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.

1. 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 (H2S) [6].

H2S is a colorless, flammable, and water-soluble gas with the characteristic smell of rotten eggs. In the past several centuries, H2S had been known only for its toxicity and environmental hazards [7, 8]. It elicits its toxic effects by reversibly inhibiting cytochrome c oxidase (CcO), preventing oxidative phosphorylation and lowering the production of adenosine triphosphate (ATP). Recently, there has been growing evidence that H2S plays a broad range of physiological and pathophysiological functions [9, 10], including induction of angiogenesis [11], regulation of neuronal activity [9], vascular relaxation [12], glucose homeostatic regulation [13], and protection against I/R injury in heart, liver, kidney, lung, and brain [1418]. The abnormal metabolism of H2S could result in an array of pathological disturbances in the form of hypertension, diabetes, atherosclerosis, heart failure, sepsis, inflammation, erectile dysfunction, cataracts, asthma, and neurodegenerative diseases [10]. In addition, H2S can also interact with other specific molecules, including NO [19], CcO [20], catalase [21], myoglobin [21, 22], hemoglobin [21, 22], Kelch-like ECH-associated protein 1 (Keap1) [23], cysteine residues on ATP-sensitive potassium (KATP) channels [24], epidermal growth factor receptor [25], and vascular endothelial growth factor receptor 2 [25, 26]. Considering H2S is involved in numerous biological processes, it is now widely accepted that H2S functions as the third signaling gasotransmitter, along with NO and CO [9].

With the deepening of research on H2S and I/R injury, the role that H2S plays in attenuating I/R injury has begun to be elucidated. In this review, we highlight recent studies that provide new insight into the production and metabolism of H2S and discuss the role and mechanism of H2S on I/R injury.

2. Production and Metabolism of H2S

2.1. Endogenous Production of H2S

H2S is endogenously generated in mammalian cells via both enzymatic and nonenzymatic pathways, although the nonenzymatic pathway is less important in H2S production [27]. With regard to the enzymatic pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two pyridoxal-5′-phosphate- (PLP-) dependent enzymes, which use either L-cysteine or L-cysteine together with homocysteine as their principal substrates to produce H2S [9]. Unlike CBS and CSE, 3-mercaptopyruvate sulfurtransferase (3-MST) is a PLP-independent enzyme, which uses 3-mercaptopyruvate (3MP) as a substrate to produce H2S. 3MP is a metabolite of L-cysteine and α-ketoglutarate by cysteine aminotransferase (CAT) [9]. CSE and CBS are cytosolic enzymes with tissue-specific distributions. CBS is predominantly expressed in the central nervous system and is also found in liver, kidney, ileum, uterus, placenta, and pancreatic islets. CSE is abundant in heart, liver, kidney, uterus, ileum, placenta, and vascular smooth muscle. CSE is the most relevant H2S-producing enzyme in the cardiovascular system [9, 27]. CAT and 3-MST are localized both in cytosol and mitochondria, but the majority of these two enzymes are present in the mitochondria [9]. They have been found in the heart, kidney, liver, lung, thymus, testis, brain, and thoracic aorta and are apparently important for H2S production in the brain and vasculature [9, 27, 28]. Furthermore, a recent study has demonstrated that D-cysteine (a negative control of L-cysteine) can be metabolized to achiral 3MP by D-amino acid oxidase and can be used as a substrate for 3-MST to produce H2S in both kidney and brain [29]. During the enzymatic pathway, H2S can be immediately released or stored in a form of bound or acid-labile sulfur in the cells [30].

Apart from enzymatic pathway, endogenous H2S can also be produced through nonenzymatic processes that are less well understood [27, 30, 31]. Nonenzymatic production of H2S occurs through glucose, inorganic, and organic polysulfides (present in garlic), glutathione, and elemental sulfur [30, 31]. H2S can be generated from glucose either via glycolysis (>90%) or from phosphogluconate via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (<10%) [7, 27, 30]. Glucose could react with cysteine, methionine, or homocysteine to produce gaseous sulfur compounds such as H2S and methanethiol [7, 8, 30]. H2S is also produced through direct reduction of glutathione and elemental sulfur. Reduction of elemental sulfur to H2S is mediated through reducing equivalents of the glucose oxidation pathways such as nicotinamide adenine dinucleotide and NADPH [7, 8]. Thiosulfate is an intermediate of sulfur metabolism from cysteine and H2S formation from thiosulfate through a reductive reaction involving pyruvate, which acts as a hydrogen donor [7, 8, 32, 33]. In addition, garlic and garlic-derived organic polysulfides could induce H2S production in a thiol-dependent manner, such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and S-allyl cysteine (SAC) [3034].

2.2. Exogenous Source of H2S

H2S gas has been considered as the authentic resource of exogenous H2S [35]. Recent studies have shown that H2S gas plays important roles in promoting angiogenesis [11], ameliorating type II diabetes [13], and protecting against myocardial I/R injury [36]. However, H2S gas is not an ideal resource due to a possible toxic impact of H2S excess and difficulty in obtaining precisely controlled concentration [35]. Currently, a number of H2S-releasing compounds have already been successfully designed and developed. These compounds could be mainly divided into two types, including the “H2S donors,” which release H2S as the only mechanism of action, and the “H2S-releasing hybrid drugs,” also known as “dirty drugs” in which H2S 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 (Na2S), and calcium sulfide, have been widely used as H2S donors [7, 8, 35]. As the maximum concentration of H2S released from these salts can be reached within seconds, they have been called fast-releasing H2S donors [35]. However, the effective residence time of these donors in tissues may be very short because H2S is highly volatile in solutions [35]. Ideal H2S donors for therapeutic purposes should generate H2S with relatively slow-releasing rates and longer periods of treating time. Recently, many slow-releasing H2S donors (Table 1) and H2S-releasing hybrid drugs (Table 2) have been designed and synthesized to increase the treatment efficacy of H2S.

2.3. Metabolism of H2S

In order to maintain a proper physiological balance of its metabolism, H2S can be broken down through several enzymatic and nonenzymatic processes [7, 10, 37]. The main pathway of H2S catabolism occurs in mitochondria. Mitochondrial oxidative modification converts H2S 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 H2S metabolism under physiological conditions [7, 10, 37, 38]. The secondary mechanism of H2S catabolism is the methylation to methanethiol and dimethylsulfide via thiol S-methyltransferase in the cytosol [10, 37, 38]. The third pathway of H2S metabolism is the interaction of H2S with methemoglobin that leads to sulfhemoglobin, which is considered as a possible biomarker of plasma H2S [10, 37, 38]. These three pathways are considered the main processes of H2S catabolism in mammals. Furthermore, recent studies have shown that H2S could be converted into sulfite via minor oxidative routes in activated neutrophils [10, 37].

3. H2S and I/R Injury

3.1. H2S 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]. H2S is now considered as an endogenous signaling molecule which plays an important role in the cardiovascular system [6, 15, 27]. In the heart, H2S 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 H2S 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 H2S and its donors-induced cardioprotection against MIRI is shown in Table 3.

3.2. H2S 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 [4143]. 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, anti-inflammatory, and cytoprotective activities may be possible candidates for protecting the liver from I/R injury [46]. Recent studies have shown that H2S could significantly attenuate hepatic I/R injury in several ways, including inflammation, apoptosis, oxidation, and AKT activation (Table 4). The results suggest that H2S has a protective effect against hepatic I/R injury, and targeting H2S may present a promising approach against I/R-induced liver injury.

3.3. H2S 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 [4749]. H2S 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 H2S production and increase severity of damage and mortality after renal I/R injury, which indicates that H2S may play a role in alleviating renal I/R injury [14]. More recently, there is growing evidence regarding the beneficial effects of H2S on ameliorating renal I/R injury mainly via a variety of antioxidant, antiapoptotic, and anti-inflammatory effects (Table 5). These studies indicate that H2S and its donors may be of benefit in conditions associated with renal I/R injury, such as renal transplantation.

3.4. H2S 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 [5254]. Emerging evidences indicate that H2S functions not only as a neuromodulator, but also as a neuroprotectant in the central nervous system [18, 5557]. In an in vivo model of cerebral I/R injury, treatment with low concentration of H2S decreased the infarct size and improved the neurological function via antiapoptotic effect, implying that H2S has a therapeutic role in cerebral ischemic stroke [18, 57]. DAS, an H2S donor, could also protect the brain from I/R injury partly via its antiapoptotic effects [58]. ADT, another H2S 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 H2S on cerebral I/R injury are controversial [56]. Treatment with a higher dose of exogenous H2S donor could deteriorate the effects of cerebral I/R injury [18, 59]. These opposite effects of H2S on cerebral I/R injury may be partially associated with the concentration of H2S in brain. This research offers a novel insight for future studies on the cytoprotective effects of a proper dose of H2S on central nervous system degenerative diseases, such as Alzheimer’s disease and Parkinson’s disease.

3.5. H2S 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 bypass, strangulated hernias, liver and intestinal transplantation, mesenteric artery occlusion, shock, and severe trauma [6064]. 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 H2S 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 H2S 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/R-induced damage of intestinal tissues both in vitro and in vivo [65]. These observations provide new insight regarding the potential use of H2S as a therapeutic agent to limit intestinal I/R injury.

3.6. H2S 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]. H2S has been found to play an important role in protecting against gastric I/R injury. Endogenous H2S 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 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 mitogen-activated protein kinase dependent antiapoptosis pathway [66]. Thus, H2S and its donors may have potential therapeutic value in acute gastric mucosal lesion, which is often caused by I/R.

3.7. H2S 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 H2S could protect against limb I/R injury in a dose- and time-dependent manner needs further investigation.

3.8. H2S 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, 7477]. 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 H2S could attenuate the lung I/R injury by reducing lung oxidative stress [16], which suggests that administration of H2S or its donors might be a novel preventive and therapeutic strategy for lung I/R injury.

3.9. H2S 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 H2S can mediate antiapoptotic effects and thus protect the rat retina against I/R injury [84]. ACS67, a H2S-releasing 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 H2S 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 H2S in retinal I/R injury using a postconditioning approach.

4. Concluding Remarks

H2S 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. H2S can be endogenously generated via both enzymatic and nonenzymatic pathways and mainly metabolized through three pathways in mammals. However, whether H2S 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 H2S-generating enzymes in different organ and tissue. In order to increase the treatment efficacy of H2S, a number of slow-releasing H2S donors and H2S-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 H2S donor. Agents/drugs with antiapoptotic, antioxidative, anti-inflammatory, and antitumor effects could be conjugated with H2S donor to enhance their therapeutic effects. Furthermore, new drug targeting carrier systems should be designed to effectively transport the H2S 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 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. Whether H2S could exert protection against I/R injury in other organs and/or tissues need to be further demonstrated. In addition, the molecular targets of H2S 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 H2S-releasing compounds. Considering different doses of H2S-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 H2S 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 H2S in I/R injury. In addition, longer-term studies are required to determine whether H2S 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 H2S in I/R injury, treatment with H2S 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.


This work was supported by Grant 132300410012 (Yanzhang Li) from Henan Provincial Science & Technology, China, and National Natural Science Foundation of China Grant 81471174 (Mengzhou Xue) and Grant 31300884 (Jun Wang). Drs. Ji, Li, and Xue are Yellow River Scholars in Biology, Biochemistry and Neurology, respectively. The authors apologize to all colleagues whose relevant contributions could not be cited due to space limitations.