Effects and mechanism of baicalin on wound healing of full-thickness skin defects in diabetic mice
10.3760/cma.j.cn501225-20231104-00179
- VernacularTitle:黄芩素对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制
- Author:
Yan SHI
1
;
Liang YI
;
Weiqiang ZHANG
;
Nike LIU
;
Huicai WEN
;
Ronghua YANG
Author Information
1. 南昌大学第一附属医院烧伤整形与创面修复医学中心，南昌 330006
- Keywords:
Diabetes mellitus;
Macrophages;
Reactive oxygen species;
Baicalin;
Wound repair;
Inflammatory response
- From:
Chinese Journal of Burns
2024;40(11):1085-1094
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects and mechanism of baicalin on the wound healing of full-thickness skin defects in diabetic mice.Methods:This study was an experimental research. Mononuclear cells were isolated from five male C57BL/6J mice aged 8-12 weeks and induced to differentiate into macrophages for conducting the subsequent experiments. According to the random number table (the same grouping method below), macrophages in a high-glucose environment were divided into 0 μmol/L baicalin group (no baicalin was added), 5 μmol/L baicalin group, 15 μmol/L baicalin group, 25 μmol/L baicalin group, 50 μmol/L baicalin group, and 75 μmol/L baicalin group treated with the corresponding final molarity of baicalin and 1 μg/mL endotoxin/lipopolysaccharide (LPS). After treatment for 48 hours, the cell proliferation activity was detected using a microplate reader. Macrophages in a high-glucose environment were divided into LPS group treated with 1 μg/mL LPS and LPS+baicalin group treated with 50 μmol/L baicalin+1 μg/mL LPS. After treatment for 48 hours, the percentage of double-positive cells for inducible nitric oxide synthase (iNOS) and CD80, as well as that for arginase 1 (Arg1) and CD206 among the cells, were detected using immunofluorescence method, the secretion levels of interleukin 1β (IL-1β), IL-6, IL-23, IL-10, insulin-like growth factor (IGF), and transforming growth factor β 1 (TGF-β 1) by the cells were detected using enzyme-linked immunosorbent assay, the expression of reactive oxygen species in the cells was detected using a fluorescent probe method, the protein expression of nuclear factor κB in the cells were detected using Western blotting, and the expression of nuclear factor 2 in the cells was observed using immunofluorescence method. The number of cell experimental samples was 3. Twenty-four 8-week-old male db/db mice were selected. After preparing full-thickness skin defect wounds on their backs, they were divided into baicalin group and normal saline group (with 12 mice in each group). On the third day after injury, 50 μmol/L baicalin and normal saline were injected into the wounds of mice, respectively. The wound healing situation was observed and the percentage of the residual wound area was calculated on the 4 th, 8 th, and 12 th day after injury. The wound tissue was sampled on the 8 th day after injury, hematoxylin-eosin staining was performed to observe the epithelial regeneration and inflammatory cell infiltration, the protein expression of CD31 was detected by Western blotting, and the expression of reactive oxygen species was detected by a microplate reader. The number of animal experimental samples was 6. Results:After treatment for 48 hours, only the proliferation activity of macrophages in 50 μmol/L baicalin group was significantly higher than that in 0 μmol/L baicalin group ( P<0.05). After treatment for 48 hours, the percentage of double-positive cells for iNOS and CD80 among the macrophages in LPS+baicalin group was (21.0±2.4)%, which was significantly lower than (66.6±4.5)% in LPS group ( t=15.63, P<0.05); the percentage of double-positive cells for Arg1 and CD206 among the macrophages in LPS+baicalin group was (59.1±2.1)%, which was significantly higher than (18.6±1.7)% in LPS group ( t=25.38, P<0.05); compared with those in LPS group, the secretion levels of IL-1β, IL-6, and IL-23 by the macrophages in LPS+baicalin group were significantly decreased (with t values of 14.26, 15.95, and 12.23, respectively, P<0.05), while the secretion levels of IL-10, IGF, and TGF-β 1 were significantly increased (with t values of 8.49, 11.98, and 13.84, respectively, P<0.05); the expression of reactive oxygen species in the macrophages in LPS+baicalin group was significantly lower than that in LPS group ( t=5.54, P<0.05); compared with those in LPS group, the protein expression of nuclear factor κB in the nucleus of the macrophages in LPS+baicalin group was significantly decreased ( t=36.22, P<0.05), while that in the cytoplasm was significantly increased ( t=14.47, P<0.05), and the expression of nuclear factor 2 in the nucleus was increased. On the 4 th and 8 th day after injury, the wound area of mice in baicalin group was significantly smaller than that in normal saline group, and the wounds of mice in baicalin group completely healed on the 12 th day after injury. On the 4 th, 8 th, and 12 th day after injury, the residual wound area percentage of mice in baicalin group was significantly lower than that in normal saline group (with t values of 13.29, 10.08, and 11.72, respectively, P<0.05). On the 8 th day after injury, compared with those in normal saline group, the wound tissue of mice in baicalin group showed significant re-epithelization, the infiltration of inflammatory cells was reduced, the expression of CD31 protein was significantly increased ( t=17.23, P<0.05), and the expression of reactive oxygen species was significantly reduced ( t=5.78, P<0.05). Conclusions:Baicalin alleviates the inflammatory response of macrophages by lowering the level of reactive oxygen species in cells and promoting the polarization of macrophages to the M2 type, thereby facilitating the healing of full-thickness skin defect wounds in diabetic mice.
