Fig. 4

Potential mechanism of the antifibrotic effect of metformin on CLAD. a Concentration of fibrotic cytokines (TGF-β1, IL-1β, and TNF-α) in lung was measured using ELISA (pg/ml), which was shown in boxplots. b Western blot analysis of p-Smad2, Smad2, p-Smad3, and Smad3 expression in lung of Syngeneic group (lanes 1–2), Allo-CsA group (lanes 3–4), Allo-NS group (lanes 5–6), Allo-Metformin group (lanes 7–8). β-actin was used as the internal reference. Three PVDF membranes that represent samples of different rats in each group were shown. The gel-distributor scheme of different target proteins and internal reference was based on the molecular weight. Values of protein expression were expressed as fold increase of gray values over that of the syngeneic group (lane 1 in each PVDF membranes) were shown in the boxplots, including p-Smad2, Smad2, p-Smad2/Smad2 ratio, p-Smad3, Smad3, and p-Smad3/Smad3 ratio. c Western blot analysis of p-AMPKα and AMPKα expression in lung homogenate of Syngeneic group (lanes 1–2), Allo-CsA group (lanes 3–4), Allo-NS group (lanes 5–6), Allo-Metformin group (lanes 7–8). β-actin was used as the internal reference. Three PVDF membranes that represent samples of different rats in each group was shown. Values of protein expression were expressed as fold increase of gray values over that of the syngeneic group (lane 1 in each PVDF membranes) were shown in the boxplots, including p-AMPKα, AMPKα, and p-AMPKα/AMPKα ratio. d Representative double-immunofluorescence staining images of lung parenchyma of four groups. Red fluorescence represented p-AMPKα expression, green fluorescence represented α-SMA expression, and blue fluorescence represented nuclei. The objective magnification was 10 × or 40 ×. Scar bar, 100 μm or 20 μm. Gamma: 1.4. The relative expression value of p-AMPK in α-SMA⁺ cells was shown in boxplots. The relative expression value of p-AMPK was represented as mean fluorescence intensity (MFI) of p-AMPKα in the α-SMA⁺ region/ MFI of DAPI in each field of view. One-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple test, *P, 0.05, **P, 0.01, ***P, 0.01. (n = 6 rats / group)