Effect and mechanism of Andrias davidianus skin mucopolysaccharides on full-thickness skin defect wound healing in diabetic mice
10.3760/cma.j.cn501225-20240725-00280
- VernacularTitle:大鲵皮肤黏液多糖对糖尿病小鼠全层皮肤缺损创面愈合的作用及其机制
- Author:
Weiming GOU
1
;
Peng YANG
;
Yifei LU
;
Xiaorong ZHANG
;
Yiming QIN
;
Jingyuan LI
;
Yong HUANG
;
Qing ZHANG
;
Gaoxing LUO
Author Information
1. 陆军军医大学（第三军医大学）第一附属医院全军烧伤研究所，创伤与化学中毒全国重点实验室，重庆 400038
- Publication Type:Journal Article
- Keywords:
Polysaccharides;
Macrophages;
Neovascularization, physiologic;
Diabetes mellitus, experimental;
Andrias davidianus;
Wound repair
- From:
Chinese Journal of Burns
2025;41(2):127-136
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect and mechanism of Andrias davidianus skin mucopolysaccharides (ASMP) on full-thickness skin defect wound healing in diabetic mice. Methods:This study was an experimental study. The ASMP with polysaccharide content of (70.0±0.3)% was prepared; the proliferation activity of human umbilical vein endothelial cells (HUVECs) was detected by cell counting kit-8, showing that the optimal concentration of ASMP was 0.05 mg/mL. The HUVECs were taken and divided into blank control group, vascular endothelial growth factor (VEGF) group, and ASMP group according to the random number table method (the same grouping method below), which were cultured with conventional medium and the media containing 50 ng/mL VEGF and 0.05 mg/mL ASMP, respectively, and then cultured under hypoxic (with volume fraction of oxygen being 5%) and normal-oxygen conditions for 12 hours, and the length of tube formation was observed. Human monocytic leukemia cells were induced with phorbol ester to differentiate into M0 macrophages. These cells were then divided into blank control group, lipopolysaccharide (LPS) group, and ASMP group, which were cultured respectively using conventional medium, LPS-containing medium followed by conventional medium, and LPS-containing medium followed by 0.05 mg/mL ASMP-containing medium. After 48 hours of culture, the expressions of CD86 and CD206 proteins (expressed as relative fluorescence intensity, the same below) were measured by immunofluorescence, and the mRNA expression levels of arginase-1 (Arg1) and CD206 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Eighteen male C57 mice aged 8-10 weeks were used, and diabetic model was successfully established using streptozotocin combined with a high-fat and high-sugar diet. Full-thickness skin defect wounds were created on the backs of the mice, and the mice were divided into blank control group, alginate dressing group, and ASMP group (with 6 mice in each group), which were treated with physiological saline, alginate dressing, and ASMP, respectively. Wound healing was observed on post injury day (PID) 3, 7, 10, and 14, and the wound healing rates of mice were calculated. On PID 7, the expressions of CD31 and CD206 proteins in the wound tissue of mice were observed by immunofluorescence. On PID 14, the thickness of granulation tissue in wounds of mice was observed by hematoxylin-eosin staining. The sample size for all experiments was 3.Results:After 12 hours of culture in normal-oxygen condition, compared with that in blank control group, the tube formation length of HUVECs in VEGF and ASMP groups was significantly increased (with q values of 10.08 and 16.91, respectively, P<0.05). After 12 hours of culture in hypoxic condition, compared with that in blank control group, the tube formation length of HUVECs in VEGF and ASMP groups was significantly increased (with q values of 11.61 and 16.91, respectively, P<0.05); compared with that in VEGF group, the tube formation length of HUVECs in ASMP group was significantly increased ( q=5.30, P<0.05). After 48 hours of culture, the relative fluorescence intensity of CD206 protein in M0 macrophages in ASMP group was 31.90±1.76, significantly higher than 1.00±0.25 in blank control group and 2.21±0.42 in LPS group (with q values of 50.75 and 48.75, respectively, both P values <0.05); the relative fluorescence intensity of CD86 protein was 5.82±0.63, significantly lower than 53.73±4.61 in LPS group ( q=30.90, P<0.05). After 48 hours of culture, the mRNA expressions of Arg1 and CD206 in M0 macrophages in ASMP group were significantly higher than those in blank control group (with q values of 35.02 and 13.09, respectively, P<0.05) and LPS group (with q values of 32.24 and 11.24, respectively, P<0.05). On PID 3, there was no statistically significant difference in intercomparison in the wound healing rate of mice among the blank control, alginate dressing, and ASMP groups ( P>0.05). Compared with those in blank control group, the wound healing rates of mice in alginate dressing group on PID 10 and 14 were significantly increased (with q values of 11.76 and 12.50, respectively, P<0.05), and the wound healing rates of mice in ASMP group on PID 7, 10, and 14 were significantly increased (with q values of 5.84, 15.90, and 14.96, respectively, P<0.05); compared with those in alginate dressing group, the wound healing rates of mice in ASMP group on PID 7 and 10 were significantly increased (with q values of 4.77 and 4.14, respectively, P<0.05). On PID 7, the relative fluorescence intensity of CD31 protein in wound tissue of mice in alginate dressing and ASMP groups was significantly stronger than that in blank control group (with q values of 7.63 and 16.85, respectively, P<0.05); the relative fluorescence intensity of CD31 protein in wound tissue of mice in ASMP group was significantly stronger than that in alginate dressing group ( q=9.22, P<0.05). On PID 7, the relative fluorescence intensity of CD206 protein in wound tissue of mice in alginate dressing and ASMP groups was significantly stronger than that in blank control group (with q values of 8.76 and 29.36, respectively, P<0.05), and the relative fluorescence intensity of CD206 protein in wound tissue of mice in ASMP group was significantly stronger than that in alginate dressing group ( q=20.61, P<0.05). On PID 14, the wound granulation tissue of mice in ASMP group was thicker compared with that in blank control group and alginate dressing group. Conclusions:ASMP can significantly enhance the ability of new blood vessel formation and optimize the immune microenvironment by promoting HUVEC tube formation as well as inducing macrophages to polarize toward the M2 type, thereby accelerating full-thickness skin defect wound healing in diabetic mice.
