Blocking TG2 attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting EMT

https://doi.org/10.1016/j.resp.2020.103402Get rights and content

Highlights

  • The effect that blocking TG2 attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting EMT was firstly found.

  • This effect is mainly achieved by affecting the process of epithelial-mesenchymal transformation induced by bleomycin.

  • Our study supported the application of TG2 inhibitors in treatment for pulmonary fibrosis.

Abstract

Background

Epithelial-mesenchymal transformation (EMT) is a central mechanism for the occurrence and development of pulmonary fibrosis. Therefore, to identify the key target molecules regulating the EMT process is considered as an important direction for the prevention and treatment of pulmonary fibrosis. Transglutaminase 2 (TG2) has been recently found to play an important role in the regulation of inflammation and the generation of extracellular matrix. Here, our study focuses on the roles of TG2 in pulmonary fibrosis and EMT.

Methods

at first, the expression of TG2 and the EMT-related markers like E-cadherin, Vimentin, and α-SMA were detected with Western Blotting, immunohistochemistry and other methods in the mice with pulmonary fibrosis induced by bleomycin. Further, MLE 12 cells were used to study the effects on EMT of the inhibition of TG2 in vitro. Finally, GK921, an inhibitor against TG2, was used to show its function in both prevention and treatment of pulmonary fibrosis induced by bleomycin in mice.

Results

bleomycin succeeded to induce pulmonary fibrosis in mice, with increased TG2 expression, EMT and Akt activation. Knock-down of TG2 by siRNA technique in MLE 12 cell (a mouse alveolar epithelial cell line) and GK921 (an inhibitor of TG2) all inhibited the EMT process, however SC79, an activator of Akt rescued above inhibition. Finally, GK921 alleviated pulmonary fibrosis in mice induced by bleomycin.

Conclusion

Blocking TG2 reduces bleomycin-induced pulmonary fibrosis in mice via inhibiting EMT.

Introduction

Lung fibrosis, highlighted with the parenchymal cell reduction and fiber connective tissue hyperplasia, progressively causes the structural destruction and function decrease of the lung tissue, which results in lung failure and severely threatens patients' health. Worldwide, tissue fibrosis is a main cause of major morbidity and mortality. Concerned statistic data indicates a high mortality in lung fibrosis patients, with only 2–5 years survival after the diagnosis of idiopathic pulmonary fibrosis and 5-year survival rate low to 20 %. Besides, the mechanism of the development of lung fibrosis is very complicated (Raghu et al., 2006). Previous reports have suggested that dysregulation of inflammatory factors (Goodwin and Jenkins, 2009; Xin et al., 2019), extracellular matrix density increase (Shinde et al., 2017), and oxidative stress (Yang et al., 2018) were involved in this process. In recent years, the role of epithelial-mesenchymal transition (EMT) in lung fibrosis was widely studied. EMT is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, acquire migratory and invasive properties, and finally turn into mesenchymal cells. And most researches showed that alveolar epithelial cells underwent EMT during the development of lung fibrosis, leading to an increase of Interpulmonary fibers (Li et al., 2017; Ma et al., 2017; Rout-Pitt et al., 2018). Thus, EMT was thought to be pivotal to the occurrence and development of pulmonary fibrosis and finding the key targets regulating EMT is of great significance for the treatment of pulmonary fibrosis.

Transglutaminase is widely expressed in various tissues of mammals, among which Transglutaminase 2(TG2) has been most studied and its function is the most important. Recent studies showed that TG2 was involved in many diseases such as neurodegenerative diseases, autoimmune diseases, etc (Lai and Greenberg, 2013). Notably, TG2 was also reported to play an important role in the occurrence and development of pulmonary fibrosis (Olsen et al., 2011). However, it is still unknown whether it has a regulatory effect on EMT during the process of pulmonary fibrosis. Therefore, this study aimed to investigate the role of TG2 in pulmonary fibrosis and EMT in the bleomycin-treated mice and try to reveal its molecular mechanism.

Section snippets

Animal experiments

6-8 weeks old male C57BL/6 mice were intraperitoneally injected with bleomycin (Sigma, product number BP971) at 1.5 mg/kg once every 2 days for 1 week. Lung tissues of the mice were collected for pathological examination at 1, 2 and 3 weeks after drug withdrawal to confirm the establishment of pulmonary fibrosis model.

18 mice were randomly divided into3 groups: #control group (intraperitoneal injection of normal saline at 10 mL/kg), #model group (intraperitoneal injection of bleomycin at 1.5

Pulmonary fibrosis was induced by bleomycin in mice

Mice were intraperitoneally injected with bleomycin at 1.5 mg/kg every 2 days for 1 week. Mouse lung tissues were then subjected to H&E staining and Masson staining at 1week, 2 weeks, and 3 weeks post bleomycin induction. H&E staining data showed that mice lungs appeared thick septa with infiltration of immune cells at 2nd week post bleomycin treatment, which came to the most significant at 3rd week (Fig. 1A, B). In addition, Masson staining results showed that no significant positive area was

Discussion

EMT is most studied in pulmonary fibrosis (Lai and Greenberg, 2013). After suffering hypoxia and inflammation, epithelial cells lose their cell polarity and cell-cell adhesion, gradually lose the epithelial morphology, and gain the characteristics of the mesenchymal cells (Higgins et al., 2007). During EMT process, the expression of epithelial marker E-cadherin decreases while those of mesenchymal markers such as α-SMA, Vimentin is upregulated, which induces cytoskeleton reorganization into

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the grants from National Natural Science Foundation of China (Grant number 81700078) and Natural Science Foundation of Jiangsu Province (Grant number BK20171172)

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