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.

这项研究的目的是确认二甲双胍是否可以通过抑制转谷氨酰胺酶2（TG2）和随后的TGF-β途径来减轻TGF-β1诱导的肺纤维化。在体外，进行了MTT测定和膜联蛋白V-FITC / PI染色测定，以确定二甲双胍对人胎儿肺成纤维细胞（HFL-1细胞）增殖和凋亡的影响。通过蛋白质印迹法测定TG2，胶原蛋白I（Col I）和α-平滑肌肌动蛋白（α-SMA）的蛋白表达。为了进一步证实TG2与二甲双胍的抗纤维化作用之间的关系，使用了TG2 siRNA和TG2过表达质粒来干扰TG2的表达。使用博来霉素诱导的肺纤维化模型确定二甲双胍的体内抑制作用。大鼠细胞上清液和血清中TG2的浓度，通过ELISA测定法测定。我们的研究结果表明二甲双胍浓度依赖性地抑制TGF-β1刺激的HFL-1细胞的增殖并促进其凋亡。二甲双胍干预后，TGF-β1刺激的TG2，Col I和α-SMA的蛋白表达降低，这在siRNA和质粒处理条件下均得到证实。在体内，通过H＆E和Masson染色以及Col I和α-SMA的蛋白表达证明，二甲双胍减轻了博来霉素诱导的肺纤维化。此外，博来霉素治疗后，磷酸化的SMAD2，磷酸化的SMAD3，磷酸化的Akt和磷酸化的ERK1 / 2均显着升高，而二甲双胍干预后降至正常水平。两者合计，我们的结果表明二甲双胍可以减轻TGF-β1诱导的肺纤维化，