Abstract
Background
In some developing countries, people have little knowledge about the causes of diabetic foot ulcers. Therefore, public health education for patients on these conditions is a prerequisite for effective pharmacological treatment. Diabetic foot ulcers are a complex symptom of diabetes and are hard to cure due to the lack of efficacious medicine and alternative treatment approaches. Vitamin A (VA) is known to have potent biological functions, including skin repair and immunoregulation. However, the potential pharmacological effects and molecular mechanisms of VA on foot ulcers are still to be discovered.
Methods
By using bioinformatic/computational analyses, including network pharmacology, gene ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analysis, we aimed to identify and reveal the pharmacological targets, molecular mechanisms, biological functions, and signaling pathways of VA in the treatment of diabetic foot ulcers.
Results
A total of 66 intersection genes were identified as candidate targets of VA, which are related to diabetic foot ulcers. Therein, 18 core genes/targets, namely JUN, MAPK1, THRB, MAPK14, MTNR1B, CXCR3, ESR1, AR, HDAC1, IL-10, CNR1, DRD2, EGFR, ADRA2A, CCND1, RXRB, RARA, and RXRA, were further identified. Furthermore, the biological processes, cell components, and molecular functions which may underlie the effects of VA against diabetic foot ulcers were characterized.
Conclusion
Based on our findings, we concluded that the pharmacological effects of VA on diabetic foot ulcers primarily involve the promotion of cellular regeneration and proliferation and the inhibition of inflammatory response. The core genes/targets may potentially serve as promising biomarkers for the diagnosis of diabetic foot ulcers.
1 Introduction
Globally, diabetes mellitus and the risk of developing complications are of great concern[1]. This disease and its associated symptoms are a dominant health issue and socioeconomic burden, especially in China of a large population[2]. In addition, many of the local population, including diabetes patients, do not possess enough medical knowledge to understand how to manage diabetes and its complications before they access clinical treatment[3]. Therefore, educating people about diabetes prevention, such as fat/sugar control, vitamin intake, physical exercise, and medication, is a necessary measure to maintain fitness and wellbeing[4]. Diabetic foot ulcers are believed to be one of the major complications of diabetes[5]. At the early stage, ulcers with peripheral vascular lesions may be asymptomatic before they become visible[6]. Thus, for diabetes patients, early screening and foot care are required to reduce the risk of developing ulcers[7]. In clinical management, the main treatment for diabetic foot ulcers includes ortho-plastic surgery and hypoglycemic drugs[8]. Unfortunately, side effects occur as a result of pharmacotherapy, including hypoglycemia, allergies, and gastrointestinal issues[9]. Thus, it is thought that prior screening and the development of a potential bioactive ingredient to effectively manage diabetic foot ulcers are needed. A prospective cohort study of diabetes patients showed that vitamin A (VA) deficiency was present in 10.9% of patients[10]. Physiologically, VA is a fat-soluble nutrient required for normal vision and is commonly used to prevent nyctalopia[11]. Functionally, VA modulates a series of biological processes, such as embryogenesis, cell proliferation, cytodifferentiation, skin restoration, and immunoregulation[12]. In addition, VA plays an important role in regulating metabolic disorders and contributing to the prevention and management of obesity, type 2 diabetes, and liver steatosis[13]. Recently, increasing evidence has shown that a shortage of VA is found in the development of type 2 diabetes and cardiovascular disorders in humans[14]. A case-controlled study indicated that a vitamin-rich diet, including VA, may reduce the risk of gestational diabetes in Chinese patients[15]. A cross-sectional study suggested that a low serum content of VA is positively associated with diabetic cases exhibiting retinopathy[16]. Currently, there is still no research into the pharmacological activity and mechanisms of VA against diabetic foot ulcers. In practice, an exceptional approach using a network pharmacology analysis can help disclose the predictive targets and biological pathways of an agent used for managing a disorder by database-based bioinformatics and computational analyses[17]. More importantly, in our previous studies using network pharmacology analyses, some bioactive compounds including fucoidan and plumbagin have been revealed to have pharmacological targets and signaling pathways against obesity and nasopharyngeal carcinoma[18–19]. In the current study, we intended to uncover the potential pharmacological targets and molecular mechanisms of VA in regard with its potential clinical application to treating diabetic foot ulcers, by using the network pharmacology strategy.
2 Methods
2.1 Uncovering candidate targets of VA for diabetic foot ulcer treatment
By applying the databases of TCMSP, Drugbank, SuperPred, ChemMapper, BATMAN TCM, and Swiss Target Prediction, all presumptive genes/targets of VA were screened and acquired after data revision using the UniProt database[20]. Meanwhile, the Genecard and OMIM databases were used to unveil diabetic foot ulcer-associated genes. All candidate targets of VA and diabetic foot ulcer targets were subjected to Venn graph mapping via Funrich software (v3.1.3) to identify the potential targets for VA to achieve its anti-diabetic foot ulcer action[21].
2.2 Construction of protein-protein interaction (PPI) and identification of core targets
All mapped targets of VA and diabetic foot ulcers were used to attain a PPI network and raw data (tsv.) via a STRING database (v11.0)[22]. Following Network-Analyzer setting in Cytoscape software (v3.7.1)[23], the core targets for VA action against diabetic foot ulcers were finally identified. The algorithm was based on degree values. The upper limit of filtering range was the maximum degree value in the topology data, whereas the lower limit was the median degree of freedom.
2.3 Core target-based Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses
By using R language-based packages of “ClusterProfiler”, “ReactomePA”, “org.Hs.eg.Db” and “GOplot”, the information of the GO and KEGG pathways from core targets were assayed and visualized. The GO information was created from “org.Hs.eg.Db” with a P-value cut-off = 0.05 and Q-value cut-off = 0.05 when enriched, and the data output is represented as the corresponding bubble, bar, and Circos circle charts. Further, “pathview” package was used in R language to map the relevant targets of the enriched KEGG pathway for pathway visualization.
2.4 Construction of network graph visualization
By applying Cytoscape Software (v3.7.1), the biotargets for VA against diabetic foot ulcers in the GO biological process and enriched pathway were used to construct the graph of the component-target-pathway network.
3 Results
Network pharmacology, including database searching and GO enrichment, KEGG enrichment, and PPI analyses, was used to identify the targets and mechanisms of VA for treating diabetic foot ulcers. The workflow is shown in Fig. 1.
Flowcharts illustrating the network pharmacology approaches and procedures used in the present study to reveal the therapeutic targets and mechanistic pathways of vitamin A against diabetic foot ulcers
3.1 Identification of targets of VA against diabetic foot ulcers
We collected 170 types of pharmaceutical targets for VA from the databases. Meanwhile, we also screened out 2031 diabetic foot ulcer-associated genes. The genes associated with VA and diabetic foot ulcer- were then overlapped, and 66 shared genes/targets of VA against diabetic foot ulcers were identified. Next, PPI of these 66 genes was analyzed (Fig. 2).
The Venn diagram (left) displaying the 66 intersection targets for vitamin A action against diabetic foot ulcers and protein-protein interaction network plot for the targets (right)
3.2 Collection of core targets of VA against diabetic foot ulcers
By obtaining data from Cytoscape Software (v3.7.1), the algorithm for applying topological parameters to harvest the function-associated proteins was used. Consequently, the core targets were collected from the screening qualifications, including JUN, MAPK1, THRB, MAPK14, MTNR1B, CXCR3, ESR1, AR, HDAC1, IL-10, CNR1, DRD2, EGFR, ADRA2A, CCND1, RXRB, RARA, and RXRA (Fig. 3; Supplementary Table S1).
Identification of 18 core targets for VA action against diabetic foot ulcers: JUN, MAPK1, THRB, MAPK14, MTNR1B, CXCR3, ESR1, AR, HDAC1, IL-10, CNR1, DRD2, EGFR, ADRA2A, CCND1, RXRB, RARA, and RXRA
3.3 Findings of biological processes and pathway enrichment analyses
Following the assays using R-language packages, all 18 core targets were used for functional processes and KEGG pathway enrichment analyses. The computed data were plotted as GO-and KEGG-derived bubble diagrams and histograms (Fig. 4). Here, the GO-based biological processes associated with the core targets comprised the following: gland development, response to steroid hormone, DNA-templated transcription, cellular response to steroid hormone stimulus, reproductive structure development, reproductive system development, steroid hormone mediated signaling pathway, transcription initiation from RNA polymerase II promoter, hormone-mediated signaling pathway, maternal process involved in female pregnancy, intracellular receptor signaling pathway, camera-type eye development, female pregnancy, eye development, organ growth, visual system development, sensory system development, multi-multicellular organism processes, muscle cell proliferation, and epithelial cell proliferation. The GO-based cell components contained nuclear chromatin, RNA polymerase II transcription factor complex, transcription factor complex, nuclear transcription factor complex, transcriptional repressor complex, GABA-ergic synapses, integral components of presynaptic membranes, intrinsic components of presynaptic membranes, membrane rafts, membrane microdomains, membrane regions, vesicle lumen, glutamatergic synapses, and ficolin-1-rich granule lumen. In addition, the GO-based molecular functions included nuclear receptor activity, transcription factor activity, direct ligand regulated sequence-specific DNA binding, steroid hormone receptor activity, chromatin DNA binding, transcription coactivator activity, RNA polymerase II transcription factor binding, transcription corepressor activity, MAP kinase activity, adrenergic receptor activity, hormone binding, retinoic acid binding, histone deacetylase binding, RNA polymerase II basal transcription factor binding, catecholamine binding, retinoid binding, isoprenoid binding, phosphatase binding, protein serine/threonine/tyrosine kinase activity, DNA-binding transcription activator activity, and RNA polymerase II-specific. Other detailed information is listed in Supplementary Table 2. As shown in Fig. 5, the 91 KEGG-based pathways characterized with core targets (P < 0.05) optimally contained thyroid hormone signaling pathways, endocrine resistance, Th17 cell differentiation, non-small cell lung cancer, thyroid cancer, the Forkhead box O (FOXO) signaling pathways, estrogen signaling pathways, breast cancer, gastric cancer, prolactin signaling pathway, pertussis, leishmaniasis, colorectal cancer, PD-L1 expression and PD-1 checkpoint pathways in cancer, GnRH signaling pathways, prostate cancer, AGE-RAGE signaling pathways in diabetic complications, Chagas disease (American trypanosomiasis), proteoglycans in cancer, and C-type lectin receptor signaling pathways. Other detailed information is presented in Supplementary Table S3.
Bioinformatics analyses revealing the top biological processes associated with vitamin A action against diabetic foot ulcers
KEGG analysis of the signaling pathways for the pharmacological effects of vitamin A against diabetic foot ulcers
3.4 Construction of network visualization
Using the Cytoscape software (v3.7.1), a VA-target-GO-KEGG-diabetic foot ulcers network using core targets was plotted, which is shown in Fig. 6. All genes/targets enriched in the KEGG pathway were displayed using R-language Software (v3.6.1).
The network visualization of the interactions linking VA to its potential targets, signaling pathways, and diabetic foot ulcers
4 Discussion
Diabetic foot ulcers refer to a terminal syndrome of diabetes that can be markedly lethal[24]. In the clinical setting, the medical diagnosis of diabetic foot ulcers routinely involves ulcerative determination, nervous system assessment, and vascular lesion test[25]. Common clinical practice for the treatment of diabetic foot ulcers includes control of blood sugar, suppression of infection, and supportive treatments for maintaining a stable internal environment[5]. However, the current pharmacological treatment for ulcers is insufficient, with poor efficacy and adverse side effects. Therefore, new drug discovery from bioactive ingredients that have better efficacy and safety profile for diabetic foot ulcers is highly desirable. VA is known to be an antioxidant with inflammation-suppressing and blood glucose level-stabilizing effects[26–27]. Some evidence indicates that VA supplement can relieve diabetic foot ulcers in patients[28]. However, the precise anti-diabetic foot ulcer mechanisms of VA have not yet been deciphered. The present study using network pharmacology analysis revealed the anti-diabetic foot ulcer property of VA (pharmacological activity) and the associated signaling pathways (molecular mechanism). Of note, multiple core targets for VA to produce anti-foot ulcer action were identified, including JUN, MAPK1, THRB, MAPK14, MTNR1B, CXCR3, ESR1, AR, HDAC1, IL-10, CNR1, DRD2, EGFR, ADRA2A, CCND1, RXRB, RARA, and RXRA. Some of these targets, like JUN, MAKP1, and MAKP14, have been reported to be associated with the development of diabetic foot ulcers[5]. JUN is regarded as a determinant of the inflammatory cascade, by transcriptionally controlling the expression of inflammatory cytokines[29]. A study with diabetic skin samples demonstrated that high glucose levels could stabilize the JUN protein[30]. In addition, activation of the MAPK signal transduction pathway, including the upregulation of MAKP1, was found in diabetic foot ulcer tissues[31]. Another member of the MAPK family, MAPK14, was reported as a candidate for genetic susceptibility to diabetic foot ulcers[32], and a genome-wide association study also suggested that MAPK14 was associated with the conditions[33]. Therefore, these genes might be new pharmaceutical targets for the treatment of diabetic foot ulcers.
Results of the GO enrichment analysis further highlighted that the anti-diabetic foot ulcer action of VA might be ascribed to a specific modulation of the molecular processes, cell components, and biological functions for cellular repair, epithelial proliferation and regeneration, suppression of oxidative stress and inflammation cascade action, and the activation of immunological competence. Indeed, the results from the KEGG analysis supported the association of the effects of VA on ulcers with the regulation of the FOXO signaling pathway, Th17 cell differentiation, modulation of the AGE-RAGE signaling pathway in diabetic complications, and endocrine resistance. FOXO signaling was reported to link insulin signaling with plasma lipoprotein metabolism[34]. A study on the circRNA-miRNA-mRNA axis demonstrated the role of FOXO in diabetic foot ulcers[35]. A clinical study with arterial specimens from the feet of patients with diabetes showed a close relationship between high glucose levels and disease progression with the downregulation of Th17 expression[36]. Overall, the findings from our bioinformatics analyses demonstrated that the potential suppression of the inflammatory response and enhancement of immunoregulation of cell proliferation and tissue repair might be the molecular/cellular mechanisms underlying the pharmacological effects of VA against foot ulcers. The identified 18 core biotargets might be the potential molecular markers for detection and diagnosis of diabetic foot ulcers. Yet, further functional characterization is needed to elucidate the roles of these potential VA targets. In addition, VA could be used as an adjuvant of the mainstay medications for diabetic foot ulcers to enhance the therapeutic efficacy in future clinical practice.
5 Conclusion
Collectively, the findings of the present bioinformatics analyses based on a network pharmacology approach uncover the potential biological and core targets, pharmacological functions, and signaling pathways of VA against diabetic foot ulcers. Our results suggest that VA can be used as a promising compound for the treatment of diabetic foot ulcers. However, our findings were solely based upon bioinformatics analyses, and thus, future experimental investigations, preclinical validation, and clinical verification are absolutely required before VA can be used for the treatment of diabetic foot ulcers in the clinical setting.
Conflicts of interests
The authors declare that they have no conflicts of interest.
Author contribution
Li X and Su M contributed to the conception, organization, and execution of the research project. Wan X W, Qin Q H, Xie R T contributed to the statistical analysis along with design, execution, review, and critique. Li X and Su M contributed to manuscript preparation, including the writing of the first draft, review, and critique.
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