Influences and mechanism of berberine on wound healing of full-thickness skin defects in diabetic mice
10.3760/cma.j.cn501225-20230411-00120
- VernacularTitle:小檗碱对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制
- Author:
Liming ZHENG
1
;
Zhongyuan LIU
;
Hongyu YAN
;
Hengfei LI
;
Zhiwen ZHANG
Author Information
1. 湖北中医药大学针灸骨伤学院，武汉 430061
- Keywords:
Berberine;
Diabetes mellitus, experimental;
Fibroblasts;
Cytokines;
Wound repair
- From:
Chinese Journal of Burns
2023;39(11):1072-1082
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the influences and mechanism of berberine on wound healing of full-thickness skin defects in diabetic mice.Methods:The experimental research method was adopted. Mouse dermal fibroblasts (MDF) conventional-glucose complete medium (hereinafter referred to as conventional medium) were prepared with final mass concentrations of berberine of 0 (no berberine), 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, 80.00, and 160.00 μg/mL, respectively. Primary MDF were cultured using conventional medium and MDF high-glucose (30 mmol/L glucose) complete medium (hereinafter referred to as high-glucose medium), and the 3 rd to 6 th passage cells were collected for the following experiments. Cells cultured in conventional medium were taken and subjected to starvation treatment for 12 hours, and then cultured in conventional media containing different concentrations of berberine for 48 hours to screen out the optimal working concentration of berberine using the cell counting kit 8 (CCK-8), the sample number was 6, and the selected optimal berberine concentration was used for subsequent cell culture experiments. Cells cultured in 2 media were taken, of which the cells cultured in conventional medium were included to the normal control group; cells cultured in high-glucose medium were divided into high-glucose alone group and high-glucose+berberine group according to the random number table (the same grouping method below). After 48 h of cultivation, cell viability was detected by CCK-8, cell migration capacity was evaluated by scratch test and Transwell assays, and mRNA and protein expression levels of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β 1 (TGF-β 1), matrix metalloproteinase 9 (MMP-9), and cysteine aspartic acid specific protease (caspase-3) in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting, respectively, and the sample numbers of the aforementioned experiments were 6, 3, 9, 3, and 6, respectively. Fifteen 8-week-old male BALB/c mice were used to establish diabetic mouse model, then full-thickness skin defect wounds on their backs were made and divided into diabetes alone group, diabetes+low-concentration berberine group (25 μg/mL), and diabetes+high-concentration berberine group (75 μg/mL) for corresponding treatments, with 5 mice in each group. The wound areas were measured using ImageJ software on post injury day (PID) 0 (immediately), 3, 7, 14, and 21. On PID 21, histological changes and collagen formation in the wound tissue were detected by hematoxylin-eosin and Masson staining, respectively, protein expression and mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, CD31, and caspase-3 in the wound tissue were detected by immunohistochemistry and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, the sample number of animal experiments was all 5. Data were statistically analyzed with one-way analysis of variance, independent sample t-test, Tukey's test, and factorial design analysis of variance. Results:After 48 hours of cultivation, the cell viability was the highest when the mass concentration of berberine was 20.00 μg/mL. After 48 h of cultivation, compared with that in normal control group, cell viability in both high-glucose alone group and high-glucose+berberine group reduced significantly ( P<0.05); compared with that in high-glucose alone group, the cell viability in high-glucose+berberine group was significantly enhanced ( P<0.05). After 48 h of cultivation, scratch test results showed that, the cell migration rates in 24 h in both high-glucose alone group and high-glucose+berberine group were significantly decreased than that in normal control group ( P<0.05); compared with that in high-glucose alone group, the cell migration rate in 24 h in high-glucose+berberine group was significantly enhanced ( P<0.05). After 48 h of cultivation, the results of Transwell experiments showed that, compared with (141±7) of cells migrating in 24 h in normal control group, the number of cells migrating in 24 h in high-glucose alone group and high-glucose+berberine group were 28±3 and 86±6, respectively, which were significantly decreased ( P<0.05); compared with that in high-glucose alone group, the number of cells migration in 24 h in high-glucose+berberine group was significantly increased ( P<0.05). After 48 h of cultivation, compared with those in normal control group, the mRNA levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose alone group and high-glucose+berberine group were significantly decreased ( P<0.05), the mRNA levels of caspase-3 were significantly increased ( P<0.05); compared with those in high-glucose alone group, the mRNA levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose+berberine group were significantly increased ( P<0.05), the mRNA expression level of caspase-3 was significantly decreased ( P<0.05). After 48 h of cultivation, compared with those in normal control group, the protein expression levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose group and high-glucose+berberine group were significantly decreased ( P<0.05), the protein expression levels of caspase-3 were significantly increased ( P<0.05); compared with those in high-glucose alone group, the protein expression levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose+berberine group were significantly increased ( P<0.05), and the protein expression level of caspase-3 was significantly decreased ( P<0.05). Compared with those in diabetes alone group, the wound areas of mice in diabetes+low-concentration berberine group on PID 14 and 21 and in diabetes+high-concentration berberine group on PID 3, 7, 14, and 21 were significantly decreased ( P<0.05); compared with that in diabetes+low-concentration berberine group, the wound area in diabetes+high-concentration berberine group was significantly decreased on PID 3, 7, 14, and 21 ( P<0.05). On PID 21, the wound of mice in diabetes alone group was not epithelialized with a large number of inflammatory cells and granulation tissue in the dermis; most of the wound tissue of mice in diabetes+low-concentration berberine group was already epithelialized, although there was a large number of inflammatory cells in the dermis; and most of the wound tissue of mice in diabetes+high-concentration berberine group had completed epithelialization with a small number of hair follicles and inflammatory cells in the dermis. On PID 21, compared with that in diabetes alone group, the collagen area of wound of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group was significantly increased ( P<0.05); compared with that in diabetes+low-concentration berberine group, the collagen area of wound of mice in diabetes+high-concentration berberine group was significantly increased ( P<0.05). On PID 21, compared with those in diabetes alone group, the protein expression levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group were significantly increased ( P<0.05), the protein expression levels of caspase-3 were significantly decreased ( P<0.05); compared with those in diabetes+low-concentration berberine group, the protein expression levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+high-concentration berberine group were significantly increased ( P<0.05), the protein expression level of caspase-3 was significantly decreased ( P<0.05). On PID 21, compared with those in diabetes alone group, the mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group were significantly increased ( P<0.05), the mRNA levels of caspase-3 were significantly decreased ( P<0.05); compared with those in diabetes+low-concentration berberine group, the mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+high-concentration berberine group were significantly increased ( P<0.05), the mRNA level of caspase-3 was significantly decreased ( P<0.05). Conclusions:Berberine can promote the proliferation and migration of MDF and the healing of full-thickness skin defect wounds of mice in diabetic mice by up-regulating the expression of biofactors including MMP-9, PDGF, TGF-β 1, and VEGF, and down-regulating the expression of caspase-3, a pro-apoptotic factor in wound tissue of mice.
