Zizhu Ointment Accelerates Wound-Healing of Diabetic Ulcers through Promoting M2 Macrophage Polarization via Downregulating the Notch4 Signaling Pathway

Abstract

Objective: The long-term clinical practice shows that Zizhu ointment (ZZO) is an empirical formula for the treatment of diabetic ulcers (DUs). In this study, we investigated the underlying mechanism of ZZO in the treatment of DU mice.

Methods: Through streptozotocin induction and high-fat diet, a DU mouse model was established and ZZO was given for treatment. The activation of Notch4 signaling was examined by immunofluorescence staining, RT-PCR, as well as Western blotting. Flow cytometry was performed to detect the counts of F4/80+ cells, M1 and M2 macrophages, as well as the expression of CD11c, CD206, etc. The role of Notch4 in wound healing in diabetic mice was verified by Notch4 inhibitors and agonists.

Results: Accelerated wound healing and decreased expression levels of Notch4 and its target genes and ligands were observed in diabetic mice treated with ZZO. ZZO promoted M2 macrophage polarization, downregulated the expression of proinflammatory factors, and upregulated the levels of anti-inflammatory factors. The same tendency was observed in diabetic mice after treatment with Notch4 inhibitors. Knockout of Notch4 accelerated the wound healing rate as well.

Conclusions: ZZO accelerates wound healing of diabetic mice through inhibiting the activation of Notch4 signaling, promoting M2 macrophage polarization, and alleviating inflammation.

Figure 1

Zizhu ointment promotes diabetic wound healing in STZ-induced diabetes in mice. (a) Representative photos of mice wounds on days 0, 3, 7, and 14 in different groups. (b) The rate of wound closure on days 3, 7, and 14 in different groups. (c) Pictures HE staining of the liver and kidney in different groups. ^∗∗p < 0.01 vs control group; ^#p < 0.05 vs diabetic ulcer group; ^##p < 0.01 vs diabetic ulcer group.

Figure 2

Zizhu ointment inhibiting the activation of Notch4 in skin tissue of diabetic mice. (a) Immunofluorescence of Notch4 protein in different groups. (b) Western blotting results of Notch1 and Hes1. (c) Quantitative PCR results of Notch4, Hes1, Hey1, dll4, and Jagged1. ^∗∗p < 0.01 vs control group; ^#p < 0.05 vs diabetic ulcer group; ^##p < 0.01 vs diabetic ulcer group.

Figure 3

Zizhu ointment treatment promotes M2 macrophage polarization. (a) The count of F4/80⁺ cells in different groups. (b) The M1/M2 ratio in different groups. (c) Relative expression of proinflammatory markers IL-6, MCP-1, iNOS, and TNF-α. (d) Relative expression levels of anti-inflammatory factors IL-4, CD206, ARG1, and IL-10. Data were expressed as mean ± standard deviation (SD); ^∗p < 0.01 vs control group; ^#p < 0.05 vs diabetic ulcer group; ^##p < 0.01 vs diabetic ulcer group.

Figure 4

Inhibition of Notch4 is beneficial to the healing of diabetic ulcer. (a) Representative images of wound healing in different treatment groups. (b) Wound closure analyzed on days 3, 7, and 14 by Image J (n = 10). (c) The count of F4/80⁺ cells in different groups. (d) The ratio of M1/M2 in different groups. (e) Relative expression of IL-6 MCP-1, iNOS, and TNF-α. (f) Relative expression of anti-inflammatory factors IL-4, CD206, ARG1, and IL-10. ^∗p < 0.05 vs control group; ^∗∗p < 0.01 vs control group; ^#p < 0.05 vs diabetic ulcer group; ^##p < 0.01 vs diabetic ulcer group.

Figure 5

Zizhu ointment accelerates wound healing in diabetic ulcer mice by suppressing the expression of Notch4 and promoting M2 macrophage polarization. (a) Western blotting results of Notch4 in control and Notch4 knockout groups. (b) Representative images of wounds healing after different treatments. (c) Wound closure on days 3, 7, and 14 by Image J (n = 10). (d) The count of F4/80⁺ cells in different groups. (e) The ratio of M1/M2 in different groups. (f) Relative expression of IL-6 MCP-1, iNOS, and TNF-α. (g) Relative expression of IL-4, CD206, ARG1, and IL-10. ^∗p < 0.05 vs control group; ^∗∗p < 0.01 vs control group; ^#p < 0.05 vs diabetic ulcer group; ^##p < 0.01 vs diabetic ulcer group.