Science Technology and Innovation (MOSTI), Government of Malaysia, for this work.

REFERENCES:

1. Betteridge DJ: What is oxidative stress? Metabolism 2000; 49:3-8.
2. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & cell Biology 2007; 39:44-84.
3. Touyz RM: Reactive oxygen species in vascular biology, role in arterial hypertension. Expert review of Cardiovascular Therapy 2003; 1:91-106.
4. Madamanchi NR, Vendrov A, Runge MS: Oxidative stress and vascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology 2005; 25:29-38.
5. Rodriguez-Iturbe B, Zhan CD, Quiroz Y, Sindhu RK, Vaziri ND: Antioxidant-rich diet relieves hypertension and reduces renal immune infiltration in spontaneously hypertensive rats. Hypertension 2003; 41:341-346.
6. Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, Kohno M, Abe Y: Renal sympathetic nerve responses to tempol in spontaneously hypertensive rats. Hypertension 2003; 41:266-273.
7. Sedeek M, Hebert RL, Kennedy CR, Burns KD, Touyz RM: Molecular mechanisms of hypertension: role of Nox family NADPH oxidases. Current opinion in nephrology and hypertension 2009; 18:122-127.
8. Harrison DG, Gongora MC, Guzik TJ, Widder J: Oxidative stress and hypertension. Journal of the American Society of Hypertension 2007; 1:30-44.
9. Touyz RM, Schiffrin EL: Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients: role of phospholipase D-dependent NADPH oxidase-sensitive pathways. Journal of hypertension 2001; 19:1245-1254.
10. Pacher P, Beckman JS, Liaudet L: Nitric oxide and peroxynitrite in health and disease. Physiological reviews 2007; 87:315-424.
11. Tanito M, Nakamura H, Kwon YW, Teratani A, Masutani H, Shioji K, Kishimoto C, Ohira A, Horie R, Yodoi J: Enhanced oxidative stress and impaired thioredoxin expression in spontaneously hypertensive rats. Antioxidants and Redox Signalling 2004; 6:89-97.
12. Stipanuk MH, Beck PW: Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat. Biochemical Journal 1982; 206:267-277.
13. Yan H, Du J, Tang C: The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochemical and Biophysical Research Communications 2004; 313:22-27.
14. Yang Y, Geng X, Tang C: Hydrogen sulfide system in the pathogenesis of renovascular hypertension in rats. Journal of geriatric Cardiology 2008; 5:1-5.
15. Li L, Moore PK: Putative biological roles of hydrogen sulfide in health and disease: a breath of not so fresh air? Trends in pharmacological sciences 2008; 29:84-90.
16. Ali MY, Ping CY, Mok Y: Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? British Journal of Pharmacology 2006; 149:625-634.
17. Zhao W, Zhang J, Lu Y, Wang R: The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J 2001; 20:6008-6016.
18. Beauchamp RO, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA, Leber P: A critical review of the literature on hydrogen sulfide toxicity. Critical Reviews in Toxicology 1984; 13:25-97.
19. Li L, Rose P, Moore PK: Hydrogen sulfide and cell signalling. Annual review of pharmacology and toxicology 2011; 51:169-187.
20. Muzaffar S, Shukla N, Bond M, Newby AC, Angelini GD, Sparatore A, Del Soldato P, Jeremy JY: Exogenous Hydrogen Sulfide Inhibits Superoxide Formation, NOX-1 Expression and Rac₁ Activity in Human Vascular Smooth Muscle Cells. Journal of vascular research 2008; 45:521-528.
21. Suzuki K, Olah G, Modis K, Coletta C, Kulp G, Ger D, Szoleczky P, Chang T, Zhou Z, Wu L: Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function. Proceedings of the National Academy of Sciences 2011; 108:13829-13834.
22. Whiteman M, Armstrong JS, Chu SH, JiaaLing S, Wong BS, Cheung NS, Halliwell B, Moore PK: The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite scavenger? Journal of neurochemistry 2004; 90:765-768.
23. Tyagi N, Moshal KS, Sen U, Vacek TP, Kumar M, Hughes Jr WM, Kundu S, Tyagi SC: H₂S Protects Against Methionine Induced Oxidative Stress in Brain Endothelial Cells. Antioxidants & Redox Signaling 2009; 11:25-33.
24. Muellner M, Schreier S, Laggner H, Hermann M, Esterbauer H, Exner M, Gmeiner B, Kapiotis S: Hydrogen sulfide destroys lipid hydroperoxides in oxidized LDL. Biochem. J 2009; 420:277-281.
25. Kimura Y, Kimura H: Hydrogen sulfide protects neurons from oxidative stress. The FASEB Journal 2004; 18:1165-1167.
26. Kimura Y, Goto YI, Kimura H: Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria. Antioxidants & Redox Signalling 2010; 12:1-13.
27. Yan SK, Chang T, Wang H, Wu L, Wang R, Meng QH: Effects of hydrogen sulfide on homocysteine-induced oxidative stress in vascular smooth muscle cells. Biochemical and biophysical Research Communications 2006; 351:485-491.
28. Hussain AI, Rathore HA, Sattar MZA, Chatha SAS, Ahmad A, Johns EJ: Phenolic profile and antioxidant activity of various extracts from Citrullus colocynthis (L.) from the Pakistani flora. Industrial Crops and Products 2013; 45:416-422.
29. Ahmad FD, Sattar MA, Rathore HA, Abdullah MH, Tan S, Abrahim ZO, Abdullah NA, Johns EJ: Exogeous Hydrogen Sulfide (H2S) Improves the Endothelial and Renal Excretory Functions in Streptozotocin Induced WKY rats. IJPSR 2012; 3(1):101-110.
30. Ahmad FD, Sattar MA, Rathore HA, Abdullah MH, Tan S, Abdullah NA, Johns EJ: Exogenous Hydrogen Sulfide (H2S) Reduces Blood Pressure and Prevents the Progression of Diabetic Nephropathy in Spontaneously Hypertensive Rats. Renal Failure 2012; 34 (2):203-210.
31. Hussain AI, Anwar F, Hussain Sherazi ST, Przybylski R: Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations. Food Chemistry 2008; 108:986-995.
32. Anand Swarup KR, Sattar MA, Abdullah NA, Abdulla MH, Salman IM, Rathore HA, Johns EJ: Effect of dragon fruit extract on oxidative stress and aortic stiffness in streptozotocin-induced diabetes in rats. Pharmacognosy Research 2010; 2:31-35.
33. Sanchez-Moreno C, A Larrauri J, Saura-Calixto F: Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents. Food Research International 1999; 32:407-412.
34. Kishi T, Hirooka Y, Kimura Y, Ito K, Shimokawa H, Takeshita A: Increased reactive oxygen species in rostral ventrolateral medulla contribute to neural mechanisms of hypertension in stroke-prone spontaneously hypertensive rats. Circulation 2004; 109:2357-2362.
35. Del Rio D, Stewart AJ, Pellegrini N: A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutrition, Metabolism and Cardiovascular Diseases 2005; 15:316-328.
36. Duarte J, PerezPalencia R, Vargas F, Angeles Ocete M, PerezVizcaino F, Zarzuelo A, Tamargo J: Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. British Journal of Pharmacology 2001; 133:117-124.
37. Ma N, Nna N: Effect of γ-Tocotreenol on Blood Pressure, Lipid Peroxtoation and Total Antioxtoant Status in Spontaneously Hypertensive Rats (SHR). Clinical and experimental Hypertension 1999; 21:1297-1313.
38. 38.           Chai W, Wang Y, Linm JY, Sun XD, Yao LN, Yang YH, Jiang W, Gao CJ, Ding Q: Exogenous Hydrogen Sulfide Protects Against Traumatic Hemorrhagic Shock Via Attenuation of Oxidative Stress. Journal of Surgical Research 2011; 176:210-219.
39. Pompella A, Visvikis A, Paolicchi A, Tata VD, Casini AF: The changing faces of glutathione, a cellular protagonist. Biochemical pharmacology 2003; 66:1499-1503.
40. Kimura Y, Kimura H: Hydrogen sulfide protects neurons from oxidative stress. The FASEB Journal 2004; 18:1165-1167.
41. Kojda G, Harrison D: Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovascular Research 1999; 43:652-671.
42. Li L, Hsu A, Moore PK: Actions and interactions of nitric oxide, carbon monoxide and hydrogen sulphide in the cardiovascular system and in inflammation-a tale of three gases. Pharmacology & Therapeutics 2009; 123:386-400.
43. Minamishima S, Bougaki M, Sips PY, De Yu J, Minamishima YA, Elrod JW, Lefer DJ, Bloch KD, Ichinose F: Hydrogen Sulfide Improves Survival After Cardiac Arrest and Cardiopulmonary Resuscitation via a Nitric Oxide Synthase Dependent Mechanism in Mice. Circulation 2009; 120:888-896.
44. Chunyu Z, Junbao D, Dingfang B, Hui Y, Xiuying T, Chaoshu T: The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochemical and Biophysical Research Communications 2003; 302:810-816.
    

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How to cite this article:

Ahmad F, Sattar MA, Rathore HA, Hussain AI, Chia TY, Jin OH, Pei YP, Ahmad I, Abdullah NA and Johns EJ: Exogenous hydrogen sulfide attenuates oxidative stress in spontaneously hypertensive rats. Int J Pharm Sci Res 2013: 4(8); 2916-2926. doi: 10.13040/IJPSR. 0975-8232.4(8).2916-26

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