AUTHOR=Wang Jie , Wang Yu , Huang Renyan , Li Wenhui , Fan Weijing , Hu Xiaoming , Yang Xiao , Han Qiang , Wang Hongfei , Liu Guobin 

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

JOURNAL=Frontiers in Pharmacology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.1027677

DOI=10.3389/fphar.2022.1027677

ISSN=1663-9812

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