CoQ10 enhances the efficacy of airway basal stem cell transplantation on bleomycin-induced idiopathic pulmonary fibrosis in mice

Abstract

Background: Recent studies have demonstrated that airway basal stem cells (BCs) transplantation can ameliorate bleomycin-induced idiopathic pulmonary fibrosis (IPF) through lung regeneration promotion. However, BCs under oxidative stress in the alveolar microenvironment are poor in survival, causing unsatisfied efficacy of BCs transplantation. In this study, we investigated whether Coenzyme Q10(CoQ10) counteracts oxidative stress in the alveolar microenvironment, thus improved the efficacy of BCs transplantation for IPF treatment.

Methods: The protective effects of CoQ10 on H₂O₂-induced BCs apoptosis and cytoplasmic reactive oxygen species (ROS) level were tested by flow cytometry in vitro. The therapeutic effects of BCs combined with CoQ10 were compared to a single BCs transplantation protocol in IPF treatment after 2 weeks and were evaluated by parameters including changes of body weight and survival rate, as well as various levels of pulmonary inflammation, α-SMA expression and hydroxyproline (HYP) in IPF mouse lung tissues.

Results: CoQ10 preincubation with BCs (10 mM, 24 h) significantly reduced the late apoptosis of BCs and the number of oxidative stressful BCs as a result of H₂O₂ stimulation (1 mM, 6 h) in vitro. IPF mouse model was constructed through bleomycin (5 mg/kg) intratracheal instillation. Bleomycin-induced IPF mice showed weight loss continuously and mortality increased progressively during modeling. Serious pulmonary inflammatory cell infiltration, collagen fiber proliferation, and collagen protein deposition were observed in lung tissues of IPF mice. Though BCs transplantation alone improved indicators above in bleomycin-induced IPF mice to some extent, the combination with CoQ10 improved the transplantation efficacy and obtained better therapeutic effects.

Conclusion: CoQ10 blocked H₂O₂-induced apoptosis of BCs and ROS production in vitro, and enhanced the efficacy of BCs transplantation against bleomycin-induced IPF in mice.

Keywords: Airway basal stem cells (BCs); Cell transplantation; Coenzyme Q10 (CoQ10); Idiopathic pulmonary fibrosis (IPF).

Fig. 1

Culture and identification of BCs. BCs were primarily isolated and cultured from airway mucosal biopsy samples from non-fibrotic patients, with DMEM/F12 medium supplement with Y-27632, A-83-01, DMH-1, and CHIR99021. A The cultured BCs were observed under a light microscope. B BCs was stained by two cell makers, P63 (Green) and KRT5 (Red). DAPI was used for nuclear staining of BCs. Scale bars: 50 μm

Fig. 2

CoQ10 inhibits BCs apoptosis and increased ROS level in BCs causing by H₂O₂. A, B The cells of each group were stained with DCFH-DA reactive oxygen fluorescent probe. Fluorescence representative pictures of cells in the blank group, H₂O₂ group and CoQ10 + H₂O₂ group were taken with a fluorescence microscope, Scale bars: 200 μm. C, D The cells of each group were stained with DCFH-DA reactive oxygen species fluorescent probe, and the ROS levels of cells in the blank group, H₂O₂ group and CoQ10 + H₂O₂ group were detected by flow cytometry. E, F The cells of each group were stained with Annexin-FITC/PI dye, and the apoptotic number of cells in the blank group, H₂O₂ group and CoQ10 + H₂O₂ group was detected by flow cytometry. *P < 0.05, **P < 0.01, ns no significant

Fig. 3

Changes in body weight of mice and survival curves after bleomycin instillation and cells transplantation. A Changes in body weight of mice in each group after bleomycin exposure. B The survival rate of mice in each group. The mortality of the mice during 21-day observation period was 30% (3/10), 30% (3/10), 30% (3/10), 20% (2/10), 0% (0/10) for the Model group, MSC group, BCs group, BCs + CoQ10 group, control group, respectively

Fig. 4

The HYP content in mice lung tissue after bleomycin instillation and cells transplantation. The collagen production in the lung tissue of mice was evaluated by hydroxyproline (HYP) assay kit. the amount of HYP (μg/mg) was calculated by comparison to the standard curve. Data was represented as the mean ± standard deviation (SD), n = 6 per group. ***P < 0.001 vs. control group; ^#P < 0.05 vs. model group; ^##P < 0.01 vs. model group; ^###P < 0.001 vs. model group

Fig. 5

Immunofluorescence of α-SMA, SPC, AQP5, and hM in the lung section of each group after bleomycin instillation and cells transplantation. The right lobes of lungs were cut into slices with a thickness of 5–7 μm, incubated with suitable antibodies for IFC staining. A Anti-α-SMA antibody (red), anti-SPC antibody (green), and DAPI (blue) staining showed the degree of fibrosis and expression of SPC in the lung tissues of mice. B Staining with antibodies of SPC (red) and human mitochondria (hM, green) that showing survival of MSCs or BCs, and differentiation into type II alveolar epithelial cells in the lung tissues of mice. C Staining with antibodies of AQP5 (red) and hM (green) that showing survival of MSCs or BCs, and differentiation into type I alveolar epithelial cells in the lung tissues of mice. Mouse pulmonary epithelial cells only shows green fluorescence in cytoplasm, while undifferentiated human MSCs or BCs with only red fluorescence, and differentiated MSCs or BCs displays overlapping positive staining of green and red fluorescence (white arrow). Scale bars: 100 μm. n = 6 per group

Fig. 6

Histopathological staining of the lung tissues after bleomycin instillation and cells transplantation. Pulmonary tissue sections were prepared by right lobes of lungs on day 21 and stained with H&E (A) and Masson’s trichrome staining (B). H&E staining showed the infiltration of inflammatory cells, and lung epithelial cells injury and repair disorder. Masson’s trichrome staining visualized collagen deposition in lung section of mice. Scale bars: 100 μm; ***P < 0.001 vs. control group; ^#P < 0.05 vs. model group; ^##P < 0.01 vs. model group. n = 6 per group