Compared effects of inhibition and exogenous administration of hydrogen sulphide in ischaemia-reperfusion injury

Introduction Haemorrhagic shock is associated with an inflammatory response consecutive to ischaemia-reperfusion (I/R) that leads to cardiovascular failure and organ injury. The role of and the timing of administration of hydrogen sulphide (H2S) remain uncertain. Vascular effects of H2S are mainly mediated through K+ATP-channel activation. Herein, we compared the effects of D,L-propargylglycine (PAG), an inhibitor of H2S production, as well as sodium hydrosulphide (NaHS), an H2S donor, on haemodynamics, vascular reactivity and cellular pathways in a rat model of I/R. We also compared the haemodynamic effects of NaHS administered before and 10 minutes after reperfusion. Methods Mechanically ventilated and instrumented rats were bled during 60 minutes in order to maintain mean arterial pressure at 40 ± 2 mmHg. Ten minutes prior to retransfusion, rats randomly received either an intravenous bolus of NaHS (0.2 mg/kg) or vehicle (0.9% NaCl) or PAG (50 mg/kg). PNU, a pore-forming receptor inhibitor of K+ATP channels, was used to assess the role of K+ATP channels. Results Shock and I/R induced a decrease in mean arterial pressure, lactic acidosis and ex vivo vascular hyporeactivity, which were attenuated by NaHS administered before reperfusion and PNU but not by PAG and NaHS administered 10 minutes after reperfusion. NaHS also prevented aortic inducible nitric oxide synthase expression and nitric oxide production while increasing Akt and endothelial nitric oxide synthase phosphorylation. NaHS reduced JNK activity and p-P38/P38 activation, suggesting a decrease in endothelial cell activation without variation in ERK phosphorylation. PNU + NaHS increased mean arterial pressure when compared with NaHS or PNU alone, suggesting a dual effect of NaHS on vascular reactivity. Conclusion NaHS when given before reperfusion protects against the effects of haemorrhage-induced I/R by acting primarily through a decrease in both proinflammatory cytokines and inducible nitric oxide synthase expression and an upregulation of the Akt/endothelial nitric oxide synthase pathway. Keywords: hydrogen sulphide, inflammation mediators, therapeutic use, shock, hemorrhagic/drug therapy, haemodynamics/drug effects


Introduction
The reperfusion phase of haemorrhagic shock is associated with an inflammatory response, including increased NF-B activation [1], increased inflammatory cytokine production [2], increased nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression [3,4], and increased activation of vascular K + ATP channels. These inflammatory responses are associated with hypotension, vasodilation and hyporesponsiveness to vasopressor agents and lead to ischaemia-reperfusion (I/R) organ injury [5]. Treating and/or preventing I/R-induced organ injury is therefore a major challenge.
Hydrogen sulphide (H 2 S) is recognised as a gasotransmitter, similar to NO and carbon monoxide. However, current knowledge relative to its role in physiology and pathology remains under discussion [6]. Many effects of H 2 S are the subject of controversy [7]. Depending on the chosen models, H 2 S has been reported to display opposite effects in haemorrhagic shock conditions. While inhaled H 2 S and intravenous sodium sulphide and sodium hydrosulphide (NaHS) reportedly increased survival [8], improved haemodynamics, attenuated metabolic failure in rodents [9][10][11], exerted cardioprotective effects [10,11] as well as protected against organ injury [12], sodium sulphide did not exert any beneficial effects in swine [13]. Moreover, in other studies, blocking H 2 S biosynthesis with D,L-propargylglycine (PAG), a cystathionine γ-lyase inhibitor, improved haemodynamics and attenuated systemic inflammation and organ injury [14,15].
The fact that H 2 S injection was associated with an increase in arterial pressure is intriguing. Currently available data indicate that H 2 S relaxes blood vessels [16] mostly, if not exclusively, by opening ATP-regulated potassium channels in vascular smooth muscle cells [17,18]. We hypothesised that H 2 S injected at the time of reperfusion could decrease the consequences of shock and reperfusion, that the use of an inhibitor of endogenous H 2 S production leads to opposite effects, and that adding a vascular K + ATP -channel inhibitor would improve the effects of H 2 S on systemic haemodynamics. Using a previously published model of I/R induced by haemorrhagic shock, we thus compared the effects of H 2 S and of its inhibition as well as of K + ATPchannel inhibition on haemodynamics, vascular reactivity and cellular pathways.

Materials and methods
The study protocol was approved by the Nancy Institutional Committee on Animal Care and Use. The experiments were performed in conformity with the European legislation on the use of laboratory animals.

Animals
Adult male Wistar rats, weighing 325 ± 15 g, were housed under 12-hour light/dark cycles in the animal facility of the University of Nancy 1 (France).

Surgical procedure
Animals were anaesthetised with intraperitoneal pentobarbital (50 mg/kg body weight). Rats were placed on a homeothermic blanket system to maintain rectal temperature between 36.8 and 37.8°C for the duration of the experiment. After local anaesthesia with lidocaine 1% (AstraZeneca, Rueil-Malmaison, France}), a tracheotomy was performed and animals were mechanically ventilated (Harvard Rodent 683 ventilator; Harvard Instruments, South Natick, MA, USA) throughout the experiment. The ventilator was set to maintain carbon dioxide partial pressure in the vicinity of 40 mmHg and oxygen was added in order to maintain oxygen partial pressure above 100 mmHg. The left carotid artery was exposed and a 2.0 mm transit-time ultrasound flow probe (Transonic Systems Inc., Ithaca, NY, USA) was attached to the artery to continuously measure carotid blood flow (CBF).
Under local anaesthesia, the femoral artery was canulated in order to measure the mean arterial blood pressure (MAP) and heart rate (HR) on the one hand, and to induce haemorrhagic shock on the other. The homolateral femoral vein was canulated for retransfusion of withdrawn blood, for fluid replacement and for bolus infusion of either vehicle or drugs.

Induction of haemorrhagic shock and protocol design
Surgery was followed by a 20-minute stabilisation period. Thereafter, haemorrhagic shock was induced by the graded withdrawal of blood from the femoral artery to a reservoir until MAP decreased to 40 mmHg and maintained during 60 minutes by further blood withdrawal or reinfusion of shed blood. At 60 minutes, shed blood was retransfused via the venous line within 10 minutes. Animals were continuously monitored for HR, MAP and CBF during 300 minutes. Hydration was performed with a perfusion of 0.9% NaCl at a rate of 1.2 ml/hour.
At the end of the experiment, rats were sacrificed and blood samples were collected for arterial lactate measurement, centrifuged (4,000 rpm, 15 minutes, 4°C) and plasma aliquoted and stored at -80°C until biochemical analysis. Organs (aorta, heart and liver) were also collected and stored at -80°C until biochemical analyses.

Monitoring and measurements
Arterial blood gases were controlled after the stabilisation period, in order to establish mechanical ventilation. Measurements of blood gas and blood glucose were recorded at baseline (t = 0 minutes at the beginning of haemorrhagic shock) and at two critical periods, namely at the end of reperfusion (t = 70 minutes) and at the end of the experiment (t = 300 minutes). MAP, HR, CBF and rectal temperature were recorded at baseline and every 10 minutes thereafter during the observation period.

Biochemical analyses
Plasma levels of IL-6 and TNFα were measured in duplicate with the use of rat IL-6 and TNFα ELISA kits (Quantikine ELISA; R&D Systems Europe, LILLE, France) according to the manufacturer's instructions. Results were expressed as picograms of the measured cytokine per millitre of plasma.

Measurement of nitrite/nitrate
NO 2 and NO 3 are the primary oxidised products of NO reacting with water, and therefore the total concentration of NO 2 -/NO 3 in plasma was used as an indicator of NO production in vivo. Briefly, the nitrate in the supernatant was first reduced to nitrite by incubation with nitrate reductase (10 U/ml) and NADPH (629.2 µg/ml) at room temperature for 30 minutes. Thereafter, total nitrite concentration in the samples was measured by Griess reaction following the addition of 100 µl Griess reagent to 100 µl sample in a 96-well plate with a flat transparent bottom. The optical density at 550 nm was measured by an ELISA microplate reader and normalised with the optical density at 550 nm of standard saline solutions.

RNA extraction and quantitative RT-PCR
Primers for quantitative RT-PCR were obtained from Eurogentec (Angers, France). Total RNA extraction was carried out with the RNA Plus mini kit (Qiagen, Courtaboeuf Cedex, France) according to the manufacturer's instructions. Total RNA was reverse-transcribed to cDNA using the iScript One-Step RT-PCR Kit for Probes (Biorad, Marnes-la-Coquette, France). cDNA obtained from the RT reaction was subjected to quantitative PCR using iTaq Fast SYBR Green Supermix with ROX (Biorad, Marnes-la-Coquette, France). The primer and concentrations were optimised according to the manufacturer's guidelines. Expression of, Kir6.1 mRNA and SUR2B mRNA were measured using iTaq Fast SYBR Green Supermix (Biorad).
The PCR reaction parameters were as follows: incubation at 50°C for 2 minutes, incubation at 95°C for 10 minutes, and thereafter 40 denaturation cycles at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. Each sample was determined in duplicate. To determine the relative mRNA levels, a standard curve for each gene was created using RNA isolated from the haemorrhagic shock group. Isolated RNA was reverse-transcribed, and dilution series of cDNA ranging from 1 pg to 10 ng were subjected to real-time PCR. The obtained threshold cycle values were plotted against the dilution factor to create a standard curve. Relative mRNA levels in test samples were then calculated from the standard curve.

Vascular reactivity
For in vivo determination, basal and maximal MAP values obtained after administration of 1 µg/kg bolus of norepinephrine were recorded in the sham, haemorrhagic shock, haemorrhagic shock + PNU and haemorrhagic shock + 1400W groups.
After an equilibration period (at least 20 minutes) under optimal passive tension, two successive contractions in response to the combination of KCl depolarisation (100 mM) and phenylephrine (PE) (10 µM) (Sigma-Aldrich) were used in order to test the maximal contractile capacity of the vessels. After a 20-minute washout period, concentration-response curves to PE were elicited by cumulative administration of this vasoconstrictor agonist (1 nM to 100 µM) in order to determine the same concentration producing an equal level of contraction in the different groups. To study endothelium-dependent relaxation, aortic rings with functional endothelium were precontracted with PE (1 µM) and then exposed to increasing incremental concentrations of acetylcholine (1 nM to 100 µM; Sigma, St Louis, MO, USA). The presence of functional endothelium was confirmed with acetylcholine (1 µM), which elicited a relaxation superior to 50%.

Statistical analyses
Results are expressed as the median and interquartile range for n experiments (n representing the number of animals). Difference between groups was tested using a Kruskal-Wallis test. When the relevant F values were significant at the 5% level, further pairwise comparisons were performed using a Dunn's multiple comparison test. All statistics were performed with the Statview software (version 5.0 software; SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically significant.

Model characterisation
Shock and I/R-induced hypotension, lactic acidosis and vascular hyporeactivity to norepinephrine The HR, MAP and CBF remained stable throughout the experiment in the control group (Figure 1; see Additional file 1). In animals subjected to haemorrhagic shock and retransfusion, blood withdrawal significantly decreased the MAP, HR and CBF (Figure 1; see Additional file 1). Haemorrhagic shock was associated with a marked elevation in plasma lactate (9 × 2 mmol/l) compared with the sham group (2.1 × 0.5 mmol/l) (see Additional file 2), while the increase in arterial pressure induced by a bolus of 1 µg/kg norepinephrine was significantly decreased (P < 0.01) in the shock group compared with sham animals (Figure 2).
Ischaemia-reperfusion is associated with overexpression/ activation of iNOS and vascular K + ATP and increased proinflammatory cytokines IR-induced vascular hyporeactivity to a bolus of 1 µg/kg norepinephrine was completely restored following the administration of 1400W, a selective inhibitor of iNOS, as well as PNU-37883A, a pore-forming receptor inhibitor of K + ATP channels (Figure 2). I/R was associated with an increase in aortic and mesenteric protein expression Kir6.1 and SUR2B (Table 1). Plasma nitrite/nitrate (NO x ), TNFα and IL-6 were also increased in shock-only rats (P < 0.05) (Figure 3).
Hemodynamic effects of NaHS administered 10 minutes after the end of reperfusion MAP, HR and CBF were not different when compared between the late NaHS group and animals subjected to haemorrhagic shock and retransfusion (Figure 4).

Comparative effects of NaHS and PAG
Hydrogen sulphide donor NaHS prevents I/R-induced hemodynamic and metabolic dysfunction while PAG, an inhibitor of endogenous H 2 S production, has no effects NaHS but not PAG significantly attenuated the drop in MAP induced by I/R (P < 0.05) (Figure 1) while CBF and HR (data not shown) remained unaffected ( Figure 1). All animals treated with NaHS survived the haemorrhagic shock, while haemorrhagic shocked rats and PAG-treated rats had MAP < 40 mmHg (which we considered equivalent to death) at the end of the experiment. Haemorrhagic shock-induced hyperlactataemia was attenuated by NaHS (HS-NaHS 5 × 2.3 mmol/l) (P < 0.05) but was not modified with PAG (P < 0.05) (see Additional file 2). Compared with shock-only rats and shock + PAG rats, NaHS-treated animals had a significantly improved pH (P < 0.05) at the end of the experiment (T 150 ) (see Additional file 2).

Sodium hydrosulphide improves vascular function in rat aortic and small mesenteric vessels
PE induced a dose-dependent increase in tension in aortic and small mesenteric vessels in control rats. In contrast, haemorrhagic shock blunted PE-stimulated contraction (P <0.01), whereas NaHS significantly restored the maximal contractile capacity to control levels (P < 0.05) while PAG had no effect ( Figure 5A). Acetylcholine produced a concentration-dependent relaxation of isolated aortic and small mesenteric vessels. Compared with the sham group, vascular responses to acetylcholine decreased in the aorta of shock-only rats (P < 0.05). The addition of NaHS improved vascular response to acetylcholine while the inhibitor PAG did not modify endothelial function ( Figure 5B).
NaHS restores the phosphorylated Akt-to-Akt ratio and phosphorylated eNOS-to-eNOS ratio, while reducing haemorrhagic shock-induced upregulation of iNOS expression Expression levels of Akt and phosphorylated Akt (Akt Ser473 phosphorylation) as well as phosphorylated Aktto-Akt ratio were decreased in the aorta of shock-only rats ( Figure 6). NaHS treatment blunted this decrease while PAG rather increased their expression levels (P < 0.05). Similar results were also found for phosphorylated eNOS-to-eNOS ratio The expression of iNOS protein, as assessed by western blotting, increased in shock-only rats (compared with rats from the sham group). This increase in iNOS expression was significantly reduced following the administration of NaHS (P < 0.05) but increased with PAG (P < 0.05).

Effect of NaHS on alterations in p38 MAPK and JNK1/2 phosphorylation induced by haemorrhagic shock
NaHS reduced the phosphorylation of both p38 and JNK ( Figure 6D,E). Conversely, PAG increased this phosphorylation compared with NaHS. Neither PAG nor NaHS influenced the phosphorylation of ERK ( Figure 6F).
PNU-37883A, a pore-forming receptor inhibitor of K + ATP channels, further increases the effects of NaHS PNU-NaHS was associated with a further increase in MAP when compared with NaHS alone (P < 0.05) (see Additional file 3). PNU alone did not modify arterial pH nor the lactate level, whereas PNU-NaHS was associated with a decrease in lactate level and an increase in arterial pH as opposed to no differences with NaHS alone (P < 0.05) (data not shown).

Discussion
Herein, we illustrate the major role of NaHS in protecting the body against the consequences of shock and I/R [19]. Our findings revealed that the pharmacological inhibition of the endogenous pathway of H 2 S production during global I/R following a severe and reperfused haemorrhagic shock did not improve or worsen the consequences of shock, suggesting that endogenous H 2 S production per se is an active protective mechanism during IR; and we confirm that NaHS, an exogenous donor of H 2 S, is beneficial in terms of haemodynamics, tissue oxygenation and vascular reactivity. The effects of NaHS appear to be associated with a decrease in proinflammatory cytokines and a reduced expression of iNOS concomitant with a restoration of the eNOS pathway. These beneficial effects of NaHS appear to be more related to anti-inflammatory effects rather than to any specific vascular effect secondary to vascular K + ATP activation since selective inhibition of vascular K + ATP channels further improved haemodynamics and lactate metabolism in NaHS-treated rats. Furthermore, the effects of NaHS were not due to NaHS-induced hibernation since the animal's body temperature was continuously maintained. Finally, H 2 S when given after reperfusion was not efficient.
Our model was characterised by profound and ultimately lethal hypotension, decreased blood flow, lactic acidosis and vascular hyporesponsiveness to vasopressor agents. These haemodynamic disturbances were associated with iNOS upregulation, proinflammatory cytokine production and activation/upregulation of vascular K + ATP channels. The present findings confirmed that H 2 S given prior to retransfusion limited the I/R-induced decrease in MAP without changing carotid blood flow and heart rate when compared with shock-only rats. Given that H 2 S is usually considered an endogenous vasodilator acting through activation of vascular K + ATP , the role of this activation was further assessed with the Figure 2 Mean arterial pressure after administration of norepinephrine. Mean arterial pressure (MAP) after administration of a bolus of 1 µg/kg norepinephrine in the haemorrhagic shock (HS) + saline, HS + 1400W (treated intraperitoneally with 1400W) and HS + PNU (treated with a 1-hour infusion of PNU-37883A; 1.5 mg/kg bolus followed by 1 mg/kg/hour) groups. *P <0.05, significantly different between HS + saline and all groups. selective vascular K + ATP blocker, PNU-37883A. Our results first demonstrated that K + ATP channels were overactivated and overexpressed both at the gene and protein levels in this model, indicating that vascular K + ATP is implicated in vascular hyporesponsiveness to vasopressor agents. Secondly, rats treated with H 2 S + PNU exhibited a higher mean arterial pressure and a better vasoreactivity to norepinephrine. This may explain why H 2 S, which Hemodynamic measurements with sodium hydrosulphide administered 10 minutes after the end of reperfusion. Mean arterial blood pressure (MAP), carotid blood flow (CBF) and heart rate (HR) in sham (filled circles), haemorrhagic shock + saline (crosses), and haemorrhagic shock + sodium hydrosulphide (NaHS) (triangles) groups recorded during a 300-minute monitoring period. *P <0.05, significantly between sham.  [20].
Potassium channels are critical metabolic sensors during acute metabolic changes such as hypoglycaemia or hyperglycaemia, ischaemia and hypoxia [21]. I/R-induced cardiovascular failure is traditionally ascribed to the effects of inflammatory mediators that induce circulatory changes with resulting tissue hypoxia and cell damage [22]. In the face of these deleterious signals, the body's adaptive response at the vascular level is to preserve cell survival through metabolic sensors by increasing local blood flow in the microcirculation, the so-called metabolic vasodilatation, in which the opening of K + ATP channels plays a major role [23]. This adaptive response also leads to systemic vasodilatation, hypotension and potentially multiple organ failure and death. Vascular potassium channels may thus have protective but also harmful roles during shock. Therefore, while the use of channel inhibitors might be an attractive option to counteract systemic vasodilatation, it may also act as a double-edged sword. Whether K + ATP activation is a protective phenomenon in this setting of disturbed microcirculation thus remains unknown.

Hydrogen sulphide and PAG exert opposite effects on pathways implicated in vascular failure
Ganster and colleagues demonstrated that H 2 S improved cardiovascular status in I/R by decreasing oxidative stress and inflammation through a decrease in NF-B activation [9]. Our present model was associated with an increase in proinflammatory and anti-inflammatory cytokines, an increase in iNOS expression and an alteration in eNOS phosphorylation. As for the phosphorylation pathway, JNK phosphorylation was increased without significant changes in the p-P38/P38 ratio. Indeed, JNK and P38 have been shown to be activated by TNF and IL-1 stimulation of endothelial cells [24] Figure 5 Effects of treatment on phenylephrine-induced contraction in aorta and on aortic dilatation to acetylcholine. Effects of treatment (A) on phenylephrine (Phe)-induced contraction in aorta and (B) on aortic dilatation to acetylcholine (ACh) in sham (filled squares), haemorrhagic shock + saline (crosses), haemorrhagic shock + sodium hydrosulphide (triangles) and haemorrhagic shock + D,L-propargylglycine (empty circles) groups. *P < 0.05. and to induce expression of proinflammatory effector molecules.
In the present study, H 2 S was found to decrease the cytokine storm as well as both gene and protein iNOS expression while increasing Akt and eNOS phosphorylation. Moreover, H 2 S reduced JNK activity and p-P38/ P38 activation, suggesting a decrease in endothelial cell activation [25]. Conversely, all of these parameters were either not altered or worsened with PAG injection.

Study limitations
The present model presents several limitations, first of which involves the use of a pressure-fixed and anaesthetised model of haemorrhagic shock that does not fully represent all of the specific patterns of human haemorrhagic shock.
Secondly, we used a fixed dose of NaHS that we previously found efficient without performing a dose-response study, thus leaving the possibility that potentially toxic or beneficial effects may have been missed.
Thirdly, we did not observe any differences between the shock group and the PAG-treated group with regard to haemodynamics, metabolism and proinflammatory cytokine parameters. Van de Louw and Haouzi recently demonstrated that, despite a severe cumulative oxygen debt (100 to 140 ml/kg), H 2 S blood and tissue concentrations did not change [26]. Nevertheless, despite the absence of a marked increase during H 2 S treatment, blocking endogenous H 2 S production most probably has little therapeutic benefit and may actually prove to be contraindicated [27].
Fourthly, when compared with mouse and humans, rats exhibited more iNOS activation during stress. The importance of the H 2 S-induced decrease in iNOS activation should therefore be discussed.
Lastly, the timing of H 2 S administration might be discussed. While pretreatment with inhaled H 2 S and intravenous sodium sulphide attenuated kidney, heart, and brain damage in mice undergoing I/R injury or cardiac arrest [28,29], similar post-treatment had no effect Proteins are expressed in whole lysates of aorta (n = 8) from all groups of rats. A typical western blot is shown below each histogram. Densitometric analysis was used to calculate the normalised protein ratio (protein to α-tubulin), which was set at 1 for the control group. Data are expressed as mean ± standard deviation. *P < 0.05, significantly different versus sham and all groups. **P <0.05 versus haemorrhagic shock (HS) + sodium hydrosulphide (NaHS) group. PAG, D,L-propargylglycine. [12,30]. Our findings are in agreement with these previous reports suggesting that H 2 S beneficial effects seem to be confined to a narrow timing window.

Conclusion
The present in vivo experimental study of I/R following resuscitated haemorrhagic shock in rats demonstrates that H 2 S administered exogenously before reperfusion is protective against the deleterious cardiovascular effects of haemorrhage-induced I/R. On the contrary, blocking endogenous H 2 S production or administering H 2 S after the reperfusion had no effect. More specifically, H 2 S decreases proinflammatory cytokine and iNOS expression and restores the Akt/eNOS pathway. Such beneficial effects of H 2 S donors warrant further experimental studies.

Key messages
• H 2 S, administered exogenously before reperfusion is protective against the deleterious cardiovascular effects of haemorrhage-induced I/R.
• H 2 S is not effective when given after reperfusion.
• H 2 S increased MAP through anti-inflammatory effects despite vasodilatory effects due to K + ATP -channel activation.
• H 2 S decreases proinflammatory cytokine and iNOS expression and restores the Akt/eNOS pathway.