Introduction

Hydrogen sulfide (H2S) is a colorless, gaseous molecule at room temperature. It is widely known and disliked for its pungent, rotten egg-like smell. H2S is very water-soluble and lipophilic, resulting in quick and convenient transport between cells and tissues1. It is also strongly toxic, especially to the central nervous system. In fact, H2S acts as a more potent inhibitor of mitochondrial respiration than cyanide2. The maximum concentration in the air is approximately 10 mg/m3, and high concentrations of H2S may cause an instant loss of olfactory sensation or fainting. However, H2S, resembling CO and NO, is converted from a noxious molecule to a signaling molecule that may have promise in the medical and pharmaceutical fields.

Endogenous H2S is produced from L-cysteine by both CSE and CBS, with the cofactor cysteine amino transferase, through a so-called “trans-sulfuration pathway”3,4,5. Both CSE and CBS enzymes are pyridoxal-5′-phosphate-dependent with different concentrations in various tissues. It was previously reported that CBS is predominantly located in the central nervous system (hippocampus, cerebellum, cerebral cortex, and brain stem)6, while CSE emerges more in the cardiovascular system (aorta, mesenteric artery, portal vein, and other vascular tissue)7,8. An amount of CBS or CSE is tissue-specific, and coincidence and sole incidence are both allowed. A recent investigation also suggests the presence of another enzyme besides CBS and CSE that can generate endogenous H2S: 3-MST, which generates H2S from cysteine with the help of α-ketoglutarate in the brain9. 3-MST is located in the liver, kidney, heart, lung, thymus, testis, thoracic aorta, and brain. Three pathways of H2S degradation exist: (1) desulfurization to thiosulfate by mitochondrial oxidation, then to sulfite or sulfate; (2) cytosolic methylation to dimethylsulfide; and (3) sulfhemoglobin formation by the binding to hemoglobin9,10. A previous investigation shows that after H2S is synthesized, it is quickly absorbed or stored as a form of bound or acid-labile sulfur, while free H2S is maintained at a baseline level11. Bound sulfur is stored intracellularly in neurons and astrocytes of rats and releases H2S in the presence of physiologic concentrations of the endogenous reducing substances glutathione and cysteine. Acid-labile sulfur is another form of stored H2S. It localizes in the mitochondria and the release of H2S occurs at a pH below 5.4.

Methods to study H2S conventionally included three aspects. First, changes in substances involved in the down-regulation of the H2S concentration, such as CBS and CSE antagonists and inhibitors of potent targets are observed. Second, symptoms are compared between a range of H2S concentrations by enhancing the dosage of H2S, mainly through exogenous H2S donors, such as NaHS or other H2S-releasing compounds. Third, the variance in H2S concentration in certain diseases is measured, and the function of this variability is determined. The effort put into studying the pathological and physiological functions of H2S has allowed for a general understanding of its behavior in the body, including its synthesis, metabolism, potent targets, and effects. The large amount of evidence gathered through various experiments and observations has shown that the role of H2S as a vital endogenous gasotransmitter is ubiquitous throughout the human body. The use of H2S in the central nervous system, cardiovascular system, and gastrointestinal system is promising, and the investigation of its role is important because cardiovascular and cerebrovascular diseases as well as gastrointestinal cancers are among the top causes of human mortality. Hence, a breakthrough in dealing with diseases that severely affect our health in hope of longevity to occur is necessary. Furthermore, H2S-associated drugs with effectiveness similar to that of nitroglycerin in cases of angina pectoris may be developed in the future.

Recent evidence for the therapeutic application of H2S

Since 1996, when Ade and Kimura began their investigation of H2S, the role of H2S has been gradually revealed by various contributions from scientists worldwide3. Recent discoveries regarding H2S demonstrate its critical importance, much like NO and CO, to the medical field. This small, gaseous molecule appears to be both powerful and ubiquitous.

In the central nervous system, H2S ameliorates ischemic injuries but leads to the aggravation of stroke. Enhancement of the H2S concentration in the brain induces brain infarct, while CBS or CSE inhibitors can reverse this effect. The role of H2S in neuron protection has been shown in glutamate-induced death with increasing concentrations of cysteine and γ-glutamylcysteine, which causes enhanced GSH concentrations11,12,13,14. In patients with AD, localized increases in H2S resulted in a delay of aggravation and exacerbation of symptoms13,15,16. In addition, patients with Down syndrome overproduce H2S because their urinary excretion of thiosulfate, a specific H2S end product, was increased, suggesting a positive relationship between H2S concentration and the aggravation of Down syndrome16,17,18.

In the cardiovascular system, H2S is related to hypertension, atherosclerosis, and myocardial injury. Its utility for treating hypertension may correspond to its vasodilatory effect. H2S has the ability to relax the rat thoracic aorta, portal vein, and mesenteric artery, which suggests a more fundamental role in the regulation of contractility and blood pressure19,20,21,22,23. Conversely, further study has suggested that H2S is a vasoconstrictor at low concentrations with a possible mechanism for suppressing NO, which is also involved in contractility. As plaque is destabilized and aortic smooth muscle cell proliferation is possibly reduced by H2S, atherosclerosis is also palliated owing to an enhanced H2S concentration24,25. Similarly, H2S is able to help patients recover from myocardial injury, particularly ischemia-reperfusion injury26,27,28,29,30,31. In summary, many cardiovascular diseases demonstrated a relationship with H2S modulation, prognosticating a comprehensive utility of H2S in heart diseases.

In the respiratory system, investigation into the link between H2S and pulmonary hypertension was the first research in the field of pathophysiology and H2S in the world32. In recent years, many investigations examining the role of H2S in pulmonary hypertension, in which H2S was administered to models of chronic hypoxia, have been conducted33,34.

In the gastrointestinal system, H2S remains important in the regulation of local homeostasis. H2S-releasing bacteria in the intestine are a major source of endogenous H2S. Donation of H2S contributes to chloride secretion, which aggravates certain types of gastritis35. Gastrointestinal contractility shows sensitivity to H2S, which affects the gastrointestinal smooth muscle cells as well as the neurons in the enteric nervous system15,35,36. The concentration of H2S is enhanced when abdominal sepsis or endotoxemia occurs, and administration of H2S leads to exacerbation of these conditions, mainly because of its pro-inflammatory effect36,37. Apart from these negative effects, H2S has been shown to have a protective anti-inflammatory effect on the gastrointestinal system in some types of gastritis and colitis38,39,40. An obvious contradiction, thus, emerges, leading to puzzling study results in terms of the actual pro- or anti-inflammatory effects of H2S. This conflict implies the presence of fundamental unknown mechanisms in addition to those that have been observed. The observed results may be derived from a balance of multiple mechanisms because a balanced result always changes with circumstances.

Furthermore, insulin secretion and diabetes mellitus can be affected by local H2S concentrations because the pancreas is among the targets of H2S41,42,43,44. In the pancreas, CSE is the main enzyme converting cysteine to H2S. It has also been reported that the concentration of H2S rose in response to the presence of pancreatitis, which is ascribed to its pro-inflammatory effect. Therefore, it is clear that H2S-releasing drugs may make a difference in pancreatitis or diabetes. However, the functions of H2S remain ambiguous and demand further investigation to elucidate its anfractuous signaling network. Current studies suggest that the effects of H2S are often influenced by its concentration and reaction time as well as the cell and disease type it is acting upon. Hence, the application of H2S in certain dysfunctions is bound to be difficult considering its potentially low specificity for certain organs and tissues (see Figure 1). On the other hand, various uses of H2S may be possible because of its power.

Figure 1
figure 1

Schematic of the involvement of H2S in various physiological and pathological functions.

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New therapeutic targets associated with H2S

The mechanisms and pathways of H2S signaling are not yet completely understood. The regulation of H2S is so complicated that new mechanisms are continuously discovered before the former mechanisms are fully understood. Fortunately, several fundamental mechanisms have been determined and have drawn much attention, and we have gradually begun to fully understand them (shown in Figure 2).

Figure 2
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Potential involvement and application of the targets of H2S, including observed dual effects.

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Activation of KATP channels

A hallmark role of H2S is the regulation of contractility, which is the most fundamental and valuable use of H2S. The mechanism directing this effect is a major research topic. Vasodilation has been shown to be caused by the activation of the KATP channel8,26,45. Evidence for this hypothesis comes from the observation that the vasodilatory effect of H2S is attenuated by KATP channel antagonists, such as glibenclamide, while the KATP-dependent current is increased by H2S, as shown by patch-clamp technology. In another study, the non-selective KATP channel antagonist glibenclamide, the selective KATP channel antagonist HMR-1098, and the selective mitochondrial KATP channel antagonist 5-HD were compared to show the effect of H2S on the KATP channel46,47,48. The results of this study suggest that H2S selectively activates the plasma-membrane KATP channel (only 5-HD treatment lacked mediation of neuroprotection during hypoxia). In addition to having a vasodilatory effect, the activation of the KATP channel preconditions the body against ischemia-reperfusion injury and promotes myocardial protection, which together provide a theoretical and experimental basis for the application of H2S in heart disease26,27,45.

Activation of T-type calcium channels

Research has revealed that H2S is involved in visceral pain-like nociception and somatic hyperalgesia in mice. This observation led to further investigation of the role of indirect activation of T-type calcium channels in facilitation of visceral nociception49,50,51,52. Because Zn2+ acts as an inhibitor of T-type calcium channels, especially the Cav3.2 isoform, H2S, which chelates Zn2+, is reasonably regarded to indirectly activate T-type calcium channels by inhibiting Zn2+53. This hypothesis is further proven by the observation that Zn2+ chelates similarly to dipicolinic acid, showing the aggravation of colonic pain as well as hyperalgesia.

Activation of TRPA1

The TRP superfamily is comprised of nonselective cation channels that are divided into six main subfamilies: TRPC, TRPV, TRPM, TRPP, TRPML, and TRPA54. Among them, both TRPV and TRPA are important targets of pathological research. It is well known that TRPV1 in the bladder wall produces detrusor overactivity. TRPA1 is present on unmyelinated sensory nerve fibers within the urothelium, suburothelial space, and muscle layer as well as around blood vessels throughout the bladder. Because TRPA1 coexists with TRPV1 in major loci, such as sensory neurons in rodent dorsal root and trigeminal ganglia, and because intracellular TRPA1 activators including allyl isothiocyanate and cinnamaldehyde also initiate detrusor overactivity resembling that caused by TRPV1, it is believed that TRPA1 is involved in sensory transduction55. A recent investigation demonstrated that H2S activated TRPA1 in the presence of inflammation by the induction of calcium responses in TRPA1-expressing CHO cells. Moreover, this result is suppressed by ruthenium red, which is a non-selective TRP antagonist, and HC-030031, which is a selective TRPA1 antagonist. This reaction is not observed with the selective TRPV1 antagonist iodoresiniferatoxin. These findings reveal the presence of H2S-specific activation of TRPA1 channels56. Further investigation of H2S-specific TRPA1 channel activation may provide new solutions to various bladder disorders caused by hyperreflexia, and H2S may affect nociception, which results from complicated functions of TRPA1, upon activation via the elicitation of pain, protective reflexes, and local release of neurotransmitters in the periphery.

The NF-κB signaling pathway

H2S-induced inflammation has a relationship with the NF-κB signaling pathway. This pathway is involved in cecal ligation and puncture-induced sepsis, in which H2S regulates the expression of cytokines, chemokines, and adhesion molecules57. There are data indicating that the inhibition of SFKs causes the down-regulation of H2S-induced NF-κB activity and ICAM-1 expression, suggesting that H2S is involved in regulating the activity of NF-κB by activating the phosphorylation of SFKs, eventually resulting in mediation of ICAM-1 expression58. This pathway may also be an answer to H2S-induced anti-apoptosis. Mouse embryonic fibroblasts lacking p65, which is a subunit of NF-κB, are sensitive to TNF-α-induced cell apoptosis, indicating that NF-κB is a target molecule involved in apoptosis. Further research has elucidated a possible mechanism of enhanced transcription of CSE, which generates H2S, during TNF-α-induced apoptosis6. H2S or sulfhydrates then generates the p65 subunit of NF-κB at cysteine-38 and binds to the coactivator ribosomal protein, S3. Finally, the promotion of anti-apoptotic genes occurs, including the cellular inhibitors of apoptosis, caspase-8-c-FLIP, A1, TRAF1, and TRAF259.

Cytochrome oxidase

H2S has long been regarded as a strong toxin that inhibits mitochondrial respiration by combining the cytochrome c oxidase copper and/or heme iron site, resembling hydrogen cyanide, a well-known lethal toxin2. Studies suggest that H2S also binds and reduces ferric heme in microsomal cytochrome P450, generating a state of oxidative stress60. On the other hand, an interesting phenomenon called “suspended animation,” a form of hypometabolism, results from the above mechanism. This phenomenon was first found in rodents that inhaled H2S gas, and subsequent findings suggest that it is size-dependent or species-related61,62,63. Animals in suspended animation resembled those in hibernation, with reductions in cardiac output, ventilation frequency, and core temperature, finally leading to a decreased demand for energy. Hypometabolism can make great difference, if occurring in the human body, as an emergent protection against ischemia-associated tissue damage and infarction or providing a time delay in patients with stroke.

A scavenger of reactive oxygen and nitrogen species

H2S is highly predisposed to react with cytochrome oxidase; therefore, the hypothesis that H2S is also a potent antioxidant has been theorized. Recent studies have suggested that H2S has the ability to react with ROS and RNS, including the superoxide radical anion, hydrogen peroxide, peroxynitrite, and hypochlorite9,64,65. Thus, a protective effect against organ damage caused by ROS and/or RNS may be proven. Because the concentration of H2S in patients with AD is often severely suppressed and because AD has been proven to have a relationship with the increased generation of ROS and RNS, the role of H2S in the cure of AD merits investigation6,66.

H2S is also reported to have a specific relationship with NO. They both have a synergistic effect on vasodilation as well as an inhibitory effect on the twitching of the ileum stimulated by electricity. While NO may induce CSE activity and then enhance the production of H2S, H2S also possess the ability to catalyze release of NO from S-nitrosoglutathione67. This type of relationship reveals a deeply unexplored mechanism between H2S and NO. At a low concentration, H2S displays only a weak relaxation of vessels, mainly due to the scavenging of NO. Thus, the specific concentration of H2S play a critical role in determining its usability as a biomarker or its therapeutic application68,69.

H2S donor molecules and H2S-releasing drugs

Ordinarily, H2S-related drugs are categorized as either administrators of H2S that enhance local or whole-body H2S concentrations and cause pathological and physiological changes or as inhibitors of H2S production enzymes that reduce local or whole-body H2S concentrations and cause pathological and physiological changes. H2S donors, including donor molecules and H2S-releasing drugs, can be divided into three types: inorganic substances, such as NaHS; organic compounds, represented by GYY4137, which is derived from Lawesson's reagent; and agonists of H2S-synthesized enzymes. Inhibitors of enzymes involved in the synthesis of H2S are of great importance, especially in the study of the mechanisms of H2S functions. DL-propargylglycine, which easily permeates the cell membrane without obvious damage due to its high lipophilic property, is widely used as a nonspecific inhibitor of CSE and CBS. Disproof is a basic yet practical method for scientific investigation; therefore, the study of the effect of H2S on certain targets by diminishing its concentration may be more effective than directly administering it. In fact, these inhibitors are effective agents in diseases induced by the overproduction of H2S.

Inorganic H2S donors

Simple molecules, such as NaHS and Na2S, are basic tools used for H2S research. Their donation of H2S is rapid upon reaction with water because of high solubility. Gaseous H2S is sometimes used by respiratory passages through direct absorption by pulmonary alveoli due to high bioavailability. However, there is a fundamental shortage of the use of inorganic H2S donors in both research and clinical applications because of the need to attain long-term release at a controlled rate70,71. A too rapid release implies increasing the concentration of H2S instantly, resembling a pulse cure, which is associated with a variety of problems, including concentrations that are too high for the local tissue and a lack of a long-term effect. Both problems led to imprecise experimental parameters, including dose and time, random results, and misleading causes. Thus, this data shortage may be a partial explanation for the emergence of divergent results, even when the same function is investigated.

To obtain a better H2S donor for sustained and controlled use, organic compounds containing chemically synthesized molecules and natural plant extracts have become prevalent. Some organic compounds have shown ideal therapeutic effects and a large commercial potential.

Precursors for endogenous H2S synthesis

N-acetylcysteine and L-cysteine are precursors for endogenous H2S synthesis70. Enhancing these precursors causes H2S to increase with the catalysis of CSE and CBS. This precursor increase has minimal side effects associated with their production in the body.

Synthetic H2S donors

As in other fields, synthetic drugs are predominant among H2S-releasing drugs. GYY4137, a derivative of Lawesson's reagent, acts as a synthetic H2S donor. Its sustained release of H2S in aqueous media with a concentration above baseline has been demonstrated72. GYY4137 causes the concentration-dependent suppression of cancer cells, such as MCF-7, by the promotion of apoptosis without an obvious impact in normal cells, such as IMR90. That is to say, our demand for the sustained moderate release of H2S is met. In a recent study, a series of new H2S donors based on the N-(benzoylthio)benzamide template were synthesized. The authors utilized the theory that the S-N bond is cysteine-activated under certain conditions, which needs to be demonstrated in future studies73. The release rate could be manually adjusted by the purposeful modification of the core structure.

H2S-nonsteroidal anti-inflammatory drug hybrids

NSAIDs, among which aspirin is well known, are widely used for their anti-inflammatory and anti-gout properties. Nevertheless, they are associated with side effects, such as serious gastrointestinal bleeding and heart attack. Aspirin, as a delegate of NSAIDs, is famous for its anti-inflammatory ability as well as its prevention of atherosclerosis by inhibiting COX and platelet aggregation. However, aspirin also stimulates gastric bleeding and, worse, the prevention of platelet aggregation will hinder the cessation of bleeding. H2S-NSAID hybrids have, thus, been devised based on the protective effect of H2S on the gastrointestinal and cardiovascular systems74,75. ATB 337, which is derived from diclofenac, shows significantly reduced gastrointestinal toxicity compared with diclofenac and has an enhanced anti-inflammatory effect with no effect on hematocrit or leukocyte adherence. ATB 429, which is derived from mesalamine, also shows an improvement in treating inflammatory bowel disease compared with its parent drug. ATB 346, which is derived from naproxen, remarkably reduces gastrointestinal and cardiovascular toxicity, such as the two aforementioned drugs70. The side effects of NSAIDs mainly come from the single inhibition of only COX-1 or COX-2, which generates an imbalance in the expression of COX-1 and COX-2 in different tissues and organs. Therefore, these H2S-NSAID hybrid compounds can release H2S when dissolved by enzymes, which will protect organs, such as the stomach as well as the heart, because of the anti-oxidation or other effects of H2S. It should also be noted that these types of compounds are not a simple addition of both NSAIDs and H2S but because they can affect each other when in the same place at the same time, they make a more magnified effect than a simple two part addition.

Natural plant-derived compounds

Vegetables, represented by garlic and ginger, are ordinary salubrious foods that have anti-inflammatory functions. In recent years, garlic-derived polysulfide compounds have drawn great attention due to their potential anti-inflammatory and anti-cancer effects. DATS is the first garlic-derived molecule discovered to possess vasoactivity along with the ability to specifically suppress cancer cells76. Compounds similar to DATS, such as DAS, DADS, and DATTS, have been evaluated for similar functions, but they are complicated. Different effects have been found. DAS reportedly activates the nuclear receptor CAR which induces various hepatic drug-metabolizing phase I and phase II enzymes in the mouse liver and preventive effects on some types of gastric cancer77. DADS has demonstrated vasorelaxation effect in aortic ring preparations via glucose- and thiol-dependent cellular reactions. Interestingly, DADS modified hemoglobin's β-chain at cysteine-93 or cysteine-112 in deoxygenated human red blood cells. This was the first instance of a garlic-derived compound that was shown to be able to modify an intracellular protein77. Normally, garlic-derived compounds are simply regarded as precursors of H2S when absorbed and metabolized in blood. Therefore, because DADS itself is able to generate direct protein changes, it is worthy of future study.

S-allylcysteine is believed to have a cardioprotective effect and is a potential source of H2S. However, whether it plays a role as a H2S precursor or a modulator of H2S-related enzymes is controversial76. Another novel garlic-derived compound, S-propargyl-cysteine, is now under investigation. It was recently shown to attenuate lipopolysaccharide-induced spatial learning and memory impairment through TNF signaling and the NF-κB pathway in rats, which contributes to studies about AD and other diseases related to neuronal damage6,79.

Compounds extracted from other plants display similar effects. Sulforaphane, which is derived from isothiocyanates found in cruciferous vegetables, exhibits an anti-cancer property. Sulforaphane suppresses the proliferation of prostate cancer cells and enhances the expression of CBS and CSE. In addition, sulforaphane activates p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, suggesting a potent mechanism80. Although most of the significant results have been gained from in vitro studies, the study of H2S-releasing donors in vivo is more complex. Therefore, the identification of a feasible drug for clinical use requires further research.

Future developments: challenges for translation of the toxic molecule H2S

Investigation of the role of H2S is still in its infancy. H2S has both scientific and technological value. The latter tends to dominate because of its financial value and direct applicability, which substantiates research efforts. However, the scientific investigation of H2S is also important for the elucidation of its base fundamental roles. Scientific investigation strives to address disease, enhance quality of life, and disclose the secrets of the human body. Certainly problems and challenges will arise and the limitations of experimental materials will at times constrain further H2S research. A sustained and controlled H2S-releasing donor that functions both in vitro and in vivo has not yet been found. An effective and quick method of measuring the concentration of H2S is also required, especially a non-invasive method. With an increased understanding of the various H2S mechanisms in the body, further study of H2S becomes more difficult and complicated. H2S is known as a third gaseous signaling molecule, which means that it plays the role of a messenger. The concentration of H2S has been proven to have relevance in particular diseases; for instance, it is overproduced in sepsis and found at inadequate levels in AD81. Therefore, the mechanism controlling the actual concentration of H2S in certain tissues may become the ultimate problem for H2S related research. It should be emphasized that a relevant relationship does not mean a relationship of causation. By regulating the H2S concentration in particular tissues, symptoms of a specific disease can be controlled, which implies that the origin of the disease has not been addressed. That is to say, the regulation of H2S can only provide transient protection from certain diseases, such as hypertension. The challenges of the sustained and controlled release of H2S-releasing drugs were mentioned above. Another difficulty in H2S related research comes from the multiple functions of H2S, which cause a shortage of specific effects. The effects of H2S are dose-, time-, and tissue-dependent. For patients with different diseases, H2S may need to be administered as different drugs. Therefore, a focus on the general effects of H2S, such as on cardiovascular protection, is rational.

Abbreviations

H2S, Hydrogen sulfide; CO, carbon monoxide; NO, nitric oxide; CSE, cystathionine γ-lyase; CBS, cystathionine β-synthase; 3-MST, 3-mercaptopyruvate sulfurtransferase; NaHS, sodium hydrosulfide; AD, Alzheimer's disease; KATP channels, potassium-dependent ATP-sensitive channels; TRP, transient receptor potential; TRPC, canonical transient receptor potential channel; TRPV, vanilloid transient receptor potential channel; TRPM, melastatin transient receptor potential channel; TRPP, polycystin transient receptor potential channel; TRPML, mucolipin transient receptor potential channel; TRPA, ankyrin transient receptor potential channel; CHO cells, Chinese hamster ovary cells; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SFKs, SRC family kinases; ICAM-1, intracellular adhesion molecule-1; TRAF1, TNFR-associated factor 1; TRAF2, TNFR-associated factor 2; ROS, reactive oxygen species; RNS, reactive nitrogen species; NSAIDs, nonsteroidal anti-inflammatory drugs; DATS, diallyl trisulfide; DAS, diallyl sulfide; DADS, diallyl disulfide; DATTS, diallyl tetrasulfide; CAR, orphan member of the nuclear steroid hormone receptor superfamily.