J Korean Med Sci.  2016 Sep;31(9):1419-1425. 10.3346/jkms.2016.31.9.1419.

Metformin Reduces Bleomycin-induced Pulmonary Fibrosis in Mice

• Choi SM
• Jang AH
• Kim H
• Lee KH
• Kim YW

• KMID: 2468274
• DOI: http://doi.org/10.3346/jkms.2016.31.9.1419

Abstract

Metformin has anti-inflammatory and anti-fibrotic effects. We investigated whether metformin has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis in a murine model. A total of 62 mice were divided into 5 groups: control, metformin (100 mg/kg), BLM, and BLM with metformin (50 mg/kg or 100 mg/kg). Metformin was administered to the mice orally once a day from day 1. We sacrificed half of the mice on day 10 and collected the bronchoalveolar lavage fluid (BALF) from their left lungs. The remaining mice were sacrificed and analyzed on day 21. The right lungs were harvested for histological analyses. The messenger RNA (mRNA) levels of epithelial-mesenchymal transition markers were determined via analysis of the harvested lungs on day 21. The mice treated with BLM and metformin (50 mg/kg or 100 mg/kg) showed significantly lower levels of inflammatory cells in the BALF compared with the BLM-only mice on days 10 and 21. The histological examination revealed that the metformin treatment led to a greater reduction in inflammation than the treatment with BLM alone. The mRNA levels of collagen, collagen-1, procollagen, fibronectin, and transforming growth factor-β in the metformin-treated mice were lower than those in the BLM-only mice on day 21, although statistical significance was observed only in the case of procollagen due to the small number of live mice in the BLM-only group. Additionally, treatment with metformin reduced fibrosis to a greater extent than treatment with BLM alone. Metformin suppresses the inflammatory and fibrotic processes of BLM-induced pulmonary fibrosis in a murine model.

Keyword

Metformin; Bleomycin; Pulmonary Fibrosis; Mice

MeSH Terms

Animals
Bleomycin/toxicity
Bronchoalveolar Lavage Fluid/chemistry
Collagen Type I/genetics/metabolism
Dose-Response Relationship, Drug
Female
Fibronectins/genetics/metabolism
Lung/pathology
Metformin/*therapeutic use
Mice
Mice, Inbred C57BL
Pulmonary Fibrosis/chemically induced/*prevention & control
RNA, Messenger/metabolism
Tumor Necrosis Factor-alpha/metabolism
Collagen Type I
Fibronectins
RNA, Messenger
Tumor Necrosis Factor-alpha
Bleomycin
Metformin

Figure

• 

  Fig. 1 Experimental protocol of our study. Female C57BL/6 mice were treated with intratracheal instillation of bleomycin (BLM) or saline on day 0. Metformin (50 mg/kg or 100 mg/kg, according to group assignment) or PBS was administered orally once a day from day 1 onward. Half the mice were sacrificed on day 10 and the remaining mice were sacrificed on day 21.

• 

  Fig. 2 Effect of metformin treatment on the total and differential cell counts in the bronchoalveolar fluid. The total and differential cell counts in the bronchoalveolar fluid were determined on days 10 and 21 after the instillation of bleomycin (BLM). Metformin (MFM) was administered orally to mice once a day from day 1 to either day 10 or 21. Data are shown as mean ± SEM. * P < 0.05. (A) Total cell count on day 10. (B) Number of inflammatory cells on day 10. (C) Total cell count on day 21. (D) Number of inflammatory cells on day 21. Day 10: control, MFM, n = 5; BLM, BLM + MFM 50 mg/kg, n = 7; BLM + MFM 100 mg/kg, n = 6 (1 dead). Day 21: control, MFM-only, n = 5; BLM-only, n = 2 (5 dead); BLM + MFM 50 mg/kg, n = 4 (3 dead); BLM + MFM 100 mg/kg, n = 6 (1 dead).

• 

  Fig. 3 Effect of metformin on the infiltration of inflammatory cells and collagen deposition in the lungs of BLM-treated mice. (A) Histologic findings with hematoxylin & eosin (H & E) staining on day 10. (B) Histologic findings with Masson’s trichrome staining on day 10. (C) Histologic findings with H & E staining on day 21. (D) Histologic findings with Masson’s trichrome staining on day 21. (E) Ashcroft fibrosis scores on day 10. (F) Ashcroft fibrosis score on day 21. Representative magnification, × 200.

• 

  Fig. 4 mRNA levels of collagen, collagen-1, procollagen, fibronectin, and TGF-β in lung tissue on day 21. (A) Collagen, (B) collagen-1, (C) procollagen, (D) fibronectin, (E) TGF-β. Day 21: control, MFM, n = 5; BLM, n = 2 (5 dead); BLM + MFM 50 mg/kg, n = 4 (3 dead); BLM + MFM 100 mg/kg, n = 6 (1 dead). *P < 0.05.

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