Uncovering the pharmacological mechanisms of Zizhu ointment against diabetic ulcer by integrating network analysis and experimental evaluation in vivo and in vitro

Abstract

Diabetic ulcer (DU) has been recognized as one of the most prevalent and serious complications of diabetes. However, the clinical efficacy of standard treatments for DU remains poor. Traditional Chinese medicine (TCM) shows a positive therapeutic effect on DU. Specifically, Zizhu ointment (ZZO) has been widely used to treat DU in long-term clinical practice, but the exact mechanism by which it promotes DU wound healing remains unknown. In this study, network analysis and high-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS) were conducted to identify the active compounds of ZZO. We detected isovalerylshikonin (ISO), mandenol, daidzein, kaempferol, and formononetin in both network analysis and UPLC-HRMS. Moreover, ZZO could ameliorate DU by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and inflammation signaling pathways, according to the results of KEGG analysis. We established a DU mouse model with a high-fat diet and streptozotocin injection in vivo to evaluate the network analysis result. The experimental results showed that ZZO could inhibit inflammation, remodel fibrous tissue, and promote angiogenesis in the DU area, facilitating wound healing in DU mice. Moreover, the PI3K/AKT signaling pathway was indeed activated by ZZO treatment, promoting macrophage M2 polarization. In addition, we used molecular docking technology to evaluate the binding sites between ZZO and the PI3K/AKT pathway. The results showed that ISO has a good binding interaction with AKT. Moreover, ISO promoted M2 polarization in macrophages in a dose-dependent manner in vitro. Our study found that ZZO could promote DU wound healing by inhibiting inflammation, which was achieved by macrophage M2 polarization through activating the PI3K/AKT pathway. Further studies have demonstrated that ISO plays major role in the above process. These findings provide a theoretical basis for further preclinical evaluation and lay a foundation for nano-gel compound treatment with ZZO.

Identifying and Validating GSTM5 as an Immunogenic Gene in Diabetic Foot Ulcer Using Bioinformatics and Machine Learning

“…They were maintained on a 12-hour light/dark cycle and provided unrestricted access to both water and food. 27 The mice were allocated randomly into distinct groups (n=4), which included the non-diabetic group treated with phosphate-buffered saline (PBS), and the DFU group. For a duration of three weeks, the DFU group of mice were fed a high-fat diet constituting 60% of their total calorie intake (FB-D12451, obtained from Wuxi Fan Bo Biotechnology Co., Ltd.), and within one week, they were subjected to intraperitoneal injections of STZ (40 mg/kg, Cat.…”

“… 31 Quantification of wound closure rate was performed using ImageJ software (Bethesda, MD) and calculated as follows: t-t 0 /t 0 × 100% (t: evaluation of wound healing, t0: initial injury). 27 …”