Diabetic foot (DF) is defined as wounds that manifest on the feet of individuals suffering from type 1 or type 2 diabetes (1). It is typified by infections, ulcers, and/or deep - tissue damage stemming from peripheral neuropathy and vascular complications in the lower limbs of diabetic patients. The pathogenesis of DF is intricate and multi-factorial, encompassing interrelated mechanisms like infection, micro-and macrovascular diseases, and neurological impairment (2). Globally, patients suffering from diabetic foot ulcers (DFU) encounter a five - year mortality rate that is as high as 34%. Moreover, non - traumatic lower limb amputations caused by this condition make up more than half of all cases, which poses a significant public health challenge (3, 4). Current clinical management guidelines, such as those recommended by the International Working Group on the Diabetic Foot, mainly focus on infection control, revascularization for ischemia, and mechanical offloading. Although these interventions are effective in saving limbs in critical situations, they frequently fail to fundamentally tackle the root challenge of chronic non-healing wounds (5, 6).

DFUs represent the most severe chronic complication of diabetes, with their pathophysiology primarily rooted in impaired wound healing caused by hyperglycemia-induced tissue damage (7, 8). A growing body of evidence indicates that persistent hyperglycemia creates an unfavorable microenvironment, which impairs re-epithelialization, angiogenesis, and extracellular matrix remodeling via multiple mechanisms, including the accumulation of advanced glycation end products, heightened mitochondrial oxidative stress, and continuous inflammatory responses (9, 10). Therefore, going beyond the traditional paradigms of “anti-infection” and “vascular patency”, exploring the metabolic basis of this pathological microenvironment, and identifying key endogenous regulatory factors that can restart the normal repair process have become the new frontier in surmounting current therapeutic limitations.

Metabolomics, as a scientific discipline, systematically characterizes the compositional profiles and dynamic changes of endogenous small-molecule metabolites within biological systems. The analytical targets of metabolomics span a broad spectrum of endogenous and environmentally-derived low-molecular-weight compounds, such as sugars, lipids, amino acids, fatty acids, alkaloids, and polyphenols (11). Metabolomics, through the utilization of high-throughput analysis to monitor the dynamic changes in endogenous metabolites after physiological or pathological stimuli, offers a direct portrayal of the organism’s actual physiological and biochemical state. This, in turn, facilitates the identification of potential diagnostic biomarkers and therapeutic targets (12, 13). In studies of exudate from DFU, metabolomics has identified multiple key small molecules, such as betaine, lactic acid, and carnitine, which play diverse roles in wound healing. These roles include enhancing cellular function, exerting anti-inflammatory and antioxidant effects, and promoting angiogenesis (14, 15). In the field of diabetes and its complications, existing research has indicated that abnormal changes in metabolic profiles, such as those of amino acids, fatty acids, and bile acids in blood or tissues, are closely related to insulin resistance, vascular endothelial dysfunction, and chronic inflammation (16–18). Bile acids, particularly primary bile acids like chenodeoxycholic acid (CDCA), are capable of activating key nuclear receptors and membrane receptors, including the farnesoid X receptor (FXR) and the G protein - coupled bile acid receptor (GPBAR1/TGR5). As a result, they can precisely regulate glucose homeostasis, energy expenditure, innate immune responses, and inflammatory pathways (19–21). There is compelling evidence suggesting that disruptions in bile acid metabolism are closely linked to the initiation and advancement of type 2 diabetes and non-alcoholic fatty liver disease (22, 23). Research has demonstrated that CDCA can enhance insulin sensitivity in both hepatic and peripheral tissues and exert anti-inflammatory effects (24). Although the association between perturbations in bile acid metabolism and the pathophysiology of systemic diabetes has been tentatively established, it remains uncertain whether bile acids play a crucial role in the pathological progression of DFU, which is characterized by severe local tissue damage. Specifically, it is unclear regarding the relationship between altered serum bile acid levels and ulcer risk, or whether bile acids can directly regulate wound repair processes. Numerous bioactive molecules, including chenodeoxycholic acid (CDCA), face significant challenges in complex wound environments due to limited stability, poor membrane permeability, and inadequate targeting capability. Advanced drug delivery systems—such as solid lipid nanoparticles (SLNs) that enhance dermal penetration, cubic micelles capable of co-delivering multiple therapeutic agents, and highly deformable transfersomes—present promising strategies to overcome these physiological and biochemical barriers (25, 26). By improving the local retention and enabling controlled release of active compounds, these delivery platforms hold potential to translate the pharmacological efficacy of agents like CDCA into measurable clinical outcomes, representing a key focus for future translational research within this study.

This study conducted an exploratory investigation by integrating clinical metabolomics with functional validation. Through the application of untargeted metabolomics technology, the serum metabolic profiles of patients with DFU were comprehensively characterized, and key metabolites associated with disease progression were precisely identified. Simultaneously, bioinformatics analyses and statistical modeling were utilized to accurately pinpoint endogenous metabolites with potential protective effects. The regulatory effects of candidate molecules on the functions of wound-repair cells were validated using high-glucose cellular models. The aim was to uncover novel mechanisms underlying DFU and provide a theoretical basis for the “metabolic microenvironment remodeling” therapeutic strategy.

This study was carried out as a case-control investigation in strict compliance with the principles of the Declaration of Helsinki and obtained ethical approval from the Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology (approval number: 2024 - 03 - K0048). The registration number at the Chinese Clinical Trial Registry (ChiCTR) is ChiCTR2400088055. Written informed consent was acquired from all participants before their enrollment, and the confidentiality of personal data was rigorously maintained throughout the study.

DFU represent one of the most severe complications associated with diabetes, presenting a substantial global public health burden. Epidemiological evidence indicates that the lifetime risk of foot ulcer development among individuals with diabetes may reach up to 34%, with an estimated annual global incidence of approximately 18.6 million cases (2, 27). More seriously, the mortality risk among patients with DFU is 2.5 times higher than that among diabetic patients without foot ulcers, and it is a leading cause of non-traumatic lower limb amputations in many countries (2). Approximately half of all DFUs develop secondary infections, which can potentially lead to sepsis, gangrene, and ultimately amputation. These findings jointly demonstrate the disease burden of DFU, which is marked by high incidence, high disability rate, and high mortality, along with the substantial complexity of its clinical management.

Currently, the clinical management primarily relies on a multidisciplinary collaborative approach, with its core components including vascular reconstruction, infection control, local ulcer management, and offloading therapy (28, 29). Although these strategies are crucial for limb preservation, they mainly concentrate on alleviating late-stage pathological consequences. The underlying pathophysiology of DF entails progressive tissue damage in the foot triggered by chronic hyperglycemia, which is characterized by a “neuro-vascular-immune” triad of dysfunction (30). Persistent hyperglycemia gives rise to peripheral neuropathy, which leads to the loss of protective sensation. It also facilitates the development of peripheral vascular disease, causing ischemia in the lower limbs. Coupled with immune dysfunction in a hyperglycemic milieu, these factors work in synergy to create a local microenvironment that hampers tissue repair and increases the susceptibility to infection. Therefore, transcending the traditional models of “anti-infective therapy” and “vascular revascularization” and concentrating on the analysis and targeted intervention of this pathological microenvironment has emerged as a promising avenue for enhancing outcomes in DF.

Against this backdrop, this study systematically clarifies the metabolic dysregulation characteristics of DFU by integrating clinical metabolomics and functional validation. Moreover, it identifies CDCA as a key endogenous protective factor in DF for the first time. The core finding reveals that CDCA is a disease-specific protective metabolite for DF, and its significantly decreased serum concentration is independently correlated with an increased risk of DF development. This discovery not only deepens our understanding of the metabolic basis of DF but also provides a promising approach for the development of novel therapeutic strategies based on metabolic modulation.

At the mechanistic level, this study verifies that CDCA effectively alleviates high glucose-induced cellular damage by activating the pathways related to cell proliferation and migration. In vitro experiments show that, within its physiological concentration range (5–25 nM), CDCA reverses the functional impairment in HSFs under high glucose conditions in a dose-dependent manner. This protective effect is accompanied by a significant up-regulation of the proliferation marker PCNA, the myofibroblast differentiation marker α-SMA, and the cytoskeletal protein vimentin (31, 32). These molecular alterations collectively form the mechanistic basis for CDCA-mediated enhancement of wound repair. This indicates that CDCA may expedite wound healing by facilitating entry into the proliferative phase, inducing differentiation into functional myofibroblasts, and maintaining cytoskeletal integrity. Moreover, this study demonstrated the regulatory impact of CDCA on the FXR/PKA/AMPK signaling pathway in cell models related to DF. The current results clearly showed that CDCA significantly reversed the high-glucose-induced downregulation of FXR expression and the inhibition of PKA phosphorylation. Simultaneously, it also mitigated the excessive activation of AMPK. FXR, as a central nuclear receptor for bile acids, promotes cellular proliferation and metabolic homeostasis upon activation. PKA is involved in the regulation of cell migration and gene transcription, and appropriate suppression of AMPK could contribute to alleviating the high-glucose-induced energy stress and apoptosis. These findings suggest that CDCA may enhance fibroblast proliferation, migration, and the expression of repair-related genes, and thus accelerate wound healing, by coordinately activating FXR and PKA while curbing the excessive activation of AMPK. Future studies employing gene silencing or receptor antagonists are needed to further verify the necessity of these pathways in mediating the effects of CDCA.

Although CDCA shows promising wound-repair potential in vitro, its stability, local permeability, and targeted delivery in complex wound environments require improvement. Nanocarrier-based drug delivery systems have advanced bioavailability, controlled release, and targeting capabilities. For instance, cubosomes—bicontinuous cubic liquid crystalline nanoparticles—enhance skin penetration and local retention of anti-inflammatory drugs and can be administered orally, transdermally, or intranasally, showing therapeutic potential in inflammatory conditions like rheumatoid arthritis (33). Similarly, transfersomes, as ultra-deformable lipid vesicles, effectively cross the stratum corneum and improve drug delivery to skin tissues, with proven efficacy for anti-inflammatory, antifungal, and peptide therapies (34). Therefore, encapsulating CDCA in nanocarriers such as cubosomes or transfersomes could enhance its stability, skin penetration, and sustained release at wound sites. This approach aligns with the strategy of “metabolic microenvironment remodeling” and may accelerate the translational development of CDCA-based treatments for DFU.

It should be noted that CDCA, as a crucial component of bile acids, exhibits multiple biological functions, such as the regulation of glucose and lipid metabolism homeostasis, anti - inflammatory effects, and antibacterial activity (31). This pleiotropic nature precisely meets the dual therapeutic requirements of infection control and metabolic improvement in the clinical management of DF. Previous studies have indicated that CDCA regulates glucose homeostasis and inflammatory responses by activating the FXR and TGR5 (21, 32). The current findings expand these mechanisms to the context of wound repair, providing a more comprehensive understanding of the protective role of CDCA in DF.

The innovative value of this study resides in the initial identification of CDCA as a potential therapeutic target for diabetic foot (DF) and the introduction of a novel treatment paradigm—metabolic microenvironment remodeling (35). Unlike conventional therapeutic strategies that predominantly concentrate on anti - infective therapy and vascular reconstruction, our findings indicate that enhancing the local metabolic environment at the wound site by supplementing endogenous protective metabolites could be a promising approach to surmounting the current treatment limitations in DF. This strategy directly tackles the fundamental metabolic dysfunction that is characteristic of DF and has substantial potential for clinical translation.

However, this study presents several limitations. Firstly, the single-center design and the restricted sample size might undermine the generalizability of the study’s findings. Future validation of the clinical value of CDCA necessitates multicenter, large-scale cohort studies (36). Secondly, the study mainly concentrated on cellular - level validation and was devoid of supporting evidence from animal models, thus restricting a comprehensive evaluation of CDCA’s therapeutic effects in vivo. Thirdly, the initial logistic regression model that identified CDCA as a protective factor failed to adjust for potential clinical confounders, such as the duration of diabetes or comorbid conditions. Although the comparability of the baseline groups alleviates this concern, future analyses using adjusted models are required to confirm the independent protective role of CDCA. More importantly, the specific molecular mechanisms underlying CDCA’s protective effects—particularly whether they are mediated through canonical bile acid receptors such as FXR and TGR5—remain to be thoroughly elucidated through experimental approaches including gene knockout (37, 38). Based on this, future research will focus on completing the construction of a complete evidence chain from in vitro to in vivo: 1) Utilizing gene editing technology, clarify in cell models whether CDCA exerts its effects through specific receptor pathways such as FXR and TGR5; 2) Establish an animal model of diabetic foot ulcers, and through local or systemic administration, evaluate the direct effects of CDCA on wound healing speed, tissue remodeling, and inflammatory regulation in vivo; 3) If the results are positive, further explore its preclinical efficacy and safety to lay the foundation for potential translational applications. This study also represents the first to demonstrate an independent negative association between serum CDCA levels and the risk of diabetic foot, while elucidating its direct protective effects at the cellular level—thereby providing preliminary etiological evidence supporting its potential as a novel biomarker. However, the evaluation of key performance indicators for CDCA as a diagnostic or predictive biomarker remains beyond the scope of this study, including the determination of optimal cut-off values for disease differentiation, sensitivity, specificity, and predictive value.

In conclusion, this study establishes the protective function of CDCA in DF and paves a new research path for DF treatment by targeting the metabolic microenvironment. Future endeavors ought to concentrate on clarifying the specific mechanism of action of CDCA and validating its therapeutic effectiveness in more intricate in - vivo models, with the ultimate aim of promoting the clinical translation of metabolic regulation - based therapeutic strategies for DF.

This study systematically characterizes the metabolic dysregulation features of DFU via the integration of clinical metabolomics and functional validation, and for the first time, it identifies CDCA as a crucial endogenous protective factor. The findings indicate that the serum CDCA levels in patients with DF are significantly lower compared to those in both uncomplicated diabetic patients and healthy controls. Moreover, multivariate logistic regression analysis verifies that CDCA is an independent protective factor against the progression of DF. Further cellular functional experiments reveal that CDCA effectively reverses high-glucose-induced fibroblast dysfunction in a dose-dependent manner within the physiological concentration range (5–25 nM), and the underlying mechanisms are closely related to enhanced cell proliferation, improved migratory capacity, and upregulation of repair-related genes such as PCNA and α-SMA. In conclusion, this study not only clarifies the protective role and underlying molecular mechanisms of CDCA in the pathogenesis of DF but also offers a solid experimental basis and potential therapeutic targets for the development of innovative strategies targeting the metabolic microenvironment.