Effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar fibroblasts
10.3760/cma.j.cn501225-20231220-00259
- VernacularTitle:二甲双胍对人增生性瘢痕成纤维细胞增殖及纤维化蛋白表达的影响及其机制
- Author:
Wenbo XIE
1
;
Xiaolong HU
;
Shuang WEI
;
Jihong SHI
Author Information
1. 南昌大学江西医学院临床医学系，南昌 330006
- Publication Type:Journal Article
- Keywords:
Metformin;
Cicatrix;
Fibroblasts;
Cell proliferation;
Collagen type Ⅰ;
Collagen type Ⅲ;
Hypertrophic scar;
Phosphatidylinositol 3-kinase/protein kinase B/mam
- From:
Chinese Journal of Burns
2025;41(3):268-276
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar (HS) fibroblasts (Fbs).Methods:The study was an experimental study. From June 2021 to June 2022, 5 patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University, including 3 males and 2 females, aged from 21 to 36 years. HS tissue was collected, Fbs were isolated and cultured, and Fbs of passage 5 to 7 were used for experiment. Fbs were taken and cultured in their respective media supplemented with phosphate buffered solution (PBS) or metformin at final molarities of 5, 10, 20, and 40 mmol/L for 48 hours. The cell proliferation activity was detected using the cell counting kit-8 (CCK-8), and the proliferation inhibition rate of cells was calculated. The content of hydroxyproline in the cell culture supernatant was measured using a hydroxyproline assay kit. The phosphorylation levels of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in the cells were detected by Western blotting, and the ratios of phosphorylated Akt (p-Akt) to Akt and phosphorylated mTOR (p-mTOR) to mTOR were calculated. After 24 hours of culture, the mRNA expressions of type Ⅰ collagen, type Ⅲ collagen, and α-smooth muscle actin (α-SMA) in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Another batch of Fbs were divided into control group (with conventional culture), LY294002 group, metformin group, and LY294002+metformin group. LY294002, metformin, and LY294002+metformin were added to the culture media of the last three groups, respectively, with the final molarities of LY294002 and metformin being 20 μmol/L and 10 mmol/L, respectively. CCK-8 was used to detect the cell proliferation activity at 0 (immediately), 24, and 48 hours of culture. After 48 hours of culture, Western blotting was used to detect the phosphorylation levels of Akt and mTOR in the cells, and the ratios of p-Akt to Akt and p-mTOR to mTOR were calculated. The sample size for the cell proliferation inhibition rate experiment was 4, and the sample size for the other experiments was 3.Results:After 48 hours of culture, compared with the cells treated with PBS, the proliferation inhibition rates of the cells treated with 5, 10, 20, and 40 mmol/L metformin were significantly increased (with t values of 10.69, 14.20, 19.73, and 52.54, respectively, P<0.05), the content of hydroxyproline in the culture supernatants of the cells treated with 10, 20, and 40 mmol/L metformin was significantly decreased (with t values of 8.06, 7.86, and 10.25, respectively, P<0.05), and the ratios of p-Akt to Akt in the cells treated with 10, 20, and 40 mmol/L metformin and the ratios of p-mTOR to mTOR in the cells treated with 20 and 40 mmol/L metformin were significantly decreased (with t values of 2.82, 4.28, 9.88, 5.66, and 9.08, respectively, P<0.05). After 24 hours of culture, compared with those treated with PBS, the mRNA expressions of type Ⅰ collagen and α-SMA in the cells treated with 5, 10, 20, and 40 mmol/L metformin and the mRNA expressions of type Ⅲ collagen in the cells treated with 10, 20, and 40 mmol/L metformin were significantly decreased (with t values of 4.35, 8.53, 9.57, 14.77, 4.14, 5.58, 7.89, 9.37, 5.18, 6.85, and 9.15, respectively, P<0.05). At 24 and 48 hours of culture, the proliferation activities of the cells in LY294002 group (with t values of 6.30 and 13.60, respectively) and metformin group (with t values of 6.47 and 10.69, respectively) were significantly lower than those in control group ( P<0.05). After 48 hours of culture, the ratios of p-Akt to Akt in the cells of LY294002 group and metformin group were 0.554±0.027 and 0.681±0.029, respectively, which were significantly lower than 1.053±0.193 in control group (with t values of 4.45 and 3.31, respectively, P<0.05). The ratio of p-Akt to Akt in the cells of LY294002+metformin group was 0.387±0.023, which was significantly lower than that in metformin group ( t=5.95, P<0.05). After 48 hours of culture, the ratio of p-mTOR to mTOR in the cells of LY294002 group was significantly lower than that in control group ( t=4.01, P<0.05), and the ratio of p-mTOR to mTOR in the cells of LY294002+metformin group was significantly lower than that in metformin group ( t=6.05, P<0.05). Conclusions:Metformin can inhibit the proliferation and expression of fibrotic proteins type Ⅰ collagen, type Ⅲ collagen, and α-SMA of human HS Fbs through phosphatidylinositol 3-kinase/Akt/mTOR signaling pathway.
