Objective To explore the mechanism of Wenyang Shengji Ointment (温阳生肌膏, WYSJO) in the treatment of diabetic wounds from the perspective of network pharmacology, and to verify it by animal experiments.

Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and related literature were used to screen active compounds in WYSJO and their corresponding targets. GeneCards, Online Mendelian Inheritance in Man (OMIM), DrugBank, PharmGkb, and Therapeutic Target Database (TTD) databases were employed to identify the targets associated with diabetic wounds. Cytoscape 3.9.0 was used to map the active ingredients in WYSJO, which was the diabetic wound target network. Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) platform was utilized to construct protein-protein interaction (PPI) network. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed to identify signaling pathways between WYSJO and diabetic wounds. AutoDock 1.5.6 was used for molecular docking of core components in WYSJO to their targets. Eighteen rats were randomly divided into control, model, and WYSJO groups (n = 6). The model and WYSJO groups were used to prepare the model of refractory wounds in diabetes rats. The wound healing was observed on day 0, 5, 9, and 14 after treatment, and the wound tissue morphology was observed by hematoxylin-eosin (HE) staining. The expression levels of core genes were detected by quantitative real-time polymerase chain reaction (qPCR).

Results A total of 76 active compounds in WYSJO, 206 WYSJO drug targets, 3 797 diabetic wound targets, and 167 diabetic wound associated WYSJO targets were screened out through network pharmacology. With the use of WYSJO-diabetic wound target network, core targets of seven active compounds encompassing quercetin, daidzein, kaempferol, rhamnetin, rhamnocitrin, strictosamide, and diisobutyl phthalate (DIBP) in WYSJO were found. GO enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve lipopolysaccharide, bacteria-derived molecules, metal ions, foreign stimuli, chemical stress, nutrient level, hypoxia, and oxidative stress in the biological processes. KEGG enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve advanced glycation end products (AGE-RAGE), p53, interleukin (IL)-17, tumor necrosis factor (TNF), hypoxia inducible factor-1 (HIF-1), apoptosis, lipid, atherosclerosis, etc. The results of animal experiments showed that WYSJO could significantly accelerate the healing process of diabetic wounds (P < 0.05), alleviate inflammatory response, promote the growth of granulation tissues, and down-regulate the expression levels of eight core genes histone crotonyltransferase p300 (EP300), protoc gene-oncogene c-Jun (JUN), myelocytomatosis (MYC), hypoxia inducible factor 1A (HIF1A), mitogen-activated protein kinase 14 (MAPK14), specificity protein 1 (SP1), tumor protein p53 (TP53), and estrogen receptor 1 (ESR1) predicted by the network pharmacology (P < 0.05).

Conclusion  The mechanism of WYSJO in treating diabetes wounds may be closely related to AGE-RAGE, p53, HIF-1, and other pathways. This study can provide new ideas for the pharmacological research of WYSJO, and provide a basis for its further transformation and application.