Role of Hydrogen Sulfide in Ischemia-Reperfusion Injury

Ischemia-reperfusion (I/R) injury is one of the major causes of high morbidity, disability, and mortality in the world. I/R injury remains a complicated and unresolved situation in clinical practice, especially in the field of solid organ transplantation. Hydrogen sulfide (H2S) is the third gaseous signaling molecule and plays a broad range of physiological and pathophysiological roles in mammals. H2S could protect against I/R injury in many organs and tissues, such as heart, liver, kidney, brain, intestine, stomach, hind-limb, lung, and retina. The goal of this review is to highlight recent findings regarding the role of H2S in I/R injury. In this review, we present the production and metabolism of H2S and further discuss the effect and mechanism of H2S in I/R injury.


Introduction
Ischemia-reperfusion (I/R) is a well-recognized pathological condition that is characterized by an initial deprivation of blood supply to an area or organ followed by subsequent vascular restoration and concomitant reoxygenation of downstream tissue [1]. I/R can develop as a consequence of trauma, hypertension, shock, sepsis, organ transplantation, or bypass surgery leading to end-organ failure such as acute renal tubular necrosis, bowel infarct, and liver failure. I/R can also occur under various complications of vascular diseases such as stroke and myocardial infarction [1,2]. Several pathophysiologic mechanisms have been proposed as mediators of the damage induced by I/R, such as activation of the complement system and leukocyte recruitment, endoplasmic reticulum stress, calcium overload, reduction of oxidative phosphorylation, increased free radical concentration, development of the no-reflow phenomenon, endothelial dysfunction, and activation of signaling pathways of apoptosis, necrosis, and/or autophagy [1,3]. Many studies have shown that there are three time frames in the protection against I/R injury: before the index ischemic episode (ischemic preconditioning), during ischemia (ischemic conditioning), and at the onset of reperfusion (ischemic postconditioning) [4,5]. Currently, several therapeutic gases have been shown to play a role in the treatment of I/R injury, including hydrogen, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S) [6].

Exogenous
Source of H 2 S. H 2 S gas has been considered as the authentic resource of exogenous H 2 S [35]. Recent studies have shown that H 2 S gas plays important roles in promoting angiogenesis [11], ameliorating type II diabetes [13], and protecting against myocardial I/R injury [36]. However, H 2 S gas is not an ideal resource due to a possible toxic impact of H 2 S excess and difficulty in obtaining precisely controlled concentration [35]. Currently, a number of H 2 S-releasing compounds have already been successfully designed and developed. These compounds could be mainly divided into two types, including the "H 2 S donors, " which release H 2 S as the only mechanism of action, and the "H 2 S-releasing hybrid drugs, " also known as "dirty drugs" in which H 2 S release is an ancillary property which accompanies a principal mechanism of the hybrid drugs [35]. Inorganic sulfide salts, such as sodium hydrosulfide (NaHS), sodium sulfide (Na 2 S), and calcium sulfide, have been widely used as H 2 S donors [7,8,35]. As the maximum concentration of H 2 S released from these salts can be reached within seconds, they have been called fast-releasing H 2 S donors [35]. However, the effective residence time of these donors in tissues may be very short because H 2 S is highly volatile in solutions [35]. Ideal H 2 S donors for therapeutic purposes should generate H 2 S with relatively slow-releasing rates and longer periods of treating time. Recently, many slow-releasing H 2 S donors (Table 1) and H 2 S-releasing hybrid drugs (Table 2) have been designed and synthesized to increase the treatment efficacy of H 2 S.

Metabolism of H 2 S.
In order to maintain a proper physiological balance of its metabolism, H 2 S can be broken down through several enzymatic and nonenzymatic processes [7,10,37]. The main pathway of H 2 S catabolism occurs in mitochondria. Mitochondrial oxidative modification converts H 2 S into thiosulfate through several enzymes including quinone oxidoreductase, S-dioxygenase, and S-transferase. Thiosulfate could be further converted into sulfite, which is catalyzed by thiosulfate : cyanide sulfurtransferase. Sulfite is then rapidly oxidized to sulfate by sulfite oxidase. Therefore, sulfate is a major end-product of H 2 S metabolism under physiological conditions [7,10,37,38]. The secondary mechanism of H 2 S catabolism is the methylation to methanethiol and dimethylsulfide via thiol S-methyltransferase in the cytosol [10,37,38]. The third pathway of H 2 S metabolism is the interaction of H 2 S with methemoglobin that leads to sulfhemoglobin, which is considered as a possible biomarker of plasma H 2 S [10, 37, 38]. These three pathways are considered the main processes of H 2 S catabolism in mammals. Furthermore, recent studies have shown that H 2 S could be converted into sulfite via minor oxidative routes in activated neutrophils [10,37].

H 2 S and Myocardial I/R Injury.
Myocardial ischemia is a common clinical symptom characterized by low pH values, low oxygen, and high extracellular potassium concentration, which may cause arrhythmias, cardiac dysfunction, myocardial infarction, and sudden death [3,5,6]. The damaged myocardial structure and decreased heart function induced by ischemia can be repaired with subsequent reperfusion. The effectiveness of reperfusion depends on the duration and severity of prior ischemia [6,39]. However, myocardial reperfusion could also activate a complex inflammatory response, which may finally lead to myocardial ischemia/reperfusion injury (MIRI), such as arrhythmias, myocardial stunning, microvascular dysfunction, and myocyte death [2,40]. Therefore, it is necessary to develop effective cardioprotective strategies and agents against MIRI to improve myocardial function and to reduce the risk of cardiovascular events [4]. H 2 S is now considered as an endogenous signaling molecule which plays an important role in the cardiovascular system [6,15,27]. In the heart, H 2 S is produced in the fibroblasts, myocardium, and blood vessels from L-cysteine by CSE, CBS, and 3-MST and accumulates at relatively high local concentrations [6,27,30]. An accumulating body of evidence indicates that exogenous or endogenous H 2 S could exert cardioprotection against MIRI in cardiac myocytes, isolated hearts, and intact animals. However, it is currently difficult to define the precise underlying mechanisms for this protection.
A summary of what is known about the mechanisms by which H 2 S and its donors-induced cardioprotection against MIRI is shown in Table 3.

H 2 S and Hepatic I/R Injury.
Liver I/R-induced injury represents a continuum of organic processes that could produce profound liver damage and ultimately lead to morbidity and mortality [41,42]. Hepatic I/R injury has now been considered a worldwide health problem and usually occurs in liver transplantation, hemorrhagic shock and resuscitation, trauma, liver resection surgery, and aortic injury during abdominal surgery [41][42][43]. Hepatic I/R injury can be categorized into warm I/R and cold storage reperfusion injury, which share a common mechanism in the disease aetiology [41,42]. Increasing number of experimental and clinical studies indicate that pathways/factors involved in the hepatic I/R injury include liver Kupffer cells and neutrophils, intracellular calcium overload, oxidative stress, anaerobic metabolism, mitochondria, adhesion molecules, chemokines, and proinflammatory cytokines [41,42,44,45]. Despite significant advances in surgical techniques and perioperative cares, hepatic I/R injury remains one of the major complications in hepatic resection and transplantation [46]. Novel agents/drugs exhibiting antioxidative, antiinflammatory, and cytoprotective activities may be possible candidates for protecting the liver from I/R injury [46]. Recent studies have shown that H 2 S could significantly attenuate hepatic I/R injury in several ways, including inflammation, apoptosis, oxidation, and AKT activation ( Table 4). The results suggest that H 2 S has a protective effect against hepatic I/R injury, and targeting H 2 S may present a promising approach against I/R-induced liver injury.

H 2 S and Renal I/R Injury.
Acute kidney injury (AKI) is a common and serious complication of critical illness and is associated with high morbidity, mortality, and resource utilization [25,47,48]. Renal I/R injury is one of the leading causes of AKI in many clinical settings [47,48]. Renal I/R injury often arises from shock and various surgical procedures such as kidney transplantation and resection [47][48][49]. H 2 S plays important physiological and pathological roles in the kidney [48]. For instance, it participates in the control of renal function and increases urinary sodium excretion via both tubular and vascular actions in the kidney [50]. CSE deficiency in mice could lead to reduced renal H 2 S production and increase severity of damage and mortality after renal I/R injury, which indicates that H 2 S may play a role in alleviating renal I/R injury [14]. More recently, there is growing evidence regarding the beneficial effects of H 2 S on ameliorating renal I/R injury mainly via a variety of antioxidant, antiapoptotic, and anti-inflammatory effects ( Table 5). These studies indicate that H 2 S and its donors may be of benefit in conditions associated with renal I/R injury, such as renal transplantation.

H 2 S and Cerebral I/R Injury.
Ischemic cerebrovascular disease is one of the most common disorders that greatly threaten human health with high morbidity, disability, and mortality [51]. Cerebral I/R injury is mainly characterized by a deterioration of ischemic but potentially salvageable brain tissue of an ischemic injury after reperfusion [52,53].
There are a number of risk factors involved in cerebral I/R injury, such as excitotoxicity, mitochondrial dysfunction, formation of free radicals, breakdown of the blood-brain barrier (BBB), edema, neuroinflammation, and apoptosis [52][53][54]. Emerging evidences indicate that H 2 S functions not only as a neuromodulator, but also as a neuroprotectant in the central nervous system [18,[55][56][57]. In an in vivo model of cerebral I/R injury, treatment with low concentration of H 2 S decreased the infarct size and improved the neurological function via antiapoptotic effect, implying that H 2 S has a therapeutic role in cerebral ischemic stroke [18,57]. DAS, an H 2 S donor, could also protect the brain from I/R injury partly via its antiapoptotic effects [58]. ADT, another H 2 S donor, decreased the infarct size and protected BBB integrity by suppressing local inflammation and nicotinamide adenine dinucleotide phosphate oxidase 4-derived ROS generation [55]. However, it is notable that the effects of H 2 S on cerebral I/R injury are controversial [56]. Treatment with a higher dose of exogenous H 2 S donor could deteriorate the effects of cerebral I/R injury [18,59]. These opposite effects of H 2 S on cerebral I/R injury may be partially associated with the concentration of H 2 S in brain. This research offers a novel insight for future studies on the cytoprotective effects of a proper dose of H 2 S on central nervous system degenerative diseases, such as Alzheimer's disease and Parkinson's disease.

H 2 S and Intestinal I/R Injury.
Intestinal I/R injury is considered to be a major and frequent problem in many clinical conditions, including intestinal mechanical obstruction, abdominal aortic aneurysm surgery, cardiopulmonary Myocardial I/R in vivo (mice) H 2 S (100 ppm, prior to I) has protective properties in I/R injury Reduction of myocardial ROS production and the inhibition of inflammation, necrosis, and fibrogenesis [36] Regional myocardial I/R in vivo (pig) Na 2 S (100 g/kg bolus + 1 mg/kg/hr infusion, 10 min prior to R) improves myocardial function and reduces infarct size Anti-inflammatory properties [160] Regional myocardial I/R in vivo (pig) Na 2 S (100 g/kg bolus + 1 mg/kg/hr infusion, throughout the experiment) reduces myocardial infarct size Antiapoptotic activities [161] Regional myocardial I/R in vivo (rat) NaHS (0.1-10 M, 10 min prior to I until 10 min into R) results in a concentration-dependent limitation of infarct size Mitochondrial K ATP channel opening [162] Myocardial I/R in vivo (rat) NaHS (0.2 mg/kg, prior to R) protects against the effects of haemorrhage-induced I/R Protection against oxidative stress [163] Primary cultured neonatal cardiomyocytes (rat)

NaHS (1-100 M, 30 min prior to H) shows concentration-dependent inhibitory effects on cardiomyocyte apoptosis induced by H/R
Induction of phosphorylation of GSK-3 and inhibition of mitochondrial permeability transition pore opening [164] Myocardial I/R in vivo (mice) Na 2 S (0.1 mg/kg, 7 days prior to I) attenuates myocardial I/R injury Activation of nuclear factor erythroid-2-related factor-2 signaling in an Erk-dependent manner [165] Oxidative Medicine and Cellular Longevity 7 Myocardial I/R in vivo (mice) Na 2 S (10-500 g/kg, prior to R) limits infarct size and preserves left ventricular function Inhibition of myocardial inflammation and preservation of both mitochondrial structure and function [167] Myocardial I/R in vivo (mice) Na 2 S (100 g/kg, 1 h prior to I) reduces myocardial infarct size miR-21-dependent attenuation of ischemic and inflammatory injury [168] Myocardial I/R in vivo (mice) Na 2 S (100 g/kg, 24 h prior to I) reduces myocardial infarct size Combination of antioxidant and antiapoptotic signaling [169] Isolated perfused heart ex vivo (rabbit) Allitridum ( Isolated perfused heart ex vivo (mice) Na 2 S (10 M, 40 seconds after the start of R) markedly improves the recovery of myocardial function Nitric oxide synthase 3-dependent signaling pathway [174] Myocardial I/R in vivo (rat) NaHS (14 M/kg/d, 6 d prior to I) markedly reduces heart infarct size and has great improvement in blood pressure Upregulation of survivin [175] Myocardial I/R in vivo (pig) NaHS (0.2 mg/kg, prior to R) markedly reduces myocardial infarct size and improves regional left ventricular function Higher expression of phospho-GSK-3 and lower expression of apoptosis-inducing factor [176] H/R: hypoxia/reoxygenation; SOD: superoxide dismutase; PKC: protein kinase C; ERK1/2: extracellular signal regulated kinase 1/2; PI3K (PtdIns3K): phosphatidylinositol 3-kinase; Akt (PKB): protein kinase B; COX-2: cyclooxygenase-2; ROS: reactive oxygen species; GSK-3: glycogen synthase kinase-3.
bypass, strangulated hernias, liver and intestinal transplantation, mesenteric artery occlusion, shock, and severe trauma [60][61][62][63][64]. This injury can lead to the development of systemic inflammatory response syndrome and multiple organ dysfunction syndrome [62,63]. Although many advanced treatments have been applied to clinical research, the mortality induced by intestinal I/R injury remains very high [61,63]. Therefore, it is urgent to develop new therapeutic agents/drugs for the treatment of intestinal I/R injury. Recent studies have shown that H 2 S has anti-ischemic activity in the intestinal I/R model. NaHS could significantly reduce the severity of intestinal I/R injury and dramatically increase the activities of SOD and glutathione peroxidase (GSH-Px) in both serum and intestinal tissue, which suggests that H 2 S protects against intestinal I/R injury by increasing the levels of antioxidant enzymes [63]. In addition, administration of NaHS after the onset of ischemia can attenuate I/Rinduced damage of intestinal tissues both in vitro and in vivo [65]. These observations provide new insight regarding the potential use of H 2 S as a therapeutic agent to limit intestinal I/R injury.

H 2 S and Gastric I/R Injury.
Gastric I/R injury is an important and common clinical problem which could lead to mucosal injury [66]. A number of clinical conditions contribute to gastric I/R injury, including peptic ulcer bleeding, vascular rupture or surgery, ischemia gastrointestinal disease, and hemorrhagic shock [66]. However, there are few satisfactory clinical methods in the treatment of gastric I/R injury [67]. H 2 S has been found to play an important role in protecting against gastric I/R injury. Endogenous H 2 S had a protective effect against gastric I/R in rats by enhancing the antioxidant capacity through increasing the contents of GSH and SOD [68]. Another study has shown that NaHS and L-cysteine could protect the gastric mucosa against I/R damage mainly mediated by altering mRNA expression and 8 Oxidative Medicine and Cellular Longevity Hepatic I/R in vivo (mice) NaHS (1.5 mg/kg, 1 h prior to I) protects against hepatic I/R injuries Activation of the PtdIns3K-AKT1 pathway [17] Hepatic I/R in vivo (rat) NaHS (14 M/kg, 30 min prior to I) significantly attenuates the severity of liver injury and inhibits the production of lipid peroxidation Antioxidant and antiapoptotic activities [46] Hepatic I/R in vivo (rat) DAS (1.75 mM/kg, 12-15 h prior to I) protects the liver from warm I/R injury Induction of heme oxygenase-1 and inhibition of cytochrome P450 2E1 [178] Hepatic I/R in vivo (mice) Na 2 S (1 mg/kg, 5 min prior to R) protects the murine liver against I/R injury Upregulation of intracellular antioxidant and antiapoptotic signaling pathways [179] Hepatic I/R in vivo (mice) H 2 S (100 ppm, 5 min prior to R) protects the liver against I/R injury Reduction of necrosis, apoptosis, and inflammation [180] Hepatic I/R in vivo (mice) NaHS (14 and 28 M/kg, 30 min prior to I) attenuates hepatic I/R injury Weaken the apoptosis through the inhibition of c-Jun N-terminal protein kinase 1 signaling pathway [181] Hepatic I/R in vivo (rat) NaHS (12.5, 25 and −50 M/kg, 5 min prior to I) reduces liver damage after perioperative I/R injury Inhibition of mitochondrial permeability transition pore opening, reduction of cell apoptosis, and activation of Akt-GSK-3 signaling Renal I/R in vivo (pig) Na 2 S (100 g/kg, 10 min prior to R) results in a marked reduction in kidney injury and preserves glomerular function Anti-inflammatory effects [184] Isolated perfused kidney ex vivo (pig) H 2 S (0.5 mM, 10 min before and after R) ameliorates the renal dysfunction Activation of K ATP channels [185] Renal I/R in vivo (mice) NaHS (100 M/kg, 30 min prior to I) significantly attenuates I/R injury-induced renal dysfunction The increase in expression of CSE [186] Renal I/R in vivo (rat) NaHS (100 M/kg, 15 min prior to I and 5 min prior to R) attenuates renal I/R injury Antiapoptotic and anti-inflammatory effects [187] Warm renal I/R in vivo (rat) NaHS (150 M, at time of renal pedicle clamping and during R) improves long-term renal function and decreases long-term inflammation Antiapoptotic and anti-inflammatory effects [188] Warm renal I/R in vivo (rat) NaHS (150 M, during I and R) increases renal capillary perfusion and improves acute tubular necrosis and apoptosis Decrease of leukocyte migration and inflammatory responses [189] Renal I/R in vivo (pig) Na 2 S (2 mg/kg, 2 h prior to I) attenuates tissue injury and organ dysfunction Antioxidant and anti-inflammatory effects [190] Renal I/R in vivo (rat) NaHS (100 g/kg, 20 min prior to I or 10 min prior to R) protects against renal I/R injury Antioxidant and antiapoptotic effects [191] plasma release of proinflammatory cytokines [69]. Furthermore, NaHS and L-cysteine also showed gastroprotective effects against I/R injury by Keap1 s-sulfhydration, nuclear factor-kappa B dependent anti-inflammation, and mitogenactivated protein kinase dependent antiapoptosis pathway [66]. Thus, H 2 S and its donors may have potential therapeutic value in acute gastric mucosal lesion, which is often caused by I/R.

H 2 S and
Hind-Limb I/R Injury. I/R injury can occur in skeletal muscle during elective surgery (i.e., free tissue transfer) and lower extremity arterial occlusion [70,71]. Limb I/R injury may result in a series of postreperfusion syndromes, such as crush syndrome, compartment syndrome, and myonephropathic-metabolic syndrome [72]. Currently, clinical practice mainly focuses on reducing the duration of ischemia to minimize the ischemic injury in skeletal muscle [70,71]. Therapeutic interventions that change the biochemical environment during the ischemic and/or reperfusion period may result in amelioration of subsequent cellular damage [71]. Treatment with NaHS for 20 minutes before the onset of hind-limb ischemia or reperfusion could result in significant protection against the cellular damage induced by I/R [71,73]. However, administration of NaHS for 1 minute before reperfusion did not show any protection against limb I/R Injury [73]. Whether H 2 S could protect against limb I/R injury in a dose-and time-dependent manner needs further investigation.

H 2 S and Lung I/R Injury. Lung I/R injury occurs in
various clinical conditions such as lung transplantation, cardiopulmonary bypass, trauma, cardiac bypass surgery, sleeve lobectomy, shock, pulmonary embolism, resuscitation from circulatory arrest, and reexpansion pulmonary edema [16,[74][75][76][77]. Lung I/R injury is characterized by increased pulmonary vascular resistance, worsened lung compliance, poor lung oxygenation, edema, and increased pulmonary endothelial permeability [16,78]. Currently, there is no effective therapy available for the lung I/R injury. The precise mechanism of lung I/R injury needs to be further elucidated [16,74]. A recent study has shown that preperfusion with H 2 S could attenuate the lung I/R injury by reducing lung oxidative stress [16], which suggests that administration of H 2 S or its donors might be a novel preventive and therapeutic strategy for lung I/R injury.

H 2 S and Retinal I/R Injury.
Retinal I/R injury is a common clinical condition and is associated with the loss of neurons, morphological degeneration of the retina, loss of retinal function, and ultimately vision loss [79,80]. Emerging evidence suggests that retinal I/R injury plays an important role in the pathologic processes of several ocular diseases such as diabetic retinopathy, retinopathy of prematurity, acute glaucoma, and retinal vascular occlusion [81,82].
Retinal I/R injury often results in visual impairment and blindness because of the lack of effective treatment [81,83].
One recent study has indicated that rapid preconditioning with inhaled H 2 S can mediate antiapoptotic effects and thus protect the rat retina against I/R injury [84]. ACS67, a H 2 Sreleasing derivative of latanoprost acid, possesses neuroprotective properties and could attenuate retinal ischemia in vivo and decrease the oxidative insult to RGC-5 cells (retinal ganglion cells) in vitro [85]. These results suggest that H 2 S represents a novel and promising therapeutic agent to counteract neuronal injuries in the eye [84]. Further studies are needed to prove the neuroprotective propensity of H 2 S in retinal I/R injury using a postconditioning approach.

Concluding Remarks
H 2 S is now considered as the third signaling gasotransmitter which plays a broad range of physiological and pathophysiological functions, including vascular relaxation, induction of angiogenesis, regulation of neuronal activity, and glucose homeostatic regulation. H 2 S can be endogenously generated via both enzymatic and nonenzymatic pathways and mainly metabolized through three pathways in mammals. However, whether H 2 S could be generated and metabolized via another pathway should be further studied and confirmed.
In addition, more efforts should be made to illuminate the expressions and functions of H 2 S-generating enzymes in different organ and tissue. In order to increase the treatment efficacy of H 2 S, a number of slow-releasing H 2 S donors and H 2 S-releasing hybrid drugs have been successfully designed, synthesized, and proved to be effective in vitro, ex vivo, and in vivo. Novel synthetic strategy should be developed to extend the exposure time of H 2 S donor. Agents/drugs with antiapoptotic, antioxidative, anti-inflammatory, and antitumor effects could be conjugated with H 2 S donor to enhance their therapeutic effects. Furthermore, new drug targeting carrier systems should be designed to effectively transport the H 2 S donor to the targeted organ or tissue. I/R is a pathological condition that is characterized by an initial deprivation of blood supply to an area or organ followed by the subsequent restoration of perfusion and concomitant reoxygenation. Novel mechanisms associated with I/R need to be further studied and illuminated in addition to the existing pathophysiologic mechanisms. Increasing number of studies have shown that H 2 S could protect against I/R injury in many organs and tissues, such as heart, liver, kidney, brain, intestine, stomach, hind-limb, lung, and retina. Whether H 2 S could exert protection against I/R injury in other organs and/or tissues need to be further demonstrated. In addition, the molecular targets of H 2 S in I/R injury are also needed to be clarified. Ischemic preconditioning, conditioning, and postconditioning are three time frames in the protection against I/R injury. Proper time frame and optimal duration of treatment should be confirmed according to the physicochemical property of H 2 S-releasing compounds. Considering different doses of H 2 S-releasing compounds may exert different therapeutic effects, proper dose range should also be further explored to obtain a better therapeutic efficacy. Currently, researches into the molecular mechanisms of H 2 S in I/R injury using animal experiments have made some progress. Clinical evidence-based research should also be useful in further exploring the little-understood field of the role of H 2 S in I/R injury. In addition, longer-term studies are required to determine whether H 2 S treatment permanently improves organ function following I/R injury and whether this effect reduces long-term morbidity and mortality.
In conclusion, with the rapid developments of design and synthetic strategies, as well as better understanding of the precise mechanisms behind the role of H 2 S in I/R injury, treatment with H 2 S or its donors in proper dose range and time frame will exhibit more potent therapeutic effects against I/R injury in further preclinical research and clinical application.

Conflict of Interests
The authors declare no conflict of interests related to this work.