Influence and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts

Wu Menglu; Wang Rui; Zheng Xinnan; Wu Juan; He Lin; Diao Jiansheng; Shu Maoguo; Du Huicong

doi:10.3760/cma.j.cn501225-20241216-00489

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Wu Menglu,Wang Rui,Zheng Xinnan,et al.Effects and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts[J].Chin J Burns Wounds,2025,41(4):1-9.DOI: 10.3760/cma.j.cn501225-20241216-00489.

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Influence and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts

doi: 10.3760/cma.j.cn501225-20241216-00489

Department of Plastic, Cosmetic and Maxilofacial Surgery, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China

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General Program of the Shaanxi Provincial Key Research and Development Plan in the Field of Social Development 2020SF-155

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Corresponding author: Du Huicong, Email: dhc0309@126.com
Received Date: 2024-12-16
Available Online: 2025-03-27

Abstract

Abstract

Objective To investigate the influence and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts (Fbs). Methods This study was an experimental research. The keloid tissue was collected from 7 keloid patients (2 males and 5 females, aged 20–65 years, with a disease course of more than 1 year) who underwent keloid excision surgery at the Department of Plastic, Cosmetic and Maxillofacial Surgery of the First Affiliated Hospital of Xi'an Jiaotong University from September 2020 to September 2023. The primary Fbs were isolated and cultured, and cells from passages 3 to 6 were used for experiments. The cells were divided into control group and metformin group, which were cultured in complete medium and complete medium supplemented with metformin at a final molarity of 60 mmol/L, respectively. The cell counting kit-8 was used to assess the proliferation activity of cells in two groups after 12 and 24 hours of culture, and the proliferation inhibition rate of cells in metformin group after 12 and 24 hours of culture was calculated. The apoptosis detection kit was used to detect the apoptotic distribution of cells in control group after 0 hour (immediately) of culture and in metformin group after 12 and 24 hours of culture. The cell cycle detection kit was used to detect the cycle distribution of cells in two groups after 12 and 24 hours of culture. The eukaryotic mRNA sequencing was performed on cell samples of two groups collected after 24 hours of culture, and the Kyoto encyclopedia of genes and genomes functional annotation analysis and functional enrichment analysis were performed after screening for differentially expressed genes (DEGs) with significant differential expression. Western blotting was conducted to detect the protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt), and phosphorylated mammalian target of rapamycin (p-mTOR) in the PI3K/Akt/mTOR signaling pathway of cells in two groups after 24 hours of culture. The sample size for proliferation activity detection was 6, while all other measurements were conducted with a sample size of 3. Results After 12 and 24 hours of culture, the proliferation activity of cells in metformin group was significantly lower than that in control group (with t values of 4.70 and 24.02, respectively, P<0.05); the proliferation activity of cells in metformin group after 24 hours of culture was significantly lower than that after 12 hours of culture (t=4.73, P<0.05). Compared with that after 12 hours of culture, the proliferation inhibition rate of cells in metformin group was significantly increased after 24 hours of culture (t=5.29, P<0.05). Compared with that in control group after 0 hour of culture, the proportion of early apoptotic cells in metformin group was significantly increased (with t values of 6.62 and 4.58, respectively, P<0.05), and the proportion of early and late apoptotic cells also significantly increased after 12 and 24 hours of culture (with t values of 4.84 and 3.75, respectively, P<0.05). After 24 hours of culture, the proportion of late apoptotic cells in metformin group was significantly higher than that after 12 hours of culture (t=4.55, P<0.05). After 12 hours of culture, the proportion of S-phase cells in metformin group was significantly lower than that in control group (t=5.90, P<0.05). After 24 hours of culture, compared with that in control group, the proportion of G0/G1-phase cells in metformin group was significantly increased (t=5.36, P<0.05), while the proportion of G2/M-phase cells was significantly decreased (t=17.63, P<0.05). Additionally, the proportion of S-phase cells in metformin group after 24 hours of culture was significantly higher than that after 12 hours of culture (t=7.60, P<0.05). After 24 hours of culture, a total of 4 814 DEGs were identified from cells in metformin group compared with control group. The significantly upregulated and downregulated DEGs were mainly involved in biological functions related to signal transduction, cell growth and death, transport and catabolism, the endocrine system, the digestive system, and cancer. The significantly differentially expressed DEGs were significantly enriched in pathways such as cell cycle and DNA replication, with the highest number of genes in the PI3K/Akt signaling pathway. After 24 hours of culture, the protein expressions of PI3K, p-Akt, and p-mTOR of cells in metformin group were 0.190±0.017, 0.170±0.017, and 0.247±0.005, respectively, which were significantly lower than 0.440±0.026, 0.300±0.060, and 0.547±0.025 in control group (with t values of 13.69, 3.61, and 20.12, respectively, P values all <0.05). Conclusions Metformin can significantly inhibit the proliferation of human keloid Fbs through the PI3K/Akt/mTOR signaling pathway, while effectively inducing its apoptosis process, thereby exerting antifibrotic effects.
- Keloid,
- Metformin,
- Fibroblasts,
- Cell proliferation,
- Apoptosis,
- Eukaryotic mRNA sequencing,
- Phosphatidylinositol 3-kinase /protein kinase B/mammalian target of rapamycin signaling pathway

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