Up-regulation of caveolin 1 mediated by chitosan activates Wnt/ β-catenin pathway in chronic refractory wound diabetic rat model

Diabetes mellitus (DM) can be implicated in the perturbations of vascular integrity and the dysfunction of angiogenesis. Chitosan has the advantage of promoting the vascular endothelial cell proliferation. However, the molecular mechanism of action in the promotion of wound healing by chitosan derivatives is still debated. In the current study, DM with chronic wound (CW) model rats were prepared and treated with chitosan. Vascular endothelial cells isolated from granulation tissues were conducted by RNA sequencing. Two thousand three hundred and sixteen genes were up-regulated, while 1,864 genes were down-regulated after chitosan treatment compared to CW group. Here, we observed that caveolin 1 (CAV1) was highly expressed induced by chitosan. Furthermore, we observed that CAV1 knockdown could compromise the activation of Wnt pathway by reduction of β-catenin in rat aortic endothelial cells (RAOECs) and brain endothelium four cells (RBE4s). Moreover, we determined a direct interaction between CAV1 and β-catenin by IP assay. The C-terminus of CAV1 and β-catenin (24 to 586 amino acids) contributed to the interaction of these two proteins. Finally, the protein docking analysis indicated that the fragments of β-catenin (253–261 ‘FYAITTLHN’ and 292–303 ‘KFLAITTDCLQI’) might have affected the structure by CAV1 and facilitated the resistance to degradation. Taken together, our study demonstrates that chitosan can up-regulate CAV1 expression, and CAV1 can interact with β-catenin for promotion of canonical Wnt signaling pathway activity. Our results deepens the molecular mechanism of the Wnt pathway in vascular endothelial cells and is beneficial to developing new targets to assist in enhancing the pharmacological effect of chitosan on wound healing and angiogenesis against DM.

糖尿病（Diabetes mellitus, DM）可引发血管完整性受损与血管生成功能障碍。壳聚糖（chitosan）具有促进血管内皮细胞增殖的特性。然而，壳聚糖衍生物促进伤口愈合的分子作用机制仍存在争议。
本研究构建了合并慢性伤口（Chronic wound, CW）的糖尿病模型大鼠，并采用壳聚糖进行干预处理。从该模型大鼠肉芽组织中分离的血管内皮细胞被用于RNA测序（RNA sequencing）分析。与CW组相比，壳聚糖处理后共有2316个基因上调、1864个基因下调。
本研究观察到，壳聚糖可诱导小窝蛋白1（Caveolin 1, CAV1）高表达。进一步实验证实，在大鼠主动脉内皮细胞（Rat aortic endothelial cells, RAOECs）与脑内皮细胞4系（Rat brain endothelium four cells, RBE4s）中，敲低CAV1可通过降低β-连环蛋白（β-catenin）的表达水平，削弱Wnt通路的激活。
此外，本研究通过免疫沉淀（IP）实验证实，CAV1与β-连环蛋白之间存在直接相互作用。CAV1的C端结构域与β-连环蛋白的24至586位氨基酸区段参与了二者的结合。
最后，蛋白对接（protein docking）分析结果显示，β-连环蛋白的253–261位氨基酸序列"FYAITTLHN"与292–303位氨基酸序列"KFLAITTDCLQI"片段可与CAV1结合，进而影响其蛋白结构并延缓其降解过程。
综合来看，本研究证实壳聚糖可上调CAV1的表达，而CAV1可通过与β-连环蛋白相互作用，促进经典Wnt信号通路的激活。本研究结果加深了对血管内皮细胞中Wnt通路调控机制的理解，可为开发新型靶点以增强壳聚糖在糖尿病合并慢性伤口治疗中促进伤口愈合与血管生成的药理作用提供理论依据。