Citrus alkaline extracts prevent endoplasmic reticulum stress in type II alveolar epithelial cells to ameliorate pulmonary fibrosis via the ATF3/PINK1 pathway

Background: Idiopathic pulmonary fibrosis is a chronic, progressive, fibrotic disease. Although the pathogenesis remains unclear, the effect of endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AEC IIs) is increasingly thought to be a critical mechanism. Purpose: We investigated the effects of citrus alkaline extracts (CAE) on AEC IIs and elucidated the underlying mechanism for their possible use in ameliorating pulmonary fibrosis (PF). Methods: A bleomycin-induced mouse model of PF, and an in vitro tunicamycin (TM) -induced ER stress model in A549 cells were successfully established. Accumulation of collagen in lung tissues in vivo was assessed using histological analysis and western blotting. The expression levels of the ER-stress marker BiP and other related proteins were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential was assessed to evaluate mitochondrial homeostasis. Results: CAE mitigated collagen deposition to ameliorate PF in vivo. CAE suppressed the bleomycin or TM-induced increases in ER-stress biomarker, BiP, and PERK pathway proteins, resulting in a decrease in ER stress in mouse lung tissues and A549 cells, respectively. Additionally, CAE treatment suppressed the bleomycin or TM-induced increase in the ER-stress downstream proteins, activating ATF3 and increased the levels of PINK1 in AEC IIs, both in vivo and in vitro. The reduced mitochondrial homeostasis induced by TM was restored by CAE-treatment in A549 cells. Furthermore, conditioned media from TM-treated A549 cells increased collagen deposition in MRC5 cells mainly via TGF-β1. The increased collagen deposition was not seen using conditioned media from CAE-treated A549 cells. Conclusion: These results provide novel insights into the potential mechanism of CAE in inhibiting ER stress in AEC IIs, and suggests that it has great potential to ameliorate PF via the ATF3/PINK1 pathway.