Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies have impelled researchers to reconsider the importance of choice for animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.This study aimed to follow the dynamic fibrotic features of BLM (Bleomycin)-treated mouse lungs with extended durations through a combination of the latest technologies (micro-CT imaging and histological detection of degraded collagens) with traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.As determined by classical biochemical method, total lung hydroxyproline contents reached peak at 4-week after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16-week). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration post the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and a fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10- or 16-week, rather than at 4-week post the BLM-treatment. Hence, our present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious while using hydroxyproline content assay to evaluate the severity of fibrosis.