DPP4 inhibitors have attracted much attention since their anti-fibrosis effects have been reported. Here, we further confirmed the protective effects of linagliptin on pulmonary fibrosis in SSc mouse model and revealed that suppression of EndMT, oxidative stress, inflammation are important mechanisms underlying the effects.
Intratracheal administration of BLM cause severe acute lung injury followed by pulmonary fibrosis and inflammation in mice [23], which is the most commonly used animal model for studying idiopathic pulmonary fibrosis. Subcutaneous injection of BLM also induce pulmonary fibrosis with a chronic lung injury and more insidiously progressive course, which was more similar to pulmonary fibrosis in SSc patients, including patchy interstitial fibrosis and mononuclear cell infiltration. Furthermore, Yoshizaki A et al have reported that SSc-related antibodies such as anti-Scl-70, anti-U1RNP and anti-CENP B were positive in the sera of mice treated with subcutaneous injection of BLM [23]. Therefore, subcutaneous injection of BLM may better mimic SSc-related fibrosis. And it has been commonly used to study skin and pulmonary fibrosis associated with SSc [23, 24, 25, 26, 27]. So we treated mice with BLM subcutaneously to induce pulmonary fibrosis in the present study. The results showed that subcutaneous BLM administration caused loss of normal lung structure, thickened alveolar walls and increased collagen fiber content, accompanied by significant weight loss. These BLM-induced effects were alleviated by linagliptin treatment. Lung hydroxyproline content was also remarkably reduced by linagliptin treatment. The results further validated the protective effects of linagliptin on pulmonary fibrosis, which was consistence with previous studies [13, 15].
Myofibroblasts are the main orchestrators of fibrosis by synthesizing and releasing a large amount of extracellular matrix proteins, leading to structure destruction and function loss of normal tissues and organs [28]. Myofibroblasts emerge from several sources, including expansion and activation of quiescent resident tissue fibroblasts [29], migration and tissue accumulation of bone marrow-derived circulating CD34+ fibrocytes [30] and epithelial cells or perivascular cells (pericytes) that have undergone a phenotypic transition into mesenchymal cells [31]. EndMT is another important source of myofibroblasts newly discovered in recent years [8]. In this complex biological process, endothelial cells lose their endothelial characteristics and gain mesenchymal cell characteristics, including a spindle-shaped cell morphology, loss of polarity and intercellular junctions, and enhanced migration ability [32]. EndMT also results in the initiation of expression of mesenchymal cell specific proteins, such as α-SMA, vimentin,N-cadherin. Subsequently, the involvement of EndMT in SSc related lung diseases was also confirmed [9]. Especially in SSc, the occurrence of EndMT not only makes endothelial cells an additional source of myofibroblasts, but also indirectly leads to a defective angiogenesis, an important pathogenesis in SSc [33]. Therefore, EndMT has become an interesting target for SSc treatment. Vildagliptin, a DPP4 inhibitor, have been reported to improve lipopolysaccharide-induced lung injury by inhibiting EndMT [34]. Therefore, EndMT is also likely to be a potential mechanism by which DDP4i ameliorates BLM-induced pulmonary fibrosis.
In the present study, we treated HUVECs with BLM for 48h and induced EndMT in vitro, similarly to a previous study [17]. Our results showed that linagliptin co-treatment attenuated BLM-induced EndMT in vitro. BLM-induced enhanced migration of cells was also alleviated. BLM can also induce EndMT in vivo [35]. In the study, the lungs of BLM-treated mice developed EndMT, as evidenced by the increased number of co-localized CD31/α-SMA cells observed in the lungs of the BLM group, compared with the control group. As expected, the BLM-induced EndMT was inhibited by linagliptin in vivo.
Transcription factors including Snail, Slug and Twist are key mediators of EndMT. It has been confirmed that siRNA knockdown of Snail blocked transforming growth factor (TGF)-β-induced EndMT [36], Slug silencing reversed BLM-induced EndMT [17] and Twist overexpression induced EndMT in cultured human pulmonary arterial endothelial cells [37]. Thus, we examined the effect of linagliptin on the expression of these transcription factors. BLM upregulated the expression of Snail, Slug and Twist in vitro and in vivo, while linagliptin treatment abrogated this effect. Therefore, inhibition of EndMT is another important mechanism by which linagliptin improves pulmonary fibrosis in SSc mouse model.
The mechanisms underlying the effect of linagliptin on EndMT have not been fully elucidated. Shi et al. have reported that the interaction of DPP4 with integrin β1 promotes the TGF-β-induced TGF-β receptor heterodimer formation, initiates the Smad-dependent pathway and ultimately leads to EndMT. Linagliptin inhibited the EndMT process by suppressing the expression of DPP4 and integrin β1 [38]. However, the exact mechanisms of the protective effects of linagliptin on BLM-induced EndMT need to be further studied.
Oxidative stress refers to an imbalance between oxidants and antioxidants. Although the relationship between oxidative stress and EndMT in SSc remains controversial [39], it has been validated that oxidative stress plays a pivotal role in the induction and progression of SSc [18]. Oxidants may directly activate fibroblasts or contribute to the maintenance of fibrosis by altering the balance between protease and anti-protease activity [40]. Inflammation is another important player in SSc. IL-6 and TNF-α levels have been demonstrated to be increased in the serum and skin of patients with SSc [41, 42]. Moreover, blocking IL-6 decreased collagen production in the BLM model of lung fibrosis [43]. Linagliptin has also been reported to possess anti-inflammatory and antioxidant properties [44, 45]. In our study, BLM-induced oxidative stress and inflammation were inhibited by linagliptin in vivo, indicating that the therapeutic effect of linagliptin against BLM-induced pulmonary fibrosis may also be attributed to its anti-inflammatory and antioxidant properties.
TGF-β mediated Smad-dependent and Smad-independent pathways, such as c-Abl kinase, protein kinase c-δ, are most studied in EndMT. The Akt/mTOR pathway is an important smad-independent pathway involved in EndMT [32]. BLM induces EndMT in HUVECs via activation of this pathway [17]. Studies have shown that Salvia miltiorrhiza and Geniposide suppress BLM-induced EndMT by inhibiting the Akt/mTOR pathway [16, 22]. Furthermore, this pathway may mediate oxidative stress, although further research is required [46]. Li et al have also reported that linagliptin inhibited high glucose-induced transdifferentiation of hypertrophic scar-derived fibroblasts to myofibroblasts via inactivation of the insulin-like growth factor (IGF)/Akt/mTOR pathway [47]. Here, we showed that BLM induced the activation of the Akt/mTOR pathway, and that linagliptin repressed this activation. Therefore, linagliptin may suppress EndMT by inhibiting the AKT/mTOR pathway in pulmonary fibrosis in the SSc model.
The study also has some limitations. First, the inhibitory effect of linaglitpin on EndMT in vitro was not repeated in human pulmonary endothelial cells in this study limited by objective conditions. Second, alternative models were not recruited to further validate these effects of linagliptin. These are issues that need to be addressed in future research.