Metformin attenuates motility, contraction, and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase

Figure 1 Effect of metformin in CCl₄-induced fibrotic mice. A fibrotic mouse model was induced by intraperitoneal injection of CCl₄ (1 μL/g) dissolved in olive oil (CCl₄:olive oil = 1:1, v/v) twice per week for 6 weeks. A and B: Histological changes were assessed by hematoxylin-eosin (H-E) staining and Sirius Red (S-R) staining (100 × magnification); C-E: Expression levels of α-SMA, fibronectin, and VEGF in the liver tissues were measured by immunohistochemistry (100 × magnification). Sirius Red staining was analyzed with ImageJ and immunohistochemical staining was analyzed with Image-Pro Plus 6.0. (Scale bar = 200 μm, n = 5, ^bP < 0.01 vs the control group, ^dP < 0.01 vs the CCl₄ group).

Figure 2 Effect of metformin on the activation, proliferation, and extracellular matrix secretion of hepatic stellate cells. A: Measurement of α-SMA levels in murine liver tissues by Western blot; B: Measurement of hepatic α-SMA and collagen type I mRNA expression levels by quantitative real-time PCR (n = 5, ^bP < 0.01 vs the control group, ^dP < 0.01 vs the CCl₄ group); C and D: HSCs were treated with or without 10 ng/mL PDGF-BB for 24 h, and the effect of metformin (1, 2, 5, and 10 mmol/L) on the expression levels of α-SMA, collagen type I, and fibronectin (FN) were measured by Western blot (^aP < 0.05 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB only group); E: HSCs were treated with a series of concentrations ranging from 1 mmol/L to 100 mmol/L of metformin for 24 h, and the proliferation was measured by CCK-8 assays. HSCs: Hepatic stellate cells.

Figure 3 Effect of metformin on hepatic stellate cell migration and invasion. Scratch tests were used to determine cell migration, and Transwell assays were used to evaluate cell invasion. A: HSCs were scraped and then incubated with or without PDGF-BB (10 ng/mL) and metformin (1, 2, 5, and 10 mmol/L). Images were acquired at 0 and 24 h (100 × magnification); B: HSCs were seeded in the upper chamber with a Matrigel membrane, and various concentrations of metformin (0, 1, 5, and 10 mmol/L) were added to the medium. The lower chambers were loaded with DMEM with or without 10% FBS. Cells that migrated through the membrane were fixed and stained with hematoxylin at 24 h; C: The migration ability was quantified by measuring the distance of the scratch edge; D: Cells that migrated through the membrane were counted and quantified. (^bP < 0.01 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB or FBS only groups). HSCs: Hepatic stellate cells.

Figure 4 Effect of metformin on hepatic stellate cell contraction. Collagen gels were prepared in 24-well plates. A: HSCs were seeded on the collagen gel with or without PDGF-BB (10 ng/mL) and metformin (1, 5, and 10 mmol/L); B: Metformin was changed to AICAR (500 μmol/L), LY294002 (20 μmol/L), and PD98059 (10 μmol/L); C and D: After incubation for 24 h, the areas of the collagen gel were measured for analysis. (^bP < 0.01 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB only group). HSCs: Hepatic stellate cells.

Figure 5 Effect of metformin on VEGF expression and secretion of hepatic stellate cells and angiogenesis in vitro. A and B: HSCs were incubated with or without PDGF-BB (10 ng/mL) for 24 h or CoCl₂ (150 μmol/L) for 12 h and metformin (1, 2, 5, and 10 mmol/L). The expression levels of HIF-1α and VEGF were measured by Western blot analysis, and the results were quantified; C: Cells were treated as in panel B, and the supernatant was collected. The protein level of VEGF was measured by ELISA assay; D and E: HSCs were pretreated with metformin (5 and 10 mmol/L) or AICAR (500 μmol/L) for 2 h, and then incubated with or without CoCl₂ (150 μmol/L) for 12 h. The supernatant was collected and diluted 4:1 (v/v) in DMEM with 10% FBS to form conditioned medium. HUVECs were harvested and suspended in the conditioned medium, and then seeded on Matrigel. Images were acquired at 8 h (100 × magnification), and tube lengths were calculated with ImageJ and quantified. ^aP < 0.05 and ^bP < 0.01 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB or CoCl₂ only groups. HSCs: Hepatic stellate cells.

Figure 6 Effect of metformin on AMPK, Akt/mTOR, and ERK signaling in hepatic stellate cells. A and B: HSCs were pretreated with metformin (1, 2, 5 and 10 mmol/L) for 2 h and then incubated with PDGF-BB (10 ng/mL) for 24 h or CoCl₂ (150 μmol/L) for 12 h. AMPK, Akt/mTOR, and ERK signaling pathways were assessed by Western blot analysis; C and D: The results were quantified. ^aP < 0.05 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB or CoCl₂ only groups. HSCs: Hepatic stellate cells.

Figure 7 The inhibitory effects of metformin on activated hepatic stellate cells were associated with activation of AMPK and subsequent down-regulation of the Akt/mTOR and ERK signaling pathways. A and B: HSCs were pretreated with AICAR (500 μmol/L), LY294002 (20 μmol/L), PD98059 (10 μmol/L), or rapamycin (100 nmol/L) for 2 h and then incubated with PDGF-BB (10 ng/mL) for 24 h. Expression levels of fibronectin (FN), collagen type I (COL1A2), and α-SMA were measured by Western blot analysis and the results were quantified; C and D: HSCs were pretreated with AICAR (500 μmol/L), LY294002 (20 μmol/L), PD98059 (10 μmol/L), or rapamycin (100 nmol/L) for 2 h and then incubated with or without CoCl₂ (150 μmol/L) for 12 h. Expression levels of HIF-1α and VEGF were measured by Western blot analysis; E and F: The results were quantified. (^aP < 0.05 and ^bP < 0.01 vs the control group, ^cP < 0.05 and ^dP < 0.01 vs the PDGF-BB or CoCl₂ only groups). HSCs: Hepatic stellate cells.