Pulmonary fibrosis is a common complication with low morbidity and high mortality in chronic lung diseases. Unfortunately, the conventional treatments, such as glucocorticoids combined with immuno-suppressive agents, can not effectively improve the prognosis of the patients with pulmonary fibrosis [2, 23]. Two antifibrotic agents (nintedanib and pirfenidone) have been demonstrated their efficacy in phase clinical III since 2015, but they simply slowed the progression of idiopathic pulmonary fibrosis in mild to moderate patients [8, 45]. Moreover, the cost of nintedanib and pirfenidone is too expensive for ordinary people to afford, each at a cost of almost $100 000 per patient per year [35, 38]. Therefore, to clarify its pathogenesis and find more effective and inexpensive drugs is an important issue that needs to be addressed in the treatment of pulmonary fibrosis. In the present study, we employed BMP9 to stimulate human lung fibroblast HFL-1 cells and found that metformin suppressed BMP9-induced proliferation and differentiation of HFL-1 cells through the activation of AMPK and inhibition of ALK1/Smad1/5 signal.
Metformin, a first-line drug for the clinical treatment of type 2 diabetes, is widely used in metabolic diseases due to its high effectiveness, safety and low cost. Increasing experimental and clinical studies have shown that metformin has multiple health benefits in many disorders [11, 19, 29, 36, 42, 43 ]. For instance, metformin was found to have anti-cancer, anti-aging and anti-oxidant effects. Recently, several studies demonstrated that metformin efficiently alleviated various pulmonary fibrosis, including bleomycin-, radiation- and PM2.5-induced fibrotic models [3, 7, 12, 15, 47]. In vitro experiments found that metformin inhibited TGF-β, especially TGF-β1, -induced fibroblast activation . Thus, these findings indicate that metformin might be also an important medication against fibrosis. Moreover, the existing research found that metformin exhibited pleiotropic mechanisms for alleviating lung fibrosis, such as NOX4 inhibition [18, 40], IGF-1 suppression , and mTOR inactivation [37, 41], and so on. In this study, our aims were to determine whether metformin exerted its effect on the activation of lung fibroblast by regulating AMPK and BMP9 signaling. As we expected, metformin inhibited BMP9-induced proliferation and differentiation via AMPK activation in HFL-1 cells. Furthermore, this finding was verified by the specific AMPK mutants (AMPK-CA and AMPK-DN). It suggests that metformin activates AMPK, and then restrains BMP9-mediated proliferation and differentiation of HFL-1 cells.
The activation of fibroblasts has been considered as a crucial mechanism that drives the development of pulmonary fibrosis. Abnormality of BMP9 signaling has been recently found to have closely relationship with fibroblast activation and fibrotic diseases. As an example, aberrant BMP9 signaling has been reported to induce the activation of hepatic stellate cells and promote the progression of liver fibrosis [5, 24], but to hinder cardiac fibroblast activation and delay cardiac remodeling and myocardial fibrosis [30, 31], indicating that the response of BMP9 to fibroblast activation varies with cell types. In our study, we demonstrated that BMP9 promoted the proliferation and up-regulated the expression of differentiation markers α-SMA, collagen I and collagen III in HFL-1 cells, indicating BMP9 is an effective inducer for the activation of lung fibroblasts. So far, ALK1 has been regarded as the highest affinity receptor of of BMP9 ligand in most types of cells. In most circumstances, BMP9 primarily binds to its receptor ALK1, and then phosphorylates the downstream transcriptional factor Smad1/5. A variety of studies reported that Smad1/5 was activated in fibrotic diseases. During hepatic fibrosis, for instance, ligand BMP9 induced the expression of inhibitor of differentiation 1 via the Smad1 pathway, thereby triggering hepatic stellate cells to differentiate into myofibroblasts . In scleroderma fibroblast, ALK1/Smad1/5 was reported to promote the production of extracellular matrix proteins such as collagen I and connective tissue growth factor . Consistent with these studies, our research indicated that BMP9 increased the expressions of ALK1 and p-Smad1/5, thereby activated lung fibroblasts.
A couple of evidence has shown that AMPK counter-regulates the TGF-β and BMP signaling pathway in several biological events. As an example, AMPK was reported to inhibit Smad2/3 activity, cell migration and epithelial-mesenchymal transition induced by TGF-β . Besides, one investigation confirmed that metformin suppressed the osteogenic differentiation induced by BMP6 via ALK2/Smad1/5 signaling , while another investigation found that metformin inhibited BMP9-induced tube formation of HUVECs via ALK1/Smad1/5 pathway . Additionally, adiponectin regulated the proliferation of pulmonary artery smooth muscle cells through AMPK-regulated BMP/Smad signal pathway . These results remind us that whether AMPK inhibits the TGF-β/Smad or BMP/Smad-mediated response is a general mechanism. Hence, we explored the effect of AMPK on the BMP9-mediated lung fibroblast activation in our study. Similar to the above studies, our results uncovered that an active mutant of AMPK (AMPK-CA) imitated the effect of metformin and reduced the expressions of ALK1 and p-Smad1/5 evoked by BMP9, whereas this effect of metformin were rescued by the dominant negative mutant of AMPK (AMPK-DN). Thus, the present study indicates that metformin suppresses BMP9-induced proliferation and differentiation of lung fibroblasts through activating AMPK and hindering ALK1/Smad1/5 signalling, which provides a novel explanation for anti-fibrotic mechanism of metformin.
In summary, this study shows for the first time that metformin can inhibit the proliferation and differentiation of HFL-1 cells induced by BMP9. Furthermore, the present study confirms that metformin counteracts ALK1/Smad1/5 signal pathway to regulate its inhibitory effect on activation of HFL-1 cells via activating AMPK. Therefore, our present study delineates the effect of metformin on the activation of lung fibroblasts and the relationship between AMPK and BMP9 signal during this process, strongly supporting the notion that metformin might serve as a preventive and therapeutic agent for lung fibrosis. And also, this study may reinforce our understanding of the anti-fibrotic mechanism of metformin, and provides more theoretical and experimental basis for the clinical application of metformin in the treatment of pulmonary fibrosis. To be sure, there are several limitations in the present study. For instance, the study just focused on in vitro experiment to explore the function of metformin on the activation of HFL-1 cells and its mechanism. Accordingly, we should further validate the above results in other cell lines, primary cultured cells, in vivo or clinical samples. In addition, we should continue to verify the regulation of AMPK to ALK1 by interfering ALK1 expression.