Abstract: BACKGROUND: Inflammation and oxidative stress contribute to the barriers of regeneration in chronic wound. Oxymatrine has various biological activities, such as anti-oxidation, anti-inflammation and so on, which may have the potential effect of promoting wound healing.
OBJECTIVE: To investigate the effect of oxymatrine on wound healing and the protective effect on H2O2-induced oxidative stress injury in human keratinoid cell line HaCaT cells.
METHODS: (1) In vivo experiment: Hyaluronic acid methacryloyl hydrogels containing 0, 0.05, 0.1, 0.2 g/L oxymatrine were prepared. A full-layer skin defect model with a diameter of 12 mm was made in the back of 75 diabetic mice and randomly divided into five groups for intervention, with 15 mice in each group. The wounds of the model group were bandaged and fixed. The wounds of the hydrogel group were covered with hyaluronic acid methacryloyl hydrogel. The wounds of the low-dose, moderate-dose and high-dose oxymatrine groups were covered with hyaluronic acid methacryloyl hydrogel containing 0.05, 0.1, and 
0.2 g/L oxymatrine, respectively, and then bandaged and fixed after light curing. Relevant indicators were detected within 14 days. (2) In vitro experiment: Human keratinocyte line HaCaT was divided into five groups. The normal group was cultured conventionally. H2O2 group and low-, moderate- and high-concentration oxymatrine groups were treated with H2O2 for 4 hours, and then the medium was replaced with medium containing 0, 0.05, 0.1, and 0.2 g/L oxymatrine, respectively, and the relevant indexes were detected after 24 hours of culture.
RESULTS AND CONCLUSION: (1) In vivo experiment: Compared with the model group, the wound healing rate of mice in the hydrogel group had no significant change. The wound healing rate of mice in the low-, moderate- and high-dose oxymatrine group was increased at 7 and 14 days after treatment (P < 0.05). Pathological observation of wound section 14 days after treatment showed that compared with the model group, the thickness of regenerated epidermal layer, the number of microvessels, and collagen deposition in the moderate- and high-dose oxymatrine groups were increased (P < 0.05). Western blot assay analysis of wound samples 7 days after surgery showed that compared with the model group, the protein expressions of tumor necrosis factor α and interleukin 6 in the moderate- and high-dose oxymatrine groups were decreased (P < 0.05). (2) In vitro experiment: CCK8 assay, EdU and Ki67 staining showed that compared with the H2O2 group, the cell proliferation ability of the moderate- and high-concentration oxymatrine groups was significantly increased (P < 0.05). Compared with the H2O2 group, mitochondrial membrane potential was increased (P < 0.05) and reactive oxygen species content was decreased (P < 0.05) in the moderate- and high-concentration oxymatrine groups. Western blot assay results showed that compared with the H2O2 group, the expression levels of Nrf2 nuclear protein, Nrf2 total protein, HO-1 protein, and superoxide dismutase 1 protein were increased in the high-concentration oxymatrine group (P < 0.05). (3) These findings confirm that oxymatrine can alleviate oxidative stress damage in HaCat cells and accelerate wound healing by upregulating the levels of Nrf2 and HO-1 protein. 

Key words: oxymatrine, hydrogel, antioxidant, wound healing, Nrf2/HO-1 signaling pathway, HaCat cell