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.

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Background Idiopathic pulmonary fibrosis (IPF) is an age-related disease with no cure. Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy for IPF treatment. Nevertheless, MSCs derived from patients with IPF (IPF-MSCs) become senescent, thereby reducing their beneficial effects in IPF. MicroRNAs (miRNAs) mediate the senescence of MSCs, but the underlying mechanisms are not fully understood. We investigated the mechanisms by which miR-199a-5p regulates IPF-MSC senescence and whether its inhibition could rejuvenate IPF-MSCs and enhance their therapeutic efficacy. Methods Control-MSCs and IPF-MSCs were isolated from the adipose tissue of age-matched healthy and IPF donors, respectively. Cell senescence was examined by senescence-associated β-galactosidase (SA-β-gal) staining. The level of miR-199a-5p was measured by RT-PCR. Autophagy was determined using a transmission electron microscope (TEM). The therapeutic efficacy of anti-miR-199a-5p-IPF-MSCs was assessed using a mouse model of bleomycin-induced lung fibrosis. Results Despite similar surface makers, IPF-MSCs exhibited increased cellular senescence and decreased proliferative capacity compared with control-MSCs. The expression of miR-199a-5p was significantly enhanced in the serum of IPF patients and IPF-MSCs compared with that of healthy donors and control-MSCs. The upregulation of miR-199a-5p induced senescence of control-MSCs, whereas the downregulation rescued IPF-MSC senescence. Mechanistically, miR-155-5p suppressed autophagy of MSCs via the AMPK signaling pathway by downregulating the expression of Sirtuin 1(Sirt1), resulting in cellular senescence. Accordingly, miR-155-5p inhibition promoted autophagy and ameliorated IPF-MSC senescence by activating the Sirt1/AMPK signaling pathway. Compared with IPF-MSCs, the transplantation of anti-miR-199a-5p-IPF-MSCs increased the ability to prevent progression of pulmonary fibrosis in bleomycin-treated mice. Conclusions Our study shows that miR-199a-5p regulates MSC senescence in patients with IPF by regulating the Sirt1/AMPK signaling pathway and miR-199a-5p is a novel target to rejuvenate IPF-MSCs and enhance their beneficial effects.