Therapeutic potential of BLT1 antagonist for COPD: involvement of inducing autophagy and ameliorating inflammation

Abstract

Purpose

Leukotriene B4 (LTB4) is a major pro-inflammatory mediator that leads to the persistence of chronic inflammation in chronic obstructive pulmonary disease (COPD). The purpose of this study was to evaluate therapeutic potential of BLT1 antagonist for cigarette smoke (CS)-induced COPD and to explore the underlying mechanism.

Materials and methods

In vitro, autophagy proteins were determined by Western blotting in RAW264.7 macrophages treated with U75302 (BLT1 antagonist) or autophagy inhibitor in cigarette smoke extract-induced inflammation. In vivo, C57BL/6J mice were randomly divided into three groups: Control group, CS group and CS+U75302 group. After 12-week exposure, histological analysis and lung function tests were performed to evaluate the inflammatory infiltration and emphysema. The expression of inflammatory cytokines was measured by real-time PCR and enzyme-linked immunosorbent assay. Immunohistochemical analysis and Western blotting detected the expression of autophagy-related proteins. Transmission electron microscopy (TEM) showed the alterations of autophagosomes and lysosomes.

Results

Lower levels of inflammatory factors and autophagy markers were detected in U75302-treated cells and mice after CS exposure than control. In vitro, LC3 mRNA expression was elevated when treated with U75302. Autophagy inhibition resulted in augmented inflammatory response and autophagy proteins even with U75302 treatment. Furthermore, BLT1 antagonist decreased the number of lysosomes and autophagosomes in alveolar macrophages of mice and potentially enhanced the expression of transcriptional activation of transcription factor-EB (TFEB) in vitro and vivo.

Conclusion

Insufficient autophagy of macrophages was associated with LTB4-mediated inflammation in CS-exposure models. BLT1 antagonist ameliorated inflammatory response through inducing autophagy.

Keywords:

• cigarette smoke
• inflammation
• autophagy
• BLT1 antagonist
• COPD

Acknowledgment

The study was supported by National Key R&D Program of China (granted number 2017YFC1309701 and 2017YFC1309700), National Natural Science Foundation of China (granted number 81570029), Shanghai Key Discipline for Respiratory Diseases (granted number 2017ZZ02014) and Innovative research team of high-level local universities in Shanghai. We thank the laboratory of Shanghai Ninth People’s Hospital (China) for providing professional assistances and reagent supplement.

Ethics approval

All the animal experiments were strictly conducted in accordance with the protocols approved by the Ethics Committee and Animal Care Committee of Ruijin Hospital (Shanghai Jiaotong University School of Medicine, China).

Disclosure

The authors report no conflicts of interest in this work.