Targeting gut microbiota and its associated metabolites as a potential strategy for promoting would healing in diabetes

Abstract

Impaired healing of diabetic wounds is one of the most important complications of diabetes, often leading to lower limb amputations and incurring significant economic and psychosocial costs. Unfortunately, there are currently no effective prevention or treatment strategies available. Recent research has reported that an imbalance in the gut microbiota, known as dysbiosis, was linked to the onset of type 2 diabetes, as well as the development and progression of diabetic complications. Indeed, the gut microbiota has emerged as a promising therapeutic approach for treating type 2 diabetes and related diseases. However, there is few of literatures specifically discussing the relationship between gut microbiota and diabetic wounds. This review aims to explore the potential role of the gut microbiota, especially probiotics, and its associated byproducts such as short chain fatty acids, bile acids, hydrogen sulfide, and tryptophan metabolites on wound healing to provide fresh insights and novel perspectives for the treatment of chronic wounds in diabetes.

Key Words: Type 2 diabetes; Wound healing; Gut microbiota; Short chain fatty acids; Tryptophan; Bile acids; Hydrogen sulfide

Core Tip: Recent research has reported that an imbalance in the gut microbiota was linked to the onset of type 2 diabetes, as well as the development and progression of diabetic complications. Indeed, the gut microbiota has emerged as a promising therapeutic approach for treating type 2 diabetes and related diseases. However, there is few of literatures specifically discussing the relationship between gut microbiota and diabetic wounds. In this paper, we aim to explore the potential role of the gut microbiota, especially probiotics, and its associated byproducts such as short chain fatty acids, tryptophan metabolites, bile acids, and hydrogen sulfide on wound healing to provide fresh insights and novel perspectives for the treatment of chronic wounds in diabetes.

INTRODUCTION

The prevalence of type 2 diabetes mellitus (T2DM) is escalating at an alarming rate globally. Currently, over 500 million individuals are living with T2DM worldwide, a figure projected to exceed 780 million by 2045[1]. Diabetic foot ulcers (DFUs) represent a common form of chronic wounds, affecting approximately 30% of diabetic patients[2], with 30% of these cases leading to subsequent lower limb amputation and a 70% five-year mortality rate[3]. The healing process of skin wounds comprises three main, overlapping stages. The initial stage, known as the inflammatory stage, involves various types of inflammatory cells such as neutrophils, macrophages, and lymphocytes. During this stage, macrophages release platelet-derived growth factor (PDGF), promoting the migration of fibroblasts and epithelial cells to the injury site[4]. In the proliferation and remodeling phase, fibroblasts initiate the reconstruction of the extracellular matrix and assist in angiogenesis[2], thereby accelerating wound contraction and healing. However, in DFUs characterized by nerve damage and microangiopathy[5], neutrophil function, macrophage phagocytosis, leukocyte chemotaxis, and bactericidal effects are all impaired, ultimately disrupting and stagnating the normal wound healing process[5]. The treatment of chronic wounds in diabetic patients, which are severely disabling and economically burdensome, is often protracted and unsatisfactory. Addressing the prevention and treatment of chronic wounds and ulcers in diabetic patients remains an urgent and critical challenge.

The gut microbiota, often described as the human body's "microbiome organ", provides numerous beneficial effects. These include facilitating food digestion, enhancing nutrient absorption, protecting the intestinal barrier against pathogenic bacteria, supporting the development and function of the intestinal immune system, and regulating host metabolism[6]. Multiple clinical studies have revealed a strong correlation between metabolic dysfunction and chronic inflammation in individuals with T2DM and an imbalance in their gut microbiota. T2DM patients exhibit a marked dysbiosis in gut microbiota compared to healthy controls, characterized by an increase in pathogenic bacteria such as Clostridium hathewayi, Escherichia coli (E. coli) and Clostridium symbiosum, alongside a decrease in probiotic bacteria[7]. Notably, Faecalibacterium, Akkermansia, Bifidobacterium, Bacteroides and Roseburia have been observed to decrease with T2DM progression, while Ruminococcus, Fusobacterium and Blautia demonstrate an upward trend[8]. Common findings include elevated levels of Lactobacillus species and Blautia, an increased ratio of Bacteroidetes to Firmicutes and Bacteroides Prevotella to Clostridium coccoides-Eubacterium rectale group, higher relative abundance of Firmicutes and Actinobacteria, all of which are associated with increased blood glucose levels[9]. Conversely, a reduction in the abundance of Clostridium has been linked to elevated fasting blood glucose, hemoglobin (HbA1c), and plasma triglycerides[10]. The relationship between gut microbiota and T2DM development and progression has sparked extensive discussion and deep reflection. Current research suggests that positive modulation of the gut microbiota could be advantageous in treating T2DM and its complications, including diabetic nephropathy, cerebrovascular disease, diabetic neuropathy, peripheral artery disease, coronary heart disease, and diabetic retinopathy[10]. However, existing studies have devoted limited attention to the role of gut microbiota in promoting the healing process of diabetic wounds, a topic that will be thoroughly examined in this review.

THE GUT-SKIN AXIS THEORY LAID THE FOUNDATION FOR TREATING DIABETIC WOUNDS BY TARGETING THE GUT MICROBIOTA

The intestinal barrier functions as a natural defense system, separating intestinal contents from extraintestinal tissues and organs. Compromised barrier integrity may lead to severe systemic inflammation and metabolic disorders. This barrier comprises three sequential components: The intestinal mucosal barrier, the epithelial barrier, and the vascular barrier[11] The outermost layer consists of mucus secreted by goblet cells, with an inner mucus layer containing various antimicrobial peptides (AMPs) and secretory immunoglobulin A (IgA) that effectively protect against pathogenic bacterial invasion[12]. The middle layer, composed of epithelial cells, houses immune cells in the intestinal lamina propria, including dendritic cells (DCs), Paneth cells, macrophages, and lymphocytes. These cells secrete diverse AMPs, cytokines, and IgG to maintain intestinal immune homeostasis[13]. For example, upon encountering pathogens, DCs extract bacterial antigens and activate TH17 and innate lymphoid cells 3, promoting interleukin (IL)-22 and IL-17 production. This process enhances AMP and IgA secretion, regulating the gut microbiota, resisting pathogens and inflammation, and preserving normal intestinal epithelial barrier function[11]. Notably, the skin and intestinal barriers share remarkable similarities. Both exhibit diverse microbial populations and participate in crucial functions such as immune modulation, nutrient synthesis, and barrier protection, significantly impacting immune and neuroendocrine functions[14]. Mounting evidence supports bidirectional regulation between the gut and skin, with the gut-skin axis gaining recognition in various diseases. Current research hypothesizes that the primary communication mechanisms in this axis involve metabolites, the neuroendocrine system, and the immune system. Alterations in gut microbiota and its metabolism play a particularly significant role in the relationship between gut health and skin homeostasis[14,15]. Changes in gut microbiota diversity or structure may increase host susceptibility and disrupt intestinal mucosal immune tolerance, subsequently affecting skin health. This phenomenon has been observed in inflammatory skin conditions such as acne, atopic dermatitis (AD), rosacea, and psoriasis[16-18]. For instance, Bifidobacterium longum CCFM1029 effectively suppresses abnormal helper T cell 2 type immune responses mediated by aryl hydrocarbon receptor (AhR) by converting tryptophan (Trp) into indole-3-carbaldehyde, alleviating AD symptoms[19]. Psoriasis is closely associated with gut microbiota imbalance, and probiotic and prebiotic supplementation has shown potential in mitigating psoriasis symptoms[20]. Furthermore, Lactobacillus rhamnosus (L. rhamnosus) significantly improves acne-like lesions in rats by reducing levels of inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and by targeting Trp metabolism to increase indole-3-acetic acid (IAA) and indole production[17]. These findings further corroborate the interaction within the gut-skin axis.

In T2DM, gut dysbiosis may exacerbate metabolic inflammation, diminish the production of intestinal mucus and tight junction proteins (TJP), and increase intestinal permeability, thereby compromising intestinal barrier function. This intestinal leakage could negatively impact wound healing in T2DM by allowing harmful toxins, food residues, and pathogenic microorganisms to enter the systemic circulation through the damaged intestinal epithelium. Notably, supplementation with probiotics and prebiotics, such as Bifidobacterium, Lactobacillus, and Bacteroides fragilis, could protect the intestinal barrier and mitigate distant organ inflammation by promoting goblet cell differentiation, enhancing the mucous layer, and improving tight junction function[21-23]. Additionally, gut microbiota metabolites, including hydrogen sulfide (H₂S), bile acids, indole-3-propionic acid, amino acids, trimethylamine N-oxide, and short chain fatty acids (SCFAs), are known to modulate host metabolism and maintain intestinal mucosal barrier integrity. SCFAs, often derived from beneficial gut microbes, can increase mucin synthesis[24], which provides strong physical barriers and chemical defense functions due to its abundant secretion of IgA and AMPs[25]. AMPs function as endogenous antibiotics to combat pathogens and may enhance the wound healing process[26]. Other gut metabolites, such as taurine and ellagic acid, can activate NLRP6 and the AhR-Nrf2 pathway in intestinal epithelial cells, respectively, promoting IL-18 and AMP secretion, and increasing the expression of intestinal TJP to restore damaged intestinal mucosa[27,28]. Consequently, it can be hypothesized that modulating gut microbiota through fecal microbiota transplantation (FMT) and supplementing beneficial bacteria or prebiotics may facilitate diabetic wound healing by reinforcing intestinal barrier function and maintaining immune homeostasis.

REGULATING THE GUT MICROBIOTA THROUGH DIET AND MEDICATION CAN EFFECTIVELY PROMOTE DIABETIC WOUND HEALING

Diet and nutrition play a pivotal role in wound healing, and nutritional deficiencies can lead to gut microbiota dysbiosis, which subsequently impairs immune function and wound healing capacity in diabetic patients[29]. A dietary deficiency in Trp can reduce Lactobacillus reuteri (L. reuteri) populations and affect the expansion of RORγt+ regulatory T cells (Tregs) and the reduction of Gata3+ Tregs in a microbiota-dependent manner, eliciting pro-inflammatory effects[30]. Studies in mice on methionine-restricted diets have demonstrated an increase in short-chain fatty acid-producing bacteria (Bifidobacterium, Lactobacillus, Bacteroides, Roseburia, Coprococcus, and Ruminococcus) and anti-inflammatory bacteria (Spirochaetes and Bacilli), alongside a decrease in pro-inflammatory bacteria (Desulfovibrio), contributing to improve gut health[31]. Furthermore, vitamin D deficiency has been associated with an increase in Akkermansia muciniphila, a bacterium capable of degrading intestinal mucus, leading to gut microbiota imbalance and compromised intestinal barrier function[32]. In multiple sclerosis patients, vitamin D supplementation has been shown to increase the abundance of butyrate-producing Faecalibacterium and Coprococcus in the gut, exerting anti-inflammatory effects[33]. Fatty acids exhibit a dual impact on wound healing; omega-3 polyunsaturated fatty acids from fish oil have been reported to promote wound epithelialization and reduce collagen deposition in scars[34], while also inhibiting inflammation and stimulating angiogenesis to accelerate wound healing[35,36]. These fatty acids may also confer benefits in the prevention and treatment of various conditions, including diabetes, lower limb venous ulcers, cardiovascular diseases, acute lung injury, and neurodegenerative diseases through positive modulation of the gut microbiota[35,37].

Currently, numerous anti-diabetic medications in clinical use have demonstrated efficacy in promoting diabetic wound healing through modulation of the gut microbiota and maintenance of the intestinal barrier. Recent studies have shown that metformin can increase the abundance of fiber-degrading bacteria such as Escherichia, Roseburia, Faecalibacterium, and Bifidobacterium in patients with type 2 diabetes, while reducing the abundance of Clostridium, Intestinibacter species, and Bacteroides[38-40]. In a study examining metformin treatment in adolescent patients, a decrease in Clostridium and an increase in Bifidobacterium in the gut microbiota were found to be negatively correlated with glycated HbA1c levels[41,42]. Moreover, metformin can significantly downregulate the expression of key factors in the TLR/nuclear factor κB (NF-κB) signaling pathway and upregulate the expression of tight junction factors occludin and Zonula Occludens Protein-1 (ZO-1), thereby reducing the gaps between intestinal epithelial cells and strengthening the intestinal barrier structure[43,44]. Resveratrol (RSV), another diabetes medication, also exhibits a regulatory effect on the gut microbiota, increasing the abundance of fiber-degrading bacteria in feces and downregulating the abundance of Proteobacteria, Enterococcus, and Akkermansiamuciniphila[45,46]. Furthermore, mice receiving RSV microbiota transplants display more intact intestinal morphology, with upregulation of key intestinal integrity markers and mucin gene expression, and a significant reduction in serum levels of inflammatory factors, undoubtedly enhancing the function and integrity of the intestinal barrier[46,47]. Consequently, the use of these diabetes medications not only maintains blood glucose homeostasis by positively affecting the gut microbiota but also promotes the healing of diabetic wounds by protecting the intestinal barrier.

PROBIOTICS AND FMT AS POTENTIAL OPTIONS TO PROMOTE DIABETIC WOUND HEALING

Probiotics play a crucial role in modulating the host immune system through their influence on gut microbiota, offering potential health benefits when consumed in adequate quantities. L. reuteri, a prototypical probiotic, has demonstrated both antibacterial and anti-inflammatory effects, making it suitable for treating and preventing various gastrointestinal diseases, including necrotizing enterocolitis and Clostridioides difficile (C. difficile) infections[48]. The antimicrobial activity of L. reuteri is primarily attributed to its production of 3-hydroxypropionaldehyde (also known as reuterin), an antimicrobial compound that effectively inhibits the growth of diverse gastrointestinal pathogens, including C. difficile, by inducing oxidative stress[48]. Additionally, the anti-inflammatory effect of L. reuteri is partly due to its ability to reprogram intraepithelial CD4+ T cells into immunoregulatory CD4+CD8αα+ double-positive cells by producing indole-3-lactic acid, which acts as an agonist for the AhR receptor[49]. This function is also attributed to the generation of histamine through histidine decarboxylase, a biologically active compound that can inhibit the synthesis of the pro-inflammatory cytokine TNF and regulate host mucosal immunity[48]. Several studies have demonstrated that administering three specific Trp-metabolizing Lactobacillus strains-Lactobacillus murinus CNCM 1-5020, L. reuteri CNCM I-5022, and Lactobacillus taiwanensis CNCM I-5019-to mice with gut microbiota dysbiosis significantly alleviated the severity of colonic inflammation[50]. Furthermore, research has shown that the application of L. rhamnosus GG (LGG) and LGG cell-free supernatant effectively mitigates alcohol-induced liver damage and steatosis[51]. Additionally, Lactobacillus bulgaricus and Lactobacillus plantarum (L. plantarum) have been observed to regulate the inflammatory response at wound sites in diabetic rat models, accelerate epithelial regeneration, stimulate wound contraction, and significantly enhance wound healing activity[52]. Consequently, the use of probiotics as a potential therapeutic target in treating diabetic wounds presents an attractive avenue for further research.

As anticipated, FMT demonstrates high efficacy in treating refractory or recurrent C. difficile infection[53], and its application has expanded to address ulcerative colitis and immune disorders[54,55]. Clinical trials have indicated that patients with ulcerative colitis exhibited reduced clinical activity scores and C-reactive protein levels following FMT, with endoscopic examinations revealing ulcer alleviation[55]. Existing research suggests that the administration of a water-based probiotic suspension (Symprove™, containing Lactobacillus fermentum NCIMB 30174, L. plantarum NCIMB 30173, Lactobacillus acidophilus NCIMB 30175 and Enterococcus faecium NCIMB 30176) has demonstrated a beneficial effect on gut microbiota structure in vitro[56]. Furthermore, in vivo experiments involving fecal transplantation from healthy mice to high-fat diet mice have yielded comparable outcomes. Post-transplantation, not only did microbial community diversity increase, but the compromised intestinal barrier was also restored, leading to improvements in metabolic disorders[57,58]. Additional indicators of gut health following FMT have shown positive changes, including enhanced production of lactic acid, SCFAs, and anti-inflammatory cytokines (IL-6, IL-10), as well as reduced levels of pro-inflammatory cytokines and chemokines (TNFα, MCP1, and IL-8)[59]. Moreover, FMT significantly improves blood glucose levels and demonstrates a notable effect in lowering blood lipids and blood pressure in hyperglycemic patients[52]. Considering the established link between gut microbiota and T2DM, along with the beneficial regulatory role of probiotics in immune response and intestinal metabolites, FMT shows promise in the microecological treatment of chronic wounds in diabetes.

METABOLITES OF THE GUT MICROBIOTA ARE POTENTIAL TARGETS FOR TREATING DIABETIC WOUNDS

The gut microbiota and their metabolites demonstrate a symbiotic relationship, collectively forming a complex and extensive ecological system within the host (Figure 1). The gut microbiota influences organs beyond the digestive system by producing various substances, including SCFAs, Trp, and bile acid metabolites. These substances play crucial roles in inhibiting the activation of pro-inflammatory immune cells and enhancing the differentiation and functionality of regulatory immune cells, thereby maintaining the stability of both the gut and the entire body[60]. In diabetes, indole propionic acid, a Trp metabolite synthesized by intestinal flora, has been shown to regulate the secretion of intestinal pro-insulin by modulating intestinal endocrine L cells. Additionally, it protects beta cells from damage associated with metabolism and oxidative stress due to its potent antioxidant properties[61]. These combined effects contribute to a reduced risk of developing T2DM. Exogenous SCFAs, particularly butyrate, can suppress oxidative stress and NF-κB signaling through the mediation of G protein-coupled receptors 43 (GPR43). Furthermore, they inhibit the activity of histone deacetylases (HDACs) and stimulate the expression of the Nrf2 gene, which aids in mitigating symptoms associated with diabetic nephropathy[62]. These findings suggest that targeting metabolites derived from intestinal microbiota holds significant potential for improving diabetes and its complications. The following sections will explore the therapeutic potential of gut microbiota-associated metabolites such as SCFAs, H₂S, bile acids, and Trp in promoting wound healing in diabetes (Figure 2).

Figure 1 Gut microbiota and its associated metabolites like short chain fatty acids, hydrogen sulfide, tryptophan, and bile acids. SCFA: Short chain fatty acids; BA: Bile acids; TRP: Tryptophan; H₂S: Hydrogen sulfide. This figure was created using BioRender.com.

Figure 2 Metabolites regulate the healing of diabetic wounds through various pathways. The metabolites such as chain fatty acids, hydrogen sulfide, tryptophan, and bile acids mediated by gut microbiota (GM) have a positive regulatory ability on the inhibiting inflammation and apoptosis, reducing oxidative stress, regulating blood glucose levels and promoting reepithelialization and angiogenesis. While the type 2 diabetes mellitus patients are accompanied by intestinal microbiota disorders and an increased intestinal permeability, which will cause the down-regulation of GM metabolites entering the circulatory system, and may eventually further aggravate diabetes wounds. SCFA: Short chain fatty acids; BA: Bile acids; TRP: Tryptophan; ZO-1: Zonula Occludens-1; HDACs: Histone deacetylases; MDA: Malondialdehyde; GSH: Glutathione; GM: Gut microbiota; 5-HT: 5-hydroxytryptamine; M1: M1 macrophages; M2: M2 macrophages; IDO: Indoleamine 2:3-dioxygenase; PYY: Peptide YY; GLP-1: Glucagon-like peptide-1; SOD: Superoxide dismutase; ROS: Reactive oxygen species; Ang-1: Angiopoietin-1; VEGF: Vascular endothelial growth factor; eNOS: Endothelial nitric oxide synthase; AKT: Protein Kinase B; DNMT1: DNA methyltransferase 1; NETs: Neutrophil extracellular traps; H₂S: Hydrogen sulfide. This figure was created using BioRender.com.

SCFA

SCFAs, primarily consisting of acetic, propionic, and butyric acids, are the main metabolic products of non-digestible carbohydrates fermented by intestinal microbiota[63]. Extensive research has elucidated the beneficial effects of SCFAs on host health, which operate through various biological mechanisms: (1) SCFAs, produced by gut microbiota, function as a carbon source, providing energy for colonocytes, reducing intestinal inflammation, and regulating satiety signals[64]; (2) SCFAs exhibit epigenetic effects by inhibiting HDACs[65], thus regulating the expression of inflammatory response genes, including NF-κB[66]. They also reduce macrophage secretion of pro-inflammatory cytokines such as nitric oxide, IL-6, and IL-12[67]. Moreover, SCFAs enhance the protein expression of ZO-1[68], strengthening the intestinal tight junction barrier, activating peroxisome proliferator-activated receptor gamma, and promoting the production of angiopoietin-like protein 4 in human colonic epithelial cells[67,69]; (3) SCFAs influence cellular functions by activating G protein-coupled receptors (GPR41, GPR43, and GPR109A), which facilitate the assembly of NLRP3 inflammasomes, conferring enhanced resistance to colitis[70]. Additionally, SCFAs inhibit lipid accumulation in insulin-stimulated adipocytes through FFAR2 signaling, leading to increased adipocyte activity and decreased adipose tissue inflammation[63]; (4) SCFAs influence the gut microbiome by inhibiting the colonization of harmful bacteria and promoting the growth of beneficial bacterial species, thereby positively modulating the gut environment[64]; (5) SCFAs interact with receptors in the peripheral, autonomic, and somatic nervous systems, directly affecting neuronal activity and visceral reflexes, providing additional mechanisms that contribute to overall health[71]; and (6) SCFAs actively modulate neutrophil function, influencing phagocytosis, chemotaxis, and the production of reactive oxygen species (ROS), all of which are crucial for immune defense[72].

Butyrate, a SCFA, exerts diverse positive effects on host health. It plays a vital role in regulating intestinal hormone secretion[73], inhibiting inflammatory factor expression[74], and promoting skin and mucosal ulcer recovery. By enhancing claudin-1 and ZO-1 expression and modifying occludin distribution, butyrate reinforces the intestinal barrier[75] while simultaneously increasing antioxidant levels to reduce oxidative stress[76]. It may also influence collagen breakdown by decreasing matrix metalloproteinase 9 (MMP-9) release[77], and through stimulating glucagon-like peptide-1 (GLP-1) and peptide YY secretion, it can modulate blood glucose levels[64]. Additionally, butyrate stimulates adipose tissue formation and adipokine secretion, including fibroblast growth factor, insulin-like growth factor, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF), all of which contribute to wound healing through various mechanisms[78]. Adiponectin, a key adipokine activated by butyrate, is diminished in the plasma of patients with DFU. Systemic or local adiponectin supplementation can activate AdipoR1/R2 receptors and the ERK signaling pathway, thereby enhancing keratinocyte proliferation and migration, which is essential for promoting diabetic wound healing[79,80]. Increasing SCFAs-producing bacteria in the intestines of T2DM patients could potentially serve as an advantageous approach to enhancing wound recovery.

In addition, certain gut microbes, including Faecalibacteriumprausnitzii from the Ruminococcaceae family, Eubacterium rectale and Roseburia within Lachnospiraceae, have demonstrated the capacity to produce butyrate. This production may be associated with their activation of the phosphotransbutyrylase/butyrate kinase pathway or mediation of butyryl CoA conversion to acetic acid[81]. Intestinal bacteria primarily metabolize to produce propionate through the succinate pathway and the propanediol pathway. The succinate pathway is predominantly found in Bacteroidetes and in the Negativicutes class of Firmicutes, while the propanediol pathway is mainly present in gut commensal bacteria of the family Lachnospiraceae, including Roseburiainulinivorans and Blautia species[81]. Notably, various factors influence the composition of the gut microbiota and the concentration of SCFAs in the intestine. Firstly, the gut pH value plays a role, as the proximal colon (pH around 5.6) favors fermentation of Firmicutes, while the distal colon (pH around 6.3) is conducive to the fermentation of Bacteroidetes[82]. This selective pH gradient restricts the production of propionates and promotes the formation of butyrate in the proximal part of the colon[83]. Additionally, dietary components and intake (such as the type of fiber and iron)[84], as well as the content of gut gases (such as oxygen and hydrogen) also exert an impact. Consequently, selectively addressing or treating these unfavorable factors may ameliorate gut microbiota dysbiosis, increase the concentration of SCFAs in the intestine, and ultimately promote wound healing in diabetes.

Bile acids

Primary bile acids, such as cholic acid and chenodeoxycholic acid, can be converted by intrahepatic bacteria into secondary bile acids, specifically deoxycholic acid and lithocholic acid[85]. These secondary bile acids demonstrate increased lipophilicity and cytotoxicity compared to primary bile acids, potentially explaining their detrimental effects on the intestines[86]. Bile acids possess diverse intracellular signaling functions, primarily achieved through the activation of various receptors, including nuclear receptors and GPCRs, thus playing a crucial role in inhibiting bacterial overgrowth, modulating immune function, and maintaining intestinal epithelial integrity[87]. Research indicates that the conjugate DA3-polyethyleneimine (PEI), formed from DA and PEI, significantly enhances wound healing by promoting collagen synthesis and re-epithelialization[88]. Elevated levels of DCA are associated with inflammation and cell apoptosis, impairing cell migration abilities, and the downregulation of CFTR by FXR may be a critical factor in this process[89,90]. Ursodeoxycholic acid (UDCA) exhibits cytoprotective and anti-inflammatory properties, capable of counteracting the adverse effects of DCA and promoting epithelial cell recovery[91]. The TGR5/AKT/ERK1/2 signaling pathway activated by UDCA can prevent cell apoptosis and modulate factors such as TGF-β1 and MMP-2 to facilitate wound healing[92-94]. Hyodeoxycholic acid suppresses the production of inflammatory mediators through the TGR5/AKT/NF-κB signaling pathway[95]. These findings provide a scientific foundation for the use of UDCA in treating intestinal diseases and promoting wound healing. As anticipated, the primary bacterial genera associated with bile acid metabolism in the intestine have been identified, including Bacteroides, Clostridium, Lactobacillus, Bifidobacterium and Listeria[96]. An in vitro culture experiment of live Parabacteroides distasonis demonstrated its ability to convert bile acids, resulting in increased production of lithocholic acid and UDCA, and elevating succinate salt levels in the intestines[97]. In summary, an increase in the abundance of UDCA producing bacteria may promote diabetic wound healing by correcting oxidative imbalance, reducing the expression of inflammatory mediators, enhancing collagen deposition and fibroblast migration, and inhibiting the effects of DCA.

Trp

Trp is an essential amino acid that humans cannot produce directly. It is primarily obtained through dietary intake, including sources such as cheese, bread, eggs, poultry, oats, bananas, prunes, tuna, peanuts, and chocolate[14], playing a crucial role in human health and disease. This discussion will focus on how Trp metabolites in the gastrointestinal tract exert healing effects on diabetic wounds through three main metabolic pathways. The gut microbiota can directly transform Trp into molecules with diverse biological functions through various proteases. For instance, E. coli, Clostridium, Lactobacillus, Streptococcus, Bifidobacterium, Peptococcus and Bacteroides, through their unique enzymatic systems, convert Trp into various metabolites[98]. Lactobacillus can transform Trp into indolealdehyde (IAld) and ILA through aromatic amino acid aminotransferase and indolelactic dehydrogenase 9[99]. Streptococcus thermophilus can degrade Trp into tryptamine via Trp decarboxylase. Bifidobacterium and Clostridium perfringens can produce ILA and IAA[100,101]. Moreover, many indole derivatives produced by gut microbiota metabolism, such as serotonin, tryptamine, 3-IAld, IAA, indole-3-propionic acid, indole-3-acetaldehyde and indole-3-acrylic acid, are ligands for the AhR[32]. AhR, a ligand-activated transcription factor with mixed ligand-binding characteristics, can generate a range of pleiotropic effects across different tissues. AhR-mediated signal transduction is recognized as a crucial mechanism for modulating immune responses at barrier sites and sustaining intestinal homeostasis. This regulation encompasses the modulation of epithelial renewal, barrier integrity, and the functions of various immune cells, including intraepithelial lymphocytes, Th17 cells, innate lymphoid cells, macrophages, DCs, and neutrophils[32]. The 3-IAId, an AhR ligand mentioned above, has recently been reported to have anti-inflammatory activity. The 3-IAId can significantly alleviate skin inflammation in mice with MC903 induced AD like dermatitis, and this effect can be blocked through AhR antagonists and eliminated in AhR-deficient mice[102]. Additionally, 3-IAId can improve mitochondrial dysfunction induced by high glucose, reduce oxidative stress and cell apoptosis, and promote neovascularization, although this mechanism may not depend on AhR[84]. Furthermore, the integrity and function of the epithelial barrier and metabolic complications related to HFD intake such as glucose tolerance and obesity can also be improved via 3-IAId[103]. Other AhR ligands, such as indole and IAA, may benefit T2DM by inducing L cells to secrete GLP-1 and accelerating insulin secretion[32]. Thus, indole derivatives have significant potential in enhancing mucosal and epithelial barrier function, maintaining immune homeostasis and upregulating resistance to pathogen colonization, and are expected to become effective therapeutic drugs for T2DM and its complications including impaired wound healing.

In intestinal epithelial and immune cells, Trp is predominantly metabolized into kynurenine (Kyn) by indoleamine 2,3-dioxygenase 1 (IDO1)[104]. Initially, IDO1 regulates the homeostasis of Kyn and its downstream metabolites, including quinolinic acid, xanthurenic acid, NADH, and nicotinic acid[105,106]. Under inflammatory conditions, particularly in the presence of interferons, IDO1 expression increases in various cells to metabolize Trp and interact with its mimetics, thereby restricting Trp availability[107]. Kynurenic acid (KYNA), generated by the IDO-Kyn axis, is highly sensitive to inflammation. It inhibits TNF-α at the transcription level, reduces TNF-α secretion in monocytes and CD14+ monocytes[108], and achieves mucosal protection and immune regulation through GPR35[109]. KYNA also induces autophagy degradation of NLRP3 in macrophages, leading to increased expression of host anti-inflammatory genes[110]. Recent studies indicate that KYNA-activated GPR35 enhances host energy utilization, stimulates lipid metabolism, and promotes thermogenesis[109]. KYNA may alleviate inflammatory responses and insulin resistance in skeletal muscle and adipose tissue through the GPR35/AMPK and SIRT6 signaling pathways[111], and potentially improve liver hepatic steatosis through the AMPK/autophagy pathway and AMPK/ORP150-mediated endoplasmic reticulum stress inhibition[112]. Research has demonstrated that local application of KYNA exhibits anti-scarring effects in skin wound healing models of rabbits and rats by enhancing MMP1 and MMP3 expression and inhibiting fibroblast production of type I collagen and fibronectin[113,114]. It may also influence corneal wound healing through regulating the release of cytokines such as IL-6 or IL-10[115,116]. The interaction between Kyn and gut microbiota is significant. Administration of Bifidobacterium infantis strengthens rats' intestinal mucosal immune function and resistance to fungal colonization, possibly related to the amplification of indolealdehyde producing symbiotic Lactobacillus induced by elevated serum levels of Trp and tyrosine[117]. While Kyn may benefit T2DM and its complications through these mechanisms, further research is needed to elucidate its specific effects and relationship with gut microbiota. A small portion of Trp in enterochromaffin cells is metabolized into serotonin (5-hydroxytryptamine, 5-HT) via the enzyme Trp hydroxylase 1 (TpH1), known as the 5-HT pathway. The 5-HT produces varying effects depending on the receptors expressed in different tissues and cells. For instance, the 5-HT7 receptor in intestinal DCs has anti-inflammatory effects, while 5-HT4 triggers a pro-inflammatory response in intestinal epithelial cells[118]. The 5-HT1 attenuates adenylyl cyclase to decrease cAMP levels, whereas 5-HT2 enhances phosphoinositide hydrolase activity. The serotonin transporter SERT (SLC6A4) regulates extracellular 5-HT availability by mediating its uptake into cells, where it is subsequently oxidized to 5-hydroxyindole-3-acetic acid. Research has shown that SERT decreases in colitis mouse models and patients with irritable bowel syndrome and inflammatory bowel disease. SERT-deficient mice exhibit heightened sensitivity to colitis models, suggesting that modulating available 5-HT receptor ligands through SERT may be a potential approach to regulating host inflammation[107]. Additionally, certain intestinal symbiotic bacteria can decompose dietary fiber, producing SCFAs such as butyric acid, which promote TPH1 gene expression in intestinal enterochromaffin cells, significantly impacting intestinal Trp metabolism and serotonin production. Increasing evidence supports the role of 5-HT in promoting skin wound healing. Platelet and inflammatory cell-secreted 5-HT regulates macrophage polarization and inflammation through GPCR activation[119], and stimulates tissue repair by facilitating fibroblast migration and angiogenesis. α-lactalbumin, a Trp-rich dietary protein and serotonin precursor, shows potential as a biomaterial. When loaded onto ultrafine fiber scaffolds, it effectively promotes burn wound healing and significantly reduces scar formation[120]. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, increases synaptic cleft serotonin concentration by reducing neuronal serotonin reuptake. Farahani's research demonstrates that systemic FLX administration significantly improves acute surgical wound healing in both stressed and non-stressed rat models[121]. Moreover, Bandeira found that exogenous Trp promotes skin wound healing in chronically stressed mice by inhibiting TNF-α and IDO activation[122]. The 5-HT receptors are present in various cells crucial to the wound repair microenvironment in human skin tissue, including fibroblasts, keratinocytes, platelets, neutrophils, macrophages, and mast cells[123]. Therefore, regulating 5-HT generation and transport may positively influence diabetic wound healing, although the underlying mechanisms require further investigation.

H₂S

Among the numerous gastrointestinal metabolites, H₂S is a common byproduct of metabolism present in the colon and rectum, or dissolved in the aqueous phase as H₂S and HS[124]. It is produced from the decomposition of various sulfur-containing substrates, particularly cysteine, through a variety of proteases mediated by gut microbiota. Bacillus, Clostridium, E. coli, Salmonella, Klebsiella, Streptococcus, Desulfovibrio and Enterobacter can convert cysteine into H₂S, pyruvic acid, and ammonia through cysteine desulfurase[124]. Additionally, E. coli, Salmonella, Enterobacter, Klebsiella, Bacillus, Staphylococcus, Listeria and Streptococcus may produce H₂S via thiosulfate reductase[125]. Beyond gut microbiota, mammalian tissues also generate H₂S from L-cysteine and L-homocysteine through cystathionine β-synthase, cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), with CSE appearing to be the primary source of H₂S production in the intestines[126]. Current evidence indicates that H₂S functions as a novel gasotransmitter, participating in various physiological and pathological processes, including reducing oxidative stress, regulating inflammatory responses, promoting angiogenesis, increasing vasodilation, and inhibiting apoptosis of EC and myocardial cells[127]. Furthermore, growing evidence suggests that the oxidative stress and inflammatory responses associated with diabetes can affect the endothelium of blood vessels, leading to dysregulation of coagulation and anticoagulation mechanisms, peripheral vascular disease, and peripheral circulatory dysfunction[128,129]. Thus, regulating these factors may play a role in healing diabetes and its related complications. Moreover, prolonged hyperglycemia reduces antioxidants, and the increased oxidative stress levels caused by ROS and vascular function damage ultimately lead to the occurrence of diabetes and its complications[130]. Importantly, H₂S can resist oxidative stress through two different mechanisms. First, EC-generated ROS play a crucial role in the pathogenesis of diabetic complications, and H₂S, acting as a direct scavenger of ROS, can reduce ROS and improve the functional and metabolic states of EC under hyperglycemia[131]. Second, H₂S can increase the functionality of superoxide dismutase and decrease the content of MDA to repair skin wounds in diabetic rats by enhancing the antioxidative defense system[132]. Therefore, promoting the production of antioxidants such as H₂S could stimulate the healing of diabetic ulcers.

Recent studies have confirmed the significance of endothelial progenitor cells (EPCs)-mediated angiogenesis in wound healing, while the angiogenic capacity of EPCs decreases in diabetes. This EPC-related vascular regeneration defect caused by hyperglycemic conditions may lead to chronic vascular complications, refractory wounds, and foot ulcers. Restoring EPC function could be a critical pathway to enhance the process of wound repair. In addition to mitigating oxidative stress, H₂S possesses the ability to regulate the proliferation and migration of EC[133,134]. Local transplantation of EPCs significantly improved wound healing in diabetic mice, which may be attributed to the up- regulation of angiopoietin-1 (Ang-1) expression in EPCs by H₂S donors[102]. Ang-1, a vascular growth factor, can further induce the production of VEGF, a key element in angiogenesis, and stimulate the phosphorylation of its receptor, thereby promoting endothelial cell survival and vascular stability[135]. Furthermore, H₂S could increase ICAM-1 levels in diabetic mice. This inducible transmembrane protein mediates the adhesion between leukocytes and EC and activates EC to accelerate angiogenesis[136,137]. As anticipated, H₂S effectively downregulates the protein and methylation levels of anti-angiogenic DNA methyltransferase 1 induced by high glucose, thereby enhancing the expression of miR-126-3p and subsequently alleviating the angiogenic impairment caused by high glucose[138]. Additionally, H₂S can directly enhance the transcription and protein expression of VEGF, PDGF, EGF, HIF-1α and eNOS, promote the phosphorylation of receptors VEGFR and PDGFR, accelerate angiogenesis, and improve wound healing in diabetic mice[139].

Inflammatory response plays a crucial role in the repair of skin wounds in T2DM. H₂S significantly enhances diabetic wound healing by precisely modulating immune cell activity and mitigating inflammatory responses. Specifically, H₂S reduces the polarization of M1 macrophages, the formation of neutrophil extracellular traps, and the expression of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α[105,140-142]. Moreover, H₂S activates the Nrf2-ROS-AMPK and PI3K/Akt/eNOS signaling pathways, protecting endothelial cells from inflammatory damage[131,143]. These combined effects attenuate inflammation and optimize the wound healing process in diabetic mice. Consequently, we hypothesize that the intestinal metabolite H₂S could promote wound healing in diabetes by restoring EPC function, activating angiogenesis-related factors, and regulating immune inflammation. The clinical applications of H₂S are primarily manifested in two areas. Firstly, supplementation with H₂S donors such as NaHS and GYY4137 can accelerate the healing process of diabetic wounds[144]. Secondly, H₂S donors are incorporated into innovative drug delivery systems, including nanofiber dressings, hydrogels, and sprays, to optimize the local wound environment[145,146].

The potential therapeutic effects of other gut microbiota metabolites

Recent research has revealed that, the abundance of Streptococcus bacteria capable of synthesizing imidazopyridine propionic acid (ImP) has significantly increased in the gut and skin microenvironments of T2DM mice[147]. However, Imp induces Insulin resistance and disrupts glucose metabolism via activating the mTORC1 pathway and inhibiting the AMPK signaling pathway[148,149]. Meanwhile, ImP hampers the secretion of sphingosine-1-phosphate (S1P) mediated by SPNS2, blocks the activation of the Rho signaling pathway, and thereby interferes with the angiogenesis process of human umbilical vein endothelial cells, ultimately inhibiting the healing of diabetic wounds[147]. Additionally, Lipopolysaccharide (LPS) is widely present in the gut microbiota as a key component of the cell wall of Gram-negative bacteria. While high doses of LPS typically have harmful pro-inflammatory effects, low doses of LPS can enhance the expression of TLR-4 in keratinocytes and promote wound healing[150]. Studies have shown that oral administration of LPS not only significantly improves insulin resistance and glucose intolerance but also stimulates the expression of adiponectin in adipose tissue[151]. In contrast to intravenous administration, oral intake of LPS does not induce the production of pro-inflammatory cytokines IL-1 β, IL-6, and TNF-α, but instead exerts neuroprotective effects by activating anti-inflammatory cytokine IL-10[150]. These findings provide important theoretical support for the development of FMT as a potential therapy for treating chronic non-healing diabetic wounds.

CONCLUSION

The potential of targeting gut microbiota, including FMT, probiotic supplementation, and modification of gut microbiota-associated metabolites in the treatment of diabetes and its complications has been demonstrated. This review elucidated how gut microbiota metabolites such as butyrate, ursodeoxycholic acid, Trp metabolites, and hydrogen sulfide can significantly enhance the healing process of diabetic wounds through various mechanisms. These include strengthening the intestinal epithelial barrier, promoting endothelial progenitor cell function, activating angiogenesis-related factors, regulating oxidative stress, controlling immune inflammatory responses, and facilitating fibroblast migration. Although factors such as inter-individual differences, infection risks, treatment complexity, research limitations, and drug interactions may restrict the efficacy of these metabolites in different patients and pose challenges in clinical practice, FMT still holds undeniable potential in the healing of diabetic wounds and shows promising clinical application prospects. Future research should further investigate the direct impact and mechanisms of intestinal microbiota regulation in the relationship between gut microbiota and diseases.

ACKNOWLEDGEMENTS

We express our sincere gratitude to our advisor for their support in the preparation of this manuscript.