A preliminary study on the inhibitory effect of gallic acid on the growth of human keloid fibroblasts by the transforming growth factor-β/Sma- and Mad-related proteins signaling pathway
10.35541/cjd.20230331
- VernacularTitle:五倍子酸通过TGF-β/Smads信号通路对人瘢痕成纤维细胞生长抑制效应的初步研究
- Author:
Jiaxi QIAO
1
;
Yao CHEN
;
Kun DU
;
Liuqing CHEN
;
Jinbo CHEN
;
Li WEI
Author Information
1. 武汉市第一医院皮肤科，武汉 430000
- Keywords:
Keloid;
Fibroblasts;
Cell proliferation;
Apoptosis;
Transforming growth factor beta;
Smad proteins;
Gallic acid;
TGF-β/Smads signaling pathway
- From:
Chinese Journal of Dermatology
2023;56(12):1138-1145
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of gallic acid on the morphology, proliferation and cell cycle of keloid fibroblasts, as well as on collagen contraction and the transforming growth factor-β (TGF-β) /Sma- and Mad-related proteins (Smads) signaling pathway, and to explore the role and mechanisms of action of gallic acid in the treatment of keloids.Methods:From August to December 2022, 3 keloid tissue samples were collected from 3 patients with clinically and pathologically confirmed keloids after surgery in the Department of Dermatologic Surgery, Wuhan No.1 Hospital. Primary fibroblasts were isolated and cultured by using the tissue culture method, and 3- to 8-passage fibroblasts were used for subsequent experiments. Cultured keloid fibroblasts were divided into 4 groups: low-, medium- and high-dose gallic acid groups treated with 0.025, 0.05 and 0.1 mg/ml gallic acid respectively, and a control group cultured with Dulbecco′s modified Eagle′s medium (DMEM) containing 10% fetal calf serum. After 24-, 48-, and 72-hour treatment, cellular proliferative activity was evaluated by cell counting kit 8 (CCK8) assay, and collagen contraction by using a three-dimensional culture method. After 24-hour treatment in the above groups, pictures were taken using a differential interference inverted fluorescence microscope, and changes in the cell cycle were analyzed by flow cytometry. Some keloid fibroblasts were divided into 2 groups: an experimental group (high-dose gallic acid group) treated with 0.1 mg/ml gallic acid, and a control group cultured with DMEM containing 10% fetal calf serum. After 24-hour treatment, enzyme-linked immunosorbent assay (ELISA) was performed to determine the changes in supernatant concentrations of TGF-β1, TGF-β2, and TGF-β3 in the two groups, real-time fluorescence-based quantitative PCR to detect the relative mRNA expression levels of TGF-β1, TGF-β2, TGF-β3, Smad2, Smad3, Smad4, and α-smooth muscle actin (α-SMA). Statistical analysis was carried out using t test, one-way analysis of variance and two-way analysis of variance, and least significant difference (LSD) - t test was used for multiple comparisons. Results:Compared with the control group, the gallic acid groups showed gradual changes in the shape of keloid fibroblasts under the microscope as the dose of gallic acid increased, including gradually shrinking cell bodies, enlarged intercellular spaces, cell atrophy, increased number of apoptotic cells, etc. CCK8 assay showed that the cellular proliferative activity changed significantly as the dose of gallic acid increased and the treatment time was prolonged ( Fgroup = 78.31, P < 0.001; Ftime = 4.17, P = 0.037), and the proliferative activity of keloid fibroblasts was significantly lower in the high-dose gallic acid group than in the control group at 24, 48, and 72 hours (all P < 0.05). The three-dimensional culture showed that different degrees of collagen contraction occurred in all groups over time, marked collagen contraction was observed in the control group, and a lower degree of collagen contraction in the gallic acid groups; the collagen contraction indices were significantly lower in the medium- and high-dose gallic acid groups than in the control group at 24, 48, and 72 hours (all P < 0.05). Flow cytometry showed that the cell apoptosis rates were significantly higher in the low-, medium- and high-dose gallic acid groups (38.68% ± 3.05%, 41.82% ± 2.19%, 43.56% ± 3.58%, respectively) than in the control group (12.58% ± 1.56%, all P < 0.001) after 24-hour treatment; compared with the control group, the medium- and high-dose gallic acid groups showed significantly decreased proportions of cells in the G0/G1 phase (both P < 0.01), but significantly increased proportions of cells in the S phase and G2/M phase (all P < 0.05). ELISA revealed that the TGF-β1 concentration was significantly lower in the high-dose gallic acid group (758.58 ± 31.42 pg/ml) than in the control group (1 081.30 ± 44.72 pg/ml, t = 11.81, P<0.001), there was no significant difference in the TGF-β2 concentration between the high-dose gallic acid group (71.05 ± 7.40 pg/ml) and the control group (76.43 ± 6.51 pg/ml, t = 1.09, P = 0.317), while the TGF-β3 concentration was significantly higher in the high-dose gallic acid group (5.70 ± 3.87 pg/ml) than in the control group (0.00 ± 0.00 pg/ml, t = 2.94, P = 0.026). As real-time fluorescence-based quantitative PCR revealed, the high-dose gallic acid group showed significantly decreased mRNA expression levels of TGF-β1, Smad2, Smad3, Smad4, and α-SMA (all P < 0.05), but significantly increased mRNA expression level of TGF-β3 ( t = 6.78, P = 0.002) compared with the control group; however, there was no significant difference in the TGF-β2 mRNA expression level between the above two groups ( t = 0.05, P = 0.962) . Conclusion:Gallic acid could change the cell cycle, inhibit the proliferative activity, promote apoptosis and change the shape of keloid fibroblasts, and thus inhibit scar formation and contraction, which may be related to the inhibition of TGF-β/Smads signaling pathway.
