Diabetic foot ulcers (DFUs) represent a significant complication associated with diabetes mellitus, characterized by persistent nonhealing wounds resulting from diabetic neuropathy affecting sensory, motor, and autonomic functions, along with vascular issues and bacterial infections (Armstrong et al., 2023). About 34% of people with diabetes are likely to experience a foot ulcer at some point in their lives, and those with DFUs have a mortality rate that is 2.5 times greater over 5 years compared to diabetics without foot ulcers (Armstrong et al., 2017; Rehman et al., 2023). DFUs are managed conservatively and often present early (Al-Rubeaan et al., 2015). When experiencing resting pain, surgical intervention is often required. Techniques for restoring blood circulation to the lower limbs involve arterial bypass, the placement of stents, along with various surgical and interventional methods (Bandyk, 2018). Utilizing spinal cord stimulation (SCS) may aid in relaxing the blood vessels in the lower limbs, enhancing the microcirculation in the extremities, and to some extent, preventing ulcers and gangrene, while also promoting healing and repair, which could reduce the likelihood of amputation (Li et al., 2021). Nonetheless, regardless of whether surgical intervention or SCS therapy is employed, complete reconstruction of blood vessels or treatment solely for vascular conditions is not achievable, and there is a considerable risk of long-term recurrence (Islamov et al., 2022). Consequently, there is an urgent need to identify and develop diagnostic and therapeutic biomarkers for DFUs with high specificity.

Biomarkers, described as “attributes of objective assessment and measurement that serve as indicators of typical biological functions, disease processes, or pharmacological reactions to treatment,” play a crucial role in the early diagnosis of clinical conditions, prevention of diseases, and forecasting the progression of illnesses (Wang et al., 2021). In recent years, numerous genes associated with DFUs have been identified as potential biomarkers for disease diagnosis. For example, Jeandrot et al. identified C-reactive protein as a reliable biomarker for distinguishing different DFUs grades in a study involving DFUs patients and diabetic controls (Jeandrot et al., 2008). Weigelt and colleagues indicated that the levels of IL-6 in individuals suffering from acute DFUs were elevated compared to those in diabetic patients who did not have diabetic foot issues (Weigelt et al., 2009). Additionally, Mir and his team discovered a correlation between the HSPA1B genotype, which pertains to the polymorphism of heat-shock protein (HSP) 70, and both the severity of DFUs as well as the results of surgical interventions (Mir et al., 2009). Despite the identification of numerous individual biomarkers through various methods, there is still a deficiency in model analyses that focus on multiple key genes in DFUs. Therefore, transcriptomics-based to explore novel biomarkers for the treatment and diagnosis of DFUs is imperative.

Melanin synthesis plays a crucial role in maintaining skin pigmentation. Additionally, melanin and its metabolites help maintain skin microenvironment homeostasis by scavenging reactive oxygen species, reducing oxidative damage, and modulating the release of inflammatory factors (Solano, 2020; Fu et al., 2022). While melanin is traditionally associated with skin coloration, recent studies have hinted at its role in antioxidant defense, cellular homeostasis, and stress response modulation (Braydon et al., 2024). Recent studies have shown that abnormalities in melanin synthesis may impair the proliferation and differentiation of skin cells, thereby disrupting the normal wound healing process (Sato et al., 2024). In this study, we aim to employ transcriptome analysis to uncover the possible critical molecular mechanisms involved in the development of DFUs. By integrating differential expression analysis, co-expression network construction, and functional enrichment, we aim to identify a previously unexamined class of biomarkers, potentially uncovering novel biological pathways contributing to DFU development. Furthermore, immune infiltration analysis was conducted to explore the relationship between key genes and the DFUs microenvironment. Finally, drug prediction was performed to identify potential therapeutic targets. These findings provide novel insights into the molecular landscape of DFUs, offering promising biomarkers for early diagnosis and potential therapeutic strategies for clinical intervention.

Diabetic patients often experience DFUs, a serious complication that raises the likelihood of amputation and mortality due to their challenging healing process and high rates of recurrence (McDermott et al., 2023). In recent years, research has gradually revealed that DFUs are not only caused by local circulatory disorders and persistent inflammation but are also closely related to impaired skin barrier function and dysregulated antioxidant stress responses. These factors interact, leading to a state of chronic tissue damage in DFUs, further exacerbating the difficulty of wound healing (Armstrong et al., 2023). Therefore, it is imperative to identify the relative biomarkers with high specificity for the diagnosis and treatment of DFUs. In this study, we utilized the GEO database of DFUs to conduct differential gene and pathway enrichment analysis first. Consistent with previous studies, we found that the pathogenesis of DFUs involves vascular lesions, nerve damage, and infection (Fa-Shun et al., 2022; Huang et al., 2022). Specifically, based on the outcomes of GO enrichment pathway analysis, heparin binding was identified as significantly associated with vascular lesions, growth factor binding was demonstrated to exhibit a strong correlation with nerve damage. Likewise, integrin binding, cytokine binding, and transmembrane receptor protein kinase activity were identified to be linked to the infection process. These findings highlight the distinct functional implications of these molecular interactions in the context of specific pathological conditions. After that, based on the WGCNA and LASSO modeling, three target genes (DCT, PMEL, and KIT) related to melanin synthesis in DFU were identified.

It is noted that chronic hyperglycemia and persistent inflammation lead to increased oxidative stress in the pathological environment of DFUs, which may alter melanin production (Xu et al., 2022). Therefore, investigating the role of melanin synthesis in DFUs holds significant importance. Our findings indicate that these three key genes associated with melanin synthesis have the potential to be diagnostic and therapeutic targets for DFUs. Research indicates that the DCT phenotype is linked to increased neurotization in benign nevi and is also associated with ulceration in thin malignant melanomas (Filimon et al., 2014). DCT is primarily involved in melanogenesis and antioxidant defense, where it catalyzes the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in melanocytes and enhances melanin stability (Hearing, 2000). PMEL plays a crucial role as a structural protein in the development of melanosomes, facilitating the aggregation of melanin and the creation of fibrous structures to enhance both the storage and distribution of melanin (Zhang et al., 2021). Additionally, Pan et al. demonstrated that in HaCaT cells, a reduction in melanin correlates with a decline in the levels of PMEL protein, which is essential for the formation of melanosomes (Huang et al., 2024). KIT acts as a tyrosine kinase receptor and plays a central role in regulating melanocyte proliferation, survival and differentiation (Hu et al., 2020). Research indicates that melanin and its byproducts facilitate wound healing through the scavenging of reactive oxygen species, the attenuation of inflammatory factor release, and the promotion of skin barrier restoration (Qi et al., 2023). However, in patients with DFUs, we noted a significant decline in the expression levels of DCT, PMEL, and KIT. This reduction could result in diminished melanin production, consequently compromising the protective capabilities of the skin barrier, lowering antioxidant effectiveness, and increasing the wounds’ vulnerability to additional harm caused by oxidative stress and inflammation. Moreover, based on this discovery, we constructed ROC curves to evaluate the diagnostic efficacy of DCT, PMEL, and KIT. The results showed that the AUC values of these three genes all exceeded 0.9, suggesting their robust predictive power. This finding strongly indicates that DCT, PMEL, and KIT could potentially serve as therapeutic targets for DFUs in clinical practice.

The pathogenesis of DFUs is complex, involving the joint participation of multiple signaling pathways (Hu et al., 2024). In this study, GSEA analysis revealed that the three genes could affect the MAPK and PI3K-Akt signaling pathways, which provide a new insight for prospective exploration of the molecular mechanisms and therapeutic targets of DFUs. Aligning with our findings, previous studies in the pathological environment of DFUs, the abnormalities in these two signaling pathways may exacerbate impaired wound healing. In detail, the MAPK signaling pathway regulates the proliferation and migration of keratinocytes and fibroblasts during wound repair, and the elevated levels of MAPK are linked to the development in DFUs (Phimnuan et al., 2023; Shi et al., 2023). Likewise, the abnormalities of DFUs in the PI3K-Akt pathway may lead to reduced survival capacity of keratinocytes and endothelial cells, thereby impairing angiogenesis and wound closure (Xing et al., 2017). Functionally speaking, the signaling pathways MAPK and PI3K-Akt are pivotal in numerous biological processes, including cell growth, differentiation, survival, and the regulation of immune responses, which demonstrate significant activity across different physiological and pathological states, especially when it comes to the management of melanocyte activity, the production of melanin, and processes involved in skin repair (Corrales et al., 2022; Wu et al., 2024). The signaling pathways of MAPK and PI3K-Akt are crucial in regulating the synthesis of melanin. Research indicates that Selaginellin can impede melanogenesis by blocking the MAPK signaling pathway, which subsequently leads to a reduction in the expression of the microphthalmia-associated transcription factor (MITF) and the downstream genes tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2) (Zhou et al., 2022). In addition, as a G protein-coupled receptor, melanocortin 1 receptor (MC1R) manages the synthesis quantity and quality of melanin within melanocytes (Anyamanee and Uraiwan, 2022). During the process of melanin synthesis, the activation of MC1R was accompanied by an increase in cAMP levels and the subsequent MAPK signaling cascade, leads to the activation of MITF (Po-Yuan et al., 2018). Meanwhile, the signaling pathway involving PI3K-Akt also influences melanogenesis. Specifically, when this pathway is activated, it enhances the phosphorylation of MITF, boosts the survival of melanocytes, and modulates the expression of genes that are crucial for melanin production (Zhou et al., 2021). Moreover, PI3K-Akt is important for preserving the stability of melanocytes and providing resistance to oxidative damage, with its inhibition potentially resulting in compromised melanocyte function (Mosca et al., 2021).

Prior studies point out that NK cells play a significant role in the inflammation of DFUs, while the disease activity of DFUs is associated with macrophages (Wen-Juan et al., 2024). In line with that, the immune infiltration analysis in our study demonstrated that there were differences in the compositions of macrophages, mast cells, T cells, and NK cells. Specifically, the proportion of NK cells activated were significantly decreased in disease group, which further emphasizes the significance of immune response in the development of DFUs. Moreover, irregularities within the PI3K-Akt signaling pathway result in a reduction of M2-type anti-inflammatory macrophages (Deng et al., 2024). This shift consequently favors the dominance of M1-type pro-inflammatory macrophages, potentially sustaining an inflammatory condition in DFUs, ultimately impacting the healing process of the wounds (Jere et al., 2019). These findings suggest that the MAPK and PI3K-Akt signaling pathways may play key roles in the development of DFUs, and the aberrant expression of DCT, PMEL and KIT may regulate the DFUs microenvironment by affecting these signaling pathways, which offers a potential causal relationship between immune cells and DFUs. This provides an important theoretical basis for further exploration of the pathomechanisms of DFUs as well as targeted therapeutic strategies.

Identification of valid predictive diagnosis and drug targets are crucial for DFUs (Fang Leimi et al., 2022). In this study, the drug network was constructed, and the possible target drugs were predicted for DFUs. Imatinib, a Tyrosine kinase inhibitor, was found to be a target drug for KIT. Previous studies point out that imatinib could potentially influence related immunological and metabolic pathways, and preclinical investigations demonstrate that it can both reverse and prevent diabetes (Gitelman et al., 2021). Similarly, the melanoma vaccine indirectly regulates the synthesis of melanin by participating in immunomodulation, which was found to be a target drug for PMEL in the study (Yin et al., 2023). In addition, a study has shown that amphotericin B liposomal exhibits positive therapeutic effects on the complications of patients with diabetic foot (Muhamed et al., 2021). In line with this, our finding demonstrate amphotericin B liposomal was a potential target drugs for DCT in DFUs. Nonetheless, our current research has certain limitations. Firstly, the data utilized in this investigation were primarily sourced from the GEO database. Although we applied batch effect correction and cross-validation, there remains a potential bias, which stems from sample origin, processing differences, and population heterogeneity and could impact the generalizability of our findings. Additionally, despite our findings predicting potential drug targets for key genes in DFUs, our study did not undertake a preliminary evaluation of the applicability, safety, and efficacy of these targets in patient populations. Meanwhile, individual patient factors, such as age, diabetes duration, and comorbidities, were not incorporated to explore the therapeutic potential of identified targets. Subsequent research could benefit from the incorporation of independent datasets from multicenter clinical samples, encompassing data from patients with DFUs across various geographic locations and diverse populations, enhancing the generalizability and reliability of the findings. Secondly, the downregulation of DCT, PMEL, and KIT was noted in this study solely among a cohort of DFUs patients, leading to the hypothesis that these factors might influence the progression of DFUs through the MAPK and PI3K-Akt signaling pathways; however, direct causal evidence is absent.

In summary, our detailed analysis of the transcriptome emphasizes the crucial involvement of genes associated with melanin synthesis (DCT, PMEL, and KIT) in the advancement of DFUs and their notable relationship with the infiltration of immune cells. Additionally, our predictions regarding drug targets highlight possible therapeutic options. These results not only deepen our comprehension of the fundamental mechanisms underlying DFUs but also indicate that these biomarkers might act as novel therapeutic targets Further in vitro validation assays such as gene overexpression would be performed to observe the effects of changes in the expression of the three key genes (DCT, PMEL, and KIT) on cell proliferation, migration, apoptosis, and the pathological processes related to DFUs. Additionally, single-cell RNA sequencing should also be conducted for a more in-depth analysis of DFU heterogeneity, and animal models using CRISPR/Cas9 gene editing techniques would also be established to investigate their roles in the context of the disease. Lastly, we intend to leverage various drug screening databases in future investigations to discover compounds that may modify these genes and to carry out drug intervention experiments, such as treatments involving small molecule agonists or inhibitors in DFUs cellular models, to evaluate their impact on wound healing.

In conclusion, the findings of this research indicate that there is an imbalance in the genes associated with melanin synthesis in DFUs. It offers new perspectives on the pathogenesis of DFUs and presents potential therapeutic targets for enhanced diagnosis and treatment.