Chitosan Regulates CAV1 to Facilitate M2 Macrophage Differentiation through Activation of Canonical Wnt Signaling Pathway in Diabetic Skin Trauma Model Rats

Background: Chitosan has the advantage of improving inflammatory response. However, the molecular mechanism by which chitosan derivatives promote macrophage proliferation is not fully illuminated. Here, we focused on the Caveolin 1 (CAV1)/Wnt family member (Wnt) regulatory axis to study the effect of chitosan on wound healing of diabetic skin ulcer.Methods: We prepared Sprague Dawley (SD) rat models of diabetic (DM), DM with chronic refractory wound (CW), and CW with chitosan treatment (CTS). Flow cytometric assay was used to isolate macrophage from epidermis of skin tissues with chronic wound. RNA sequencing was used to study the transcriptome of M0 and M2 macrophages. Immunoprecipitation and western blot were used to study Wnt/beta-catenin signaling pathway and the target gene Caveolin 1 (Cav1) of M0 macrophages. Molecular docking was used to mimic the protein-protein interaction between CAV1 and beta-catenin.Results: We observed that chitosan was capable of facilitating the differentiation process of M0 to M2 macrophage in wound area. Total 734 differentially expressed genes (318 elevated and 416 reduced) were found compared between CTS and CW. Cav1 was significantly up-regulated in chitosan treated M0 macrophages. Furthermore, we noticed that the delivery of chitosan particle containing with Cav1 shRNA could enhance the phosphorylation of glycogen synthase kinase 3 beta (GSK3 beta) and the followed activity of canonical Wnt pathway in M0 macrophages in vivo. Both C-terminus of CAV1 and GSK3 beta shown by protein docking remodeling indicated the interaction of these two proteins.Conclusions: Current study determines that chitosan promotes the expression of Cav1, and enhances the activity of GSK3 beta function through canonical Wnt/beta-catenin signaling pathway.