Metformin Attenuates TGF-β1-induced Pulmonary Fibrosis Through Inhibition of Transglutaminase 2 and Subsequent TGF-β Pathways

The purpose of this study was to confirm whether metformin can attenuate TGF-β1-induced pulmonary fibrosis through inhibition of transglutaminase 2 (TG2) and subsequent TGF-β pathways. In vitro, MTT assay and Annexin V-FITC/PI staining assay were performed to determine the effect of metformin on the proliferation and apoptosis of human fetal lung fibroblasts (HFL-1 cell). Protein expression of TG2, Collagen I (Col I) and α-smooth muscle actin (α-SMA) were determined by western blot. To further confirm the relationship between TG2 and the anti-fibrotic effect of metformin, TG2 siRNA and TG2 overexpression plasmid were used to interfere the expression of TG2. A bleomycin-induced pulmonary fibrosis model was employed to determine the in vivo inhibitory effect of metformin. The concentrations of TG2, both in supernatants of cells and serum of rats, were determined by ELISA assay. Our results showed that metformin concentration-dependently inhibited the proliferation and promoted the apoptosis of TGF-β1-stimulated HFL-1 cells. The protein expressions of TG2, Col I and α-SMA stimulated by TGF-β1 were decreased after metformin intervention, which was confirmed in both siRNAs and plasmids treatment conditions. In vivo, metformin attenuated bleomycin-induced pulmonary fibrosis as demonstrated by H&E and Masson staining, as well as the protein expressions of Col I and α-SMA. Besides, phosphorylated SMAD2, phosphorylated SMAD3, phosphorylated Akt and phosphorylated ERK1/2 were all significantly increased after bleomycin treatment and decreased to normal levels after metformin intervention. Taken together, our results demonstrated that metformin can attenuate TGF-β1-induced pulmonary fibrosis, at least partly, through inhibition of TG2 and subsequent TGF-β pathways.