Metformin prevents peritendinous fibrosis by inhibiting transforming growth factor-β signaling

Abstract

Injury-induced peritendinous adhesion is a critical clinical problem that leads to tendon function impairment. Therefore, it is very urgent to explore potential approaches to attenuate peritendinous adhesion formation. Recently, several studies have demonstrated the biological effect of metformin in inhibiting multiple tissue fibrosis. In this study, we performed in vitro and in vivo experiments to examine whether metformin prevents injury-induced peritendinous fibrosis. We found that tendon injury induced severe fibrosis formation in rats. However, orally administered metformin significantly alleviated the fibrosis based on macroscopic and histological evaluation. Peritendinous tissue from metformin-treated rats also showed decreased expression of fibrotic genes including col1a1, col3a1, and α-smooth muscle actin (α-SMA), and inhibition of transforming growth factor (TGF)-β1 signaling. The cell counting kit (CCK)-8, flow cytometry, and 5-ethynyl-2'-deoxyuridine (EdU) staining analyses showed that treatment of NIH/3T3 fibroblasts with metformin significantly inhibited excessive cell proliferation and promoted cell apoptosis. Metformin treatment also inhibited the expression of fibrotic genes and decreased the phosphorylation of smad2/3 and extracellular signal-regulated kinase (ERK) 1/2. Furthermore, blocking AMP-activated protein kinase (AMPK) signaling abolished the inhibitory effect of metformin on fibrosis. Our findings indicate that metformin has a protective role against peritendinous tissue fibrosis and suggest its clinical use could be a promising therapeutic approach.

Keywords: AMPK; TGF-β; metformin; peritendinous fibrosis.

Figure 1. Metformin treatment reduces peritendinous tissue adhesion in tendon-injured rats

(A) Macroscopic images showed less adhesive tissues in metformin-treated (200 mg/kg/d) rats. (B) Gross adhesion scores (left) were significantly lower in metformin-treated rats compared with control rats 3 weeks after tendon operation (2.8 vs 3.9, p = 0.005). Maximal tensile strengths (right) were comparable between control and metformin-treated rats (23 vs 21 N, p = 0.173) (n = 8 for each group). (C) HE staining of peritendinous tissues showed reduced cell proliferation and inflammation by metformin treatment. Arrows indicate adhesion formation. Scale bars (left) = 250 μm. Scale bars (right) = 100 μm. (D) Sirius-red staining revealed decreased collagen deposition by metformin treatment. Arrows indicate excessive collagen deposition. Scale bars (left) = 250 μm. Scale bars (right) = 100 μm. (E) Histological scores for adhesion decreased after metformin treatment (2.4 vs 3.8, p = 0.002). Histological healing scores were comparable between control and metformin-treated rats (2.6 vs 2.8, p = 0.667) (n =5 for each group). (F) Metformin treatment significantly deceased the hydroxyproline (Hyp) content of tendon tissues at 3 weeks (184 vs 268 ug/ml, p = 0.011) (n = 5 for each group). Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01; N.S. not significant.

Figure 2. Metformin inhibits cell proliferation and TGF-β1 signaling pathway in vivo

(A) Ki67 assay showed that metformin inhibited abnormal cell proliferation (brown) in peritendinous tissues. Scale bar = 50 μm. (B) Immunohistochemistry (IHC) for α-SMA (brown) (b) showed significantly reduced positive cells in metformin-treated rats. Scale bar (left) = 100 μm. Scale bar (right) = 50 μm. The real-time PCR (C) and western blot (D) analysis showed that both mRNA and protein levels of col1a1, col3a1 and α-SMA in peritendinous tissues decreased after metformin treatment. (E) The western blot analysis showed that metformin treatment significantly inhibited canonical (SMAD) and noncanonical (MAPK) TGF-β signaling pathways. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01.

Figure 3. Metformin induces progressive apoptosis in vitro

(A) NIH/3T3 fibroblasts were treated for 24h, 48h, 72h, and 96h, respectively. Cell Counting Kit-8 (cck8) assay showed that cell viability was repressed by metformin treatment. (B) Metformin induced a significantly increased apoptosis assessed by flow cytometry in fibroblasts. The histogram showed the percentage of apoptotic cells. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01.

Figure 4. Metformin inhibited fibroblast proliferation in vitro

(A) EdU staining was used to detect proliferative fibroblasts (red). Metformin markedly reduced the percentage of proliferative cells. (B) fibroblasts were treated as indicated for 48h and analyzed by flow cytometry to estimate the distribution of fibroblasts in each phase of the cell cycle. TGF-β1 treatment significantly increased S and G2/M phase cells, whereas metformin attenuated the transition. The histogram represents the percentage of cells in each cell cycle phase. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01.

Figure 5. Metformin decreases TGF-β1-induced adhesion in vitro

(A) Immunofluorescent (IF) staining showed that the expression of α-SMA positive (red) fibroblasts was inhibited by metformin treatment for 24 h. scale bar = 50 μm. (B) The real-time PCR analysis showed that mRNA expression levels of col1a1, col3a1 and α-SMA in NIH/3T3 fibroblasts were inhibited by metformin treatment. (C) The western blot analysis showed that protein levels of fibrotic genes were inhibited by metformin treatment. Metformin treatment increased phosphorylation of AMPK, and inhibited both SMAD and MAPK signaling pathways in NIH/3T3 fibroblasts. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01.

Figure 6. Activation of AMPK signaling pathway inhibited TGF-β1 induced fibrosis in fibroblasts

(A) AICAR (1 mM) markedly decreased the mRNA levels of col1a1, col3a1 and α-SMA induced by TGF-β1 in NIH/3T3 fibroblasts. (B) AICAR markedly decreased the protein levels of col1a1, col3a1 and α-SMA induced by TGF-β1 in NIH/3T3 fibroblasts. Both the SMAD and MAPK signaling pathways were inhibited by AICAR treatment in NIH/3T3 fibroblasts. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01.

Figure 7. Inhibitory effect of metformin on peritendinous fibrosis was blocked by Compound C

All the NIH/3T3 fibroblasts were pretreated with Compound C (1 μM) for 24 h before further treated with TGF-β1 (2 ng/ml) and/or metformin (5 mM). (A) The real-time PCR analysis showed that metformin was unbale to down-regulated the mRNA levels of col1a1, col3a1 and α-SMA in fibroblasts pretreated with Compound C. (B) The western blot analysis showed that metformin was unbale to down-regulated the protein levels of col1a1, col3a1 and α-SMA in fibroblasts pretreated with Compound C. Compound C abolished the down-regulation of metformin on SMAD and MAPK signaling pathways. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01. N.S. not significant.

Figure 8. Small interfering-RNA (siRNA) targeting AMPK blocked inhibitory effect of metformin on decreasing expression of fibrotic genes and TGF signaling pathway

All the NIH/3T3 fibroblasts were pre-transfected with scramble siRNA or AMPK siRNA. (A) Metformin was unable to down-regulated the mRNA levels of col1a1, col3a1 and α-SMA in fibroblasts pre-transfected with AMPK siRNA. (B) Metformin significantly reduced the protein levels of col1a1, col3a1 and α-SMA in fibroblasts pre-transfected with scramble siRNA. However, the inhibitory effect was abolished by AMPK siRNA. (C) In fibroblasts pretreated with AMPK siRNA, metformin was unable to inhibited SMAD or MAPK signaling pathways. Data are expressed as means ± SEM. ^* P < 0.05; ^** P < 0.01. N.S. not significant.