Novel ray of hope for diabetic wound healing: Hydrogen sulfide and its releasing agents

Graphical abstract

The role of hydrogen sulfide and its releasing agents in diabetic wound healing.

Highlights

• •This mini-review briefly introduces the causes of diabetic wound healing and the direction of clinical treatment.
• •This mini-review provides reasons why hydrogen sulfide may be considered for the treatment of diabetic wound healing.
• •This mini-review discusses the role of hydrogen sulfide in various stages of diabetic wound healing.
• •This mini-review involves ideas to improve hydrogen sulfide availability to aid future developments.
• •The mini-review suggests that hydrogen sulfide is a new hope for improving diabetic wound healing.

Abstract

Background

Diabetes mellitus (DM) is a long-term metabolic disease accompanied by difficulties in wound healing placing a severe financial and physical burden on patients. As one of the important signal transduction molecules, both endogenous and exogenous hydrogen sulfide (H₂S) was found to promote diabetic wound healing in recent studies. H₂S at physiological concentrations can not only promote cell migration and adhesion functions, but also resist inflammation, oxidative stress and inappropriate remodeling of the extracellular matrix.

Aim of Review

The purpose of this review is to summarize current research on the function of H₂S in diabetic wound healing at all stages, and propose future directions.

Key Scientific Concepts of Review

In this review, first, the various factors affecting wound healing under diabetic pathological conditions and the in vivo H₂S generation pathway are briefly introduced. Second, how H₂S may improve diabetic wound healing is categorized and described. Finally, we discuss the relevant H₂S donors and new dosage forms, analyze and reveal the characteristics of many typical H₂S donors, which may provide new ideas for the development of H₂S-released agents to improve diabetic wound healing.

Introduction

Insufficient insulin secretion, either relative or absolute, is the hallmark of diabetes mellitus [1]. Minor wounds in diabetic patients might cause major health complications resulting in wounds that heal slowly or never heal. In some cases, they can even be fatal [2]. Insulin deficiency, hyperglycemia, hyperlipidemia, proinflammatory state, peripheral neuropathy, tissue hypoxia, impaired vascular and obesity in the pathological state of diabetes can affect the wound healing process [3]. Diabetic foot ulcer (DFU) is one of the most prevalent complications of diabetes-related delayed wound healing. The occurrence and development of DFU is associated with severe morbidity and mortality, as well as large economic consequences. Therefore, reducing the risk of poor wound healing in diabetes is a worldwide issue [4]. Diabetic wound healing is slowed by many factors and its treatment is comprehensive, existing treatment strategy is mainly to control blood glucose and symptomatic treatment [5], [6]. According to the characteristics of different stages in wound healing, vasodilator drugs (propranolol, etc.), drugs for the treatment of neuropathy (pregabalin, tramadol, etc.), anti-infection drugs (antibiotics, etc.), anti-oxidative stress drugs (probucol, etc.), and nutritional support therapy are widely used [7]. And now considerable dressings and bandages loaded with collagen, growth factors and natural active products have been extensively developed to inhibit the inflammatory response, promote angiogenesis, regulate collagen deposition, and further accelerate diabetic wound healing [8], [9], [10]. Research on the management of damaged wounds may focus on combination approaches in the future, robust studies are needed to identify agents that may play a role in different stages [11].

In previous studies, H₂S was thought to be a toxic gas molecule or a byproduct of a metabolic pathway [12]. Increasing investigations in the past few years have revealed that H₂S may be qualified as the third gasotransmitter involved in mediating the physiological and pathological functions of cells [13]. H₂S is responsible for various functions such inhibiting inflammation, oxidant and tumors, regulating ion channel and protecting cardiovascular [12], [14]. The production of endogenous H₂S depends on at least three enzyme systems involved in cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfotransferase (3-MST), in addition to non-enzymatic pathways [15], [16]. All three enzymes have been shown to be present in endothelial cells (ECs) and are able to impact the angiogenic characteristics of ECs and respond to changes in angiogenic factors [17], [18]. Colorimetry, ion-selective electrodes, chromatographic methods and optical probes are commonly used for the determination of endogenous H₂S [19]. Among them, fluorescent probes of optical probes are more promising and are expected to be used to monitor endogenous short-lived H₂S, meeting the requirements of photostability, strong fluorescence signal, high sensitivity, high specificity and low cytotoxicity [20].

With the discovery of the physiological and pathological functions of H₂S, its biological effects and pathophysiological significance have become a hot issue in the field of medical research, and considerable progress has been made. A large number of research results have shown that H₂S could regulate many systemic diseases, and endogenous H₂S level has been shown to be reduced in many chronic diseases such as myocardial infarction, non-alcoholic fatty liver disease and diabetes [21]. Several clinical studies have shown that endogenous H₂S level is significantly reduced in vascular diseases [22]. The serum H₂S level and H₂S synthetase expression in patients were measured and their relationship with vascular indexes was investigated. The results indicate that endogenous H₂S may be involved in vascular remodeling, and endogenous H₂S production may be inhibited by inflammation and other factors [23], [24]. The low levels of H₂S concentration in sulfur thermal water treatments was confirmed to enhance endogenous cytoprotective activity and promote wound healing in chronic wounds that are difficult to heal [18], [25].

In recent years, there has been a growth in the number of studies on H₂S and diabetes. It is worth mentioning that in diabetic patients and animals, endogenous H₂S levels and H₂S synthase expression are decreased [26]. H₂S can regulate hemostasis, promote cell migration and adhesion functions, it can also resist inflammation, oxidative stress and regulate extracellular matrix remodeling. H₂S is a reducing substance, reducing or directly combining with the heme center of metalloprotein, and then directly or indirectly scavenging reactive oxygen species (ROS) [27]. Beyond that, Mustafa et al. (2011) first described the key molecular mechanism of H₂S signaling, namely S-sulfhydration [28]. In this H₂S-dependent post-translational modification, H₂S promotes the conversion of reactive cysteine residues to more active persulfides (-SSH) [29]. Persulfide may be involved in various reactions such as sulfur-containing molecule biosynthesis, tRNA modification, mitochondrial energy metabolism, cellular antioxidation and inhibition of Toll-like receptor-mediated inflammation [30]. S-sulfhydration modification of specific proteins by H₂S has been proven to have a significant impact on diabetes and its complications. H₂S induces Keap1 S-sulfhydration which leads to the suppression of atherosclerosis in diabetes, and also ameliorates skeletal muscle atrophy in db/db mice. Moreover, S-sulfhydration of Muscle RING finger 1 (MuRF1) prevents cardiac structural damage in diabetic cardiomyopathy, S-sulfhydration of Hrd1 reduces lipid droplet accumulation in cardiac tissues of db/db mice, S-sulfhydration of SIRT1 attenuates diabetic renal lesions via inactivation of p65 NF-κB and STAT3 phosphorylation/acetylation [31], [32], [33], [34], [35].

Therefore, in order to generate ideas for future research and innovation of H₂S in the field of diabetic wound healing management, this review will describe the function of H₂S in the healing of diabetic wounds and discuss the methods for increasing the bioavailability of H₂S.

Literature search strategy

Five electronic databases, including Science Direct, PubMed, Web of Science, Wiley Online Library and ClinicalTrials.gov were searched from 2000 to 2023 for eligible studies. We performed a literature search for Web of Science with the following search strategy: TS = ((“hydrogen sulfide” or “H₂S”) AND (“diabetic wound” or “wound”)). The strategies for other databases were similar but were adapted according to the guideline of the database. During the retrieval process, we conducted a manual search using references from the included literature to supplement the eligible studies. The duplicated literature was removed by Rayyan, and irrelevant articles were removed by browsing titles and abstracts. Finally, the conference materials, non-empirical research articles and articles related only to H₂S or wounds were removed after full-text review. The screening process was shown in Fig. 1. In the end, 61 studies on the possible mechanisms of H₂S accelerating wound healing were systematically summarized.

Fig. 1.

The flowchart of literature search strategy in this review.

H₂S in various phases of diabetic wound healing

One of the most intricate processes in multicellular organisms is the healing of wounds, which involves numerous communications within and between multiple cells. Traditional classifications of the wound healing process include 4 overlapping classical stages: coagulation and homeostasis, inflammation, cell proliferation and migration, matrix deposition and tissue remodeling [36]. The whole process of wound healing involves the platelet activation, macrophages and macrophage polarization, as well as proliferation and migration of keratinocytes, fibroblasts and ECs. These cells also secrete growth factors and cytokines that affect different stages of wound healing [10].

The abnormal metabolism of diabetes patients can further complicate the process of wound healing, and can also cause chronic wound stasis. Vascular damage and blood transport disorders make it difficult for drugs to reach the wound through blood circulation [37]. Hyperglycemia can lead to hyperfibrinogenemia in diabetic patients, activating the coagulation cascade, thereby increasing thrombin formation and fibrinogen degradation products [38]. Local tissue edema, hypoxia, and high glucose environment are suitable for bacterial growth [2]. At the same time, pathologically high glucose levels decrease the chemotaxis and phagocytosis of inflammatory cells, leading to inflammation and oxidative stress. Ischemic tissue damage is a major ischemic vascular complication that occurs in diabetic patients with insufficient oxygen supplementation [26]. Cumulative studies have shown that impaired ECs function and angiogenic properties play a key role in diabetes-induced impaired angiogenesis [39]. It is not limited to the mutual influence of the above-mentioned factors, and the joint action leads to difficult healing of diabetic wounds. In addition, cells and cytokines have different roles in 4 wound healing stages, so the development of drugs aimed at promoting the healing of diabetic wounds needs to target different stages (Fig. 2).

Fig. 2.

Features of reduced wound healing in various stages of diabetes.

H₂S in diabetic wound healing phase 1: coagulation and homeostasis

The presence of diabetes causes an imbalance between coagulation and fibrinolysis, resulting in a prothrombotic state [40]. A crucial aspect of the coagulation process involves converting of fibrinogen to fibrin, and higher fibrinogen levels have been observed in patients with DFU [41], [42]. In a study involving streptozotocin (STZ)-induced diabetic rats, it was observed that the administration of H₂S hastened wound healing while also shortening both prothrombin time (PT) and thrombin time (TT). PT and TT of the diabetic rats treated with H₂S were significantly improved. In contrast, serum fibrinogen levels were elevated in untreated diabetic rats, which were reduced after H₂S treatment [43]. Similar results were found in patients with osteoarthritis and diabetes [44][[44], [45]]. Studies have shown that H₂S can regulate thrombosis by interfering with platelet activation, H₂S at physiological concentration may hinder the clustering of platelets caused by moderate stimulation, which may be attributed to the persulfuration of platelet-associated proteins and the antioxidant activity of H₂S [45], [46], [47], [48]. Moreover, GYY4137 acts as a slow-releasing H₂S donor which was found to regulate and enhance endogenous thrombolysis by diminishing platelet aggregation and disrupting platelet activation and adhesion molecule-mediated aggregation [49], [50]. Aged garlic extract slowly releases H₂S to achieve physiological effects, which can inhibit platelet aggregation by inhibiting cAMP phosphodiesterase activity to increase cAMP levels [51].

H₂S in diabetic wound healing phase 2: inflammation

After the hemostasis phase, platelets aggregate and release pro-inflammatory mediators to enter the inflammatory phase, where leukocytes enter the wound to destroy bacteria and remove debris. Macrophages continued the process of cleaning up debris while simultaneously producing growth factors that attract immune system cells to the wound [52], [53]. Monocytes-macrophages, macrophage phenotypic transition and reduced phagocytic ability play critical roles in anti-inflammatory, tissue debridement, and cell regulatory functions [54], [55]. H₂S could reduce foamy macrophage numbers and promote macrophage phenotypes transformation [56]. In addition, there is now a wealth of data supporting the role of H₂S in promoting inflammation resolution in diabetic wound healing. H₂S treatment may abolish NETosis and neutrophil extracellular trap (NET) formation by blocking ROS mediated mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and p38 activation [57]. In a study, exogenous administration of sodium hydrosulfide (NaHS) to diabetic mice reduced neutrophil and macrophage infiltration, along with decreased interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) compared with untreated diabetic mice [58]. The administration of NaHS or S-adenosylmethionine (SAMe) could reverse the changes of Sirtuin 1 (SIRT1)-mammalian target of rapamycin (mTOR)/ nuclear factor kappa-B (NF-κB) signaling pathway induced by high glucose [59].

Antibacterial responses are often reduced in diabetic patients. The external structure of bacteria prevents the invasion of immune cells and antibiotics [60]. In addition, microbial diversity makes finding suitable and most effective treatments challenging [61]. Therefore, the development of novel dosage forms of antibacterial drugs that release H₂S has become a research hotspot [62]. A polymersome wound dressing spray acts as a bacterial inhibitor and H₂S generator can reduce by more than 50% for Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) at the concentrations of 60 and 80 μg/mL, respectively [63].

H₂S in diabetic wound healing phase 3: cell proliferation and migration

During the proliferation phase, fibroblasts are recruited from surrounding intact tissues, stimulate the migration of ECs and promote angiogenesis [64]. Activated fibroblasts are responsible for depositing collagen, secreting extracellular matrix and metalloproteinases, and providing adequate support for EC/epithelial cell adhesion, migration, growth and differentiation [65], [66]. A persistent high glucose environment in diabetic wounds leads to excessive superoxide production, which promotes oxidative stress and the production of advanced glycation end products (AGEs) [67], [68]. Modulation the level of ROS may be a prospective therapeutic method for the treatment of wounds [69].

The effects of ROS on fibroblast function are mainly reflected in inhibiting proliferation, migration and myofibroblast differentiation as well as inducing aging and apoptosis [70], [71]. H₂S donors AP39 and AP123 can protect mouse fibroblasts against photoaging by increasing collagen levels and Nrf2 nuclear translocation [72]. In addition, NaHS can protect fibroblasts from hydrogen peroxide damage by regulating autophagy related proteins LC3, p62, and Beclin 1 [73]. In other ways, H₂S inhibits high glucose-induced fibroblast senescence by triggering CSE and autophagy via the SIRT6/adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway [74]. After JK-1 (H₂S sustained-release donor) treatment, fibroblasts L929 cells migrated to the site of the wound, and JK-1 dramatically improved the degree of wound closure, granulation tissue development and collagen deposition in mouse skin wounds [75].

As a signaling molecule, ROS mediates various biological reactions in ECs, including gene expression, cell proliferation, migration, angiogenesis, apoptosis and aging [76], [77]. The adverse effect of ROS production from ECs is a key factor for the dysfunction of wound healing in diabetes [78]. Studies have found that H₂S can act as a direct scavenger of ROS or combat oxidative stress by up-regulating antioxidant-related pathways and enhancing the expression of antioxidant molecules including superoxide dismutase (SOD), HO-1, NADPH quinone dehydrogenase 1 (NQO1), catalase (CAT) and glutathione peroxidase (GSH-Px) [79], [80], [81]. Besides, NaHS (50 mol/L) could prevent ECs apoptosis induced by high glucose via upregulating SOD activity, reducing ROS generation and malondialdehyde levels, and downregulating Bax/Bcl-2 ratio [82]. NaSH reduces oxidative stress in diabetes by inhibiting NADPH oxidase isoforms, thereby restoring endogenous NO production and action in the vascular system [83]. Exogenous H₂S protects ECs from HG-induced increased inflammation and ROS, autophagy, endoplasmic reticulum stress, and angiogenesis injured by activating the Nrf2-ROS-AMPK signaling pathway and PI3K/Akt/eNOS pathway [84], [85]. Furthermore, H₂S inhibited oxidative stress and expression of ERK1/2 and p38, promoted ECs proliferation and migration and accelerated full-thickness wound healing in diabetic mice [86]. H₂S also participates in mediating the proliferation and migration of ECs, thereby accelerating blood vessels formation, and takes part in regulating vasodilation and hemodynamics [87], [88]. ECs respond to pathophysiological stimuli by releasing vasodilator substances such as NO and vasoconstrictor substances including angiotensin II, endothelin-1, thromboxane A2, regulating vasodilator tone and organ perfusion [89]. Substantial evidence suggests that H₂S promotes angiogenesis and wound healing in diabetic mice by increasing the vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF-1α) and endothelin nictric oxide synthase (eNOS) transcription, promoting granulation tissue formation [43], [90]. H₂S increases the level of miR-126-3p by down-regulating high-glucose-induced anti-angiogenic DNA methyltransferase 1 (DNMT1) protein levels and down-regulating methylation levels, thereby improving high-glucose impaired angiogenesis [91].

ROS can modify the cytoskeleton and cell junction related factors, thereby regulating the polarity, morphogenesis and functions of epithelial cells [92]. Keratinocyte damage and dysfunction is known to attribute to delayed wound healing by DM. In one study, H₂S could protect HaCaT human keratinocytes from methylglyoxal (MGO) induced damage and behavioral disorders by inhibiting apoptosis, alleviating intracellular ROS content, increasing mitochondrial membrane potential, and ultimately promoting cell adhesion and migration [93]. Meanwhile, exogenous H₂S inhibits NLRP3 inflammasome mediated inflammatory response by inhibiting high glucose induced ROS production in epithelial cells [94].

H₂S in diabetic wound healing phase 4: matrix deposition and tissue remodeling

Inflammation, angiogenesis, granulation tissue remodeling, collagen deposition and other processes involved in diabetic wound healing all require the degradation of extracellular matrix (ECM), and matrix metalloproteinases (MMPs) are important proteins for degrading ECM. H₂S and its donors have been widely demonstrated to combat adverse remodeling in diabetic nephropathy such as excessive collagen deposition, increased fibronectin and laminin expression, and decreased expression of elastin [95], [96]. H₂S can regulate ECM by up-regulating the expression of PPARγ, down-regulating the expression of plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β (TGF-β1), regulating the expression of MMPs family proteins, and inhibiting the mRNA expression of fibronectin and type IV collagen [97], [98], [99].

In brief, H₂S regulates the coagulation process, resists inflammation and oxidative stress, and provides a prerequisite for diabetic wound healing. At the same time, H₂S enhances the migration and proliferation of fibroblasts, ECs and epithelial cells in new tissues, and increases collagen synthesis and angiogenesis. During extracellular matrix remodeling, H₂S regulates the activity of MMPs and promoting granulation tissue and scar formation. Thus, H₂S contributes to all phases of diabetic wound healing (Fig. 3). At the same time, the level and action of endogenous H₂S are affected by several factors. Angiotensin Ⅱ inhibits the production of endogenous H₂S by increasing the level of ROS in vascular ECs and promoting the ubiquitination and degradation of CSE [100]. In addition, interactions between iron and H₂S affect their respective metabolism and function. Excess iron produces ROS and non-enzymatically increases the production of H₂S, whereas H₂S reverses the excess iron and ROS [101]. There is also a reciprocal effect between CBS and CSE, the two major synthetase of H₂S. CBS deficiency may up-regulate CSE expression by inducing Specific protein 1 (SP1), and CBS product cystathionine can also inhibit CSE synthesis of H₂S [102], [103]. Exploring the effect of endogenous substances on the action and level of H₂S is of key significance for H₂S to play a role in accelerating diabetic wound healing in vivo.

Fig. 3.

The role of hydrogen sulfide in various stages of diabetic wound healing.

H₂S and its releasing agents for better treatment outcomes

With the confirmation of the effect of H₂S therapy, the development of H₂S-based therapy depends on the ability to deliver H₂S to the required location under the appropriate concentration and pharmacokinetics. H₂S follows a two-step dissociation process with pKa values of approximately 6.88 and 19 (37 °C), respectively. In normal physiological conditions with a pH of 7.4, H₂S exists primarily as neutral molecules of H₂S and monoionized HS^- [104], H₂S and HS^- can reach a dynamic equilibrium [105]. On the other hand, the dose - response curve of H₂S is generally bell-shaped with a narrow therapeutic window [106], [107]. Furthermore, H₂S exhibits a multitude of distinctive characteristics that pose a set of unique obstacles for the advancement of therapeutic methods. Generally, the most significant method of delivering exogenous gasotransmitters is the inhalation of gas directly, which avoids the pain and risk of infection from local delivery [108]. However, direct inhalation of gaseous H₂S is limited by odor, irritation, toxicity, enhanced local concentrations and dosage control difficulties [109]. Thus, the development of H₂S therapeutics still faces major challenges concerning the lack of controllability and targeting specificity.

Increasing evidence suggests that H₂S has preventive or therapeutic effects on diabetic wound healing, which highlight the application prospect of H₂S-based treatment methods in the biomedical field. To investigate and comprehend the physiological functions of H₂S, H₂S donors are prescribed as the main source of exogenous H₂S to compensate for the deficiency of endogenous H₂S production, and numerous studies have demonstrated the therapeutic capacity of H₂S donors in vitro and in vivo models. Although the characteristics required for H₂S donors vary in different cases, some features are widely desirable. H₂S donors should be stable, not produce or only produce harmless by-products when functioning, and have specific release mechanisms. Although donors that rapidly release H₂S, such as NaHS and Na₂S, have been widely used, the short half-life, rapid and uncontrolled release makes them unsuitable for the sustained donation of H₂S [108]. The isothermal phase transition microparticles (NaHS@MPs) prepared by the emulsion method provide an in situ reservoir for the continuous release of exogenous H₂S under physiological conditions that attenuate the high water instability of NaSH [86].

Similar to sulfide salts, H₂S generation from Lawesson’s reagent (LR) is also rapid and uncontrolled, as well as poorly water-soluble. For ameliorating the above limitations, one of the most widely used GYY4137, a slow-releasing H₂S donor with good water solubility under physiological conditions, was developed on the basis of LR [110]. DTT is a class of H₂S donors triggered by hydrolysis, and ADT-OH is the result of demethylation of the compound ADT, which is widely utilized to convert established drug molecules into H₂S donor [111]. AP39, a DTT derivative, is a triphenylphosphonium derivatized anethole dithiolethione and mitochondria-targeted H₂S donor [112]. AP39 is effective and safer in hyperglycemic injury of endothelial cells at 1000-fold lower concentrations than Na₂S [113].

The pH-response JK-1 is a phosphorothioate based H₂S donor and protonation induced intramolecular cyclization greatly accelerates H₂S release at acidic pH. JK-1 was added into the alginate sponge obtained by crosslinking sodium alginate (SA) with Ca²⁺ to prepare a functional dressing (SA/JK-1) with H₂S release property. In comparison to the JK-1 in solution, the SA sponge’s porous structure caused the gradual release of H₂S from the JK-1 and attenuate the loss of H₂S in the system. Both in vitro and in vivo studies have shown that SA/JK-1 not only has good cytocompatibility, but also improves fibroblasts proliferation and migration, and increases the formation of granulation tissue, re-epithelialization and angiogenesis during wound healing [75].

Over the last two decades, research on dietary organosulfur compounds has received increasing attention. Among them, garlic polysulfides and isothiocyanates deriving from the Brassicaceae have been considered as H₂S donors with important pharmacological and nutritional value [114]. Furthermore, materials such as antioxidant-antibiotic polymer vesicles and antibacterial hydrogel dressings have been developed for the treatment of diabetic wounds [115], [116]. A NIR-responsive band-aid with core (MXene-loaded nanofibers)-shell (dopamine-hyaluronic acid hydrogel) structure encapsulating VEGF and H₂S donor (diallyl trisulfide, DATS) was prepared. The sustained release of DATS achieved continuous H₂S production, which induces the polarization of macrophages into an M2 phenotype, inhibits the excessive inflammatory response of wounds, facilitates the proliferation of skin cells, and promotes wound healing [117] (Fig. 4).

Fig. 4.

H₂S and its releasing agents.

Nanomaterials have the advantages of controlled release, high efficiency, and low toxicity [18], [63]. There are various substances conjugated with different anti-oxidative nanomaterials which are highly active in diabetic wound healing process [118], [119], [120]. For instance, cerium oxide nanoparticle (CNP) acts as a free radical scavenger conjugated with miR146a inhibiting the pro-inflammatory NF-κB pathway, which has a synergistic effect in regulating oxidative stress and inflammation, ultimately accelerating the healing process of diabetic wounds. CNP-miR146a inhibited the expression of proinflammatory cytokines IL6, TRAF6 and CXCL2, reduced the number of CD45 + and NOX2 + cells, and increased the expression of collagen type 1 and CD31, as well as dermal thickness [121], [122], [123]. Zinc sulfide nanoparticles (ZNS NPs) is a new type of nanomaterials that generate H₂S in an acidic microenvironment, regulate redox homeostasis and promote fibroblast viability [124]. The released Zn²⁺ can synergize with H₂S to depolarize the bacterial cell membrane, resulting in antibacterial and re-epithelialization enhancing activities of ZNS NPs [125]. The ideal gas release therapy should be biocompatible, released in a sustained and controlled manner, and maintaining a good wound environment [18]. The controlled release of H₂S from H₂S donors is triggered by cysteine, which can regulate the release rate of H₂S and improve its stability [126], [127]. Different dosage forms and administration methods of hydrogen sulfide and its donors have their own characteristics (Table 1). Due to the special gas form of H₂S, compared with injections, patches and gels, the preparation of H₂S into sprays can allow gas exchange, which is conducive to wound healing. And it can reduce the risk of contamination, making it easier to remove from wounds and thereby reducing toxicity [63], [128]. The advancement of nanotechnology has made it possible to rapidly develop stimulus responsive nanomaterials for precise gas therapy [129]. It is important to be cautious about utilizing inhaled H₂S for therapeutic purposes since it can lead to harmful side effects when present in high quantities, possibly by hindering the mitochondrial respiration process [130].

Table 1.

Comparison of several H₂S donor preparations in different dosage forms.

Dosage form	Active ingredient	Advantage	Disadvantages or adverse reactions	Reference
Microparticles	NaHS	Improved water stability and readily removed from wound bed.	A lack of a successful method for encapsulating effectively.	[86]
Hydrogels	JK-1	Absorbed wound exudate, controlled H2S release and improved biocompatibility.	Deformation caused by swelling of the spongy matrix may lead to wound tears.	[75]
Nano-fibrous	JK-1	Controlled H2S release and good cyto-compatibility.	Hard to pinpoint how H2S contribute to various phases of the wound healing process.	[128]
Fibers and hydrogels	DATS	High photothermal conversion efficiency.	Fibrous materials are often affected by the expansion of the fibrous interface, and hydrogels are easy to exchange with cells or tissues.	[117]
Microfibers	NSHD1	Prolonged the release time and achieved a tunable release curve.	Biothiols to trigger H2S release, potentially leading to unpredictable and unstable H2S release.	[131]
Spray	SATO	Substance can disperse in water and maintain its colloidal stability when diluted.		[63]

In addition to exogenous H₂S supplementation, the use of certain drugs to increase endogenous H₂S levels or activation of H₂S producing enzymes can also increase overall H₂S levels. The level of endogenous H₂S and the enzymatic activity of CSE in macrophages were significantly increased after fluvastatin treatment [132]. Metformin, a commonly used oral antidiabetic drug, has been shown to increase H₂S tissue concentration in brain, heart, kidney and liver of mice and regulate CSE expression [133], [134]. In addition, both CBS agonist S-adenosyl-L-methionine (SAM) and NaHS increased H₂S levels and inhibited intracerebral haemorrhage (ICH) - induced neutrophil infiltration and NLRP3 inflammasome activation [135]. Besides drugs that affect H₂S levels and H₂S synthase agonists, the mRNA levels of H₂S-synthetase CSE and 3-MST were significantly increased in aged animals undergoing exercise training, ultimately leading to the restoration of endogenous H₂S production to the levels observed in adults [136].

Conclusion and perspective

The above evidences suggest that endogenous or exogenous H₂S can accelerate various phases of wound healing in diabetic pathological conditions. Some important questions in the field of H₂S remain unanswered, which will require the joint efforts of researchers in disciplines such as medicinal chemistry, pharmacology, pharmaceutics, and biology. A major concern is to identify the therapeutic window of H₂S for a given target to deepen understanding of how H₂S releases from its donors and following byproducts, signaling mechanism molecules and biological functions. The application of endogenous or exogenous H₂S and its related pathways can provide new ideas for improving poor diabetic wound healing, and adjust the release rate and target of H₂S by changing the dosage form and release mode. However, many issues of H₂S in diabetic wound healing remain to be studied. H₂S plays a major role in which stage of diabetic wound healing, and the S-sulfhydrated modification of specific proteins in related pathways remains to be explored. In addition, known drugs such as non-steroidal anti-inflammatory drugs (aspirin, diclofenac, naproxen, etc.) and derivatives of sildenafil combined with H₂S have been demonstrated to have both the efficacy of the parent drug itself and the biological effects of H₂S, which can attenuate the toxic side effects of the parent drug to some extent. In the future, it is expected that H₂S will be connected to known drugs used for wound healing to constitute derivatives with better efficacy, and the specific mechanism of how they will play a biological role needs to be further studied.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Xinyi Shi is currently a graduate student in pharmacology at Shenyang Pharmaceutical University. Her research direction is the effect of natural product and hydrogen sulfide derivatives on macrophages in diabetic wound healing.

Haonan Li obtained his Ph.D. degree in natural product chemistry in 2022 from the Shenyang Pharmaceutical University. At present, as a postdoctor, he continues to study the antitumor activity of natural product derivatives and hydrogen sulfide donors.

Fengrui Guo is currently a graduate student in natural product chemistry at Shenyang Pharmaceutical University. Her research direction is the mechanisms of natural product and hydrogen sulfide derivatives protecting endothelial cells in diabetic wound healing.

Dahong Li received his Ph.D. degree in 2013 under the supervision of Prof. Jinyi Xu from China Pharmaceutical University. Then he joined the group of professor Huiming Hua in Shenyang Pharmaceutical University after graduation. He is a professor now and the main research directions are the discovery of lead compounds from natural sources and the pharmacochemical biology based on specific skeleton and activity of natural molecules.

Dr. Fanxing Xu received his Ph.D. degree in biochemical engineering from Dalian University of Technology in 2014. Then he joined Shenyang Pharmaceutical University as a lecturer and was promoted to associate professor in 2018. His current research interests focus on the pathophysiology and treatment of diabetes and its complications.