Chemicals and reagents
Bleomycin was purchased from Hanhui Pharma Co., Ltd. (Hangzhou, China). 4% paraformaldehyde was purchased from Wuhan Saiweier Biological Technology Co., Ltd (Wuhan, China). Hydroxyproline assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). CoQ10 (purity > 98%, using HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in DMSO (St. Louis, MO, USA), and diluted into the culture medium. The final concentration of the DMSO was 0.1%. Annexin V-FITC apoptosis detection kit and total ROS/superoxide detection kit were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).
Cell culture
BCs were obtained from the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China), which were primarily isolated and cultured from airway mucosal biopsy samples from non-fibrotic patients (adult male, diagnosed with chronic cough) undergoing fiberoptic bronchoscopy based on previously published method [28, 29]. BCs were cultured in DMEM/F12 medium containing 5 μM ROCK inhibitor Y-27632 (Sigma, Cat. SCM075), 1 μM A-83-01 (Sigma, Cat.SML0788), 1 μM DMH-1 (MedChemExpress, Cat. HY-12273), 1 μM CHIR99021 (Sigma, Cat.SML1046), and 1× Antibiotic–Antimycotic medium (ThermoFisher, Cat. 15240062) at 37 °C with 5% CO2 in a humidified atmosphere. The culture medium was changed every 3–4 days. Cells with 80% confluency were passaged and the generation between P2 to P8 were used for the experiment. MSCs were obtained from Southern Medical University (Guangzhou, China).
Cell groups and treatments
Groupings: (1) blank group: human BCs were cultured in medium without CoQ10 and H2O2 before measurement, and the other steps were the same; (2) H2O2 group: BCs were cultured with 1 mM H2O2 for 6 h before measurement; (3) CoQ10 + H2O2 group: BCs were incubated with 10 mM CoQ10 for 24 h, and then changed into medium with 1 mM H2O2 for 6 h before measurement.
Determination of intracellular ROS levels
ROS assay kit (DCFH-DA, MedChemExpress, Cat. HY-D0940) was used to detect the level of reactive oxygen species in human BCs of each group. Cells were treated by 10 mM CoQ10 and/or 1 mM H2O2 as the preceding steps, followed by PBS wash to remove the residual H2O2. Each cell was then added in 100 μL serum-free medium containing 2 μL of 5 mM DCFH-DA, incubate the microplate for 30 min at 36 °C in the dark with occasional shaking. Then carefully discard the liquid within each well and wash the cells twice with PBS. The ROS concentration in each well was detected by flow cytometry (BD Biosciences, NJ) or Nikon microscope (Tokyo, Japan).
Determination of cells apoptosis
After pretreatment by CoQ10 and H2O2, BCs were harvested through trypsinization and washed with cold PBS. BCs were resuspended by PBS and centrifuged at 3000 r/min for 5 min, then the supernatant was discarded and the pellet was resuspended in 1× binding buffer at a density of 1.0 × 106 cells/mL. 100 μL of the sample solution was transferred to a 5 mL culture tube, and incubated in the photophobic environment with 5 μL of FITC-conjugated annexin V (Pharmingen) and 10 μL of PI for 15 min and resuspended every 3 min. 400 μL of 1× binding buffer was added to each sample tube, and the samples were detected by flow cytometry within 1 h.
Animals
Fifty male C57/B6 mice (weighing between 20 and 30 g) were purchased from Laboratory Animal Center of Sun Yat-sen University (SYXK 2016-0112). The animals were acclimated to the laboratory for at least 7 days before experiments. The animal care and use complied with the Provisions and General Recommendation of the Chinese Experimental Animals Administration Legislation. Animal experiment protocol was approved by Laboratory Animal Center of Sun Yat-sen University.
Animals’ groupings and treatments
Mice were randomly divided into five groups (n = 10): blank group, model group, BCs group, MSCs group, and CoQ10 + BCs group. On day 0, bleomycin (5 mg/kg) was infused into the trachea to induce pulmonary injury and fibrosis. Briefly, the mice were anesthetized by continuous inhalation of diethyl ether, their airways were kept in a straight and horizontal state by placing their backs against a circular water bottle with their stomachs facing up. Then a laryngoscope was used to open their mouth to expose the glottis and a micro-sprayer (HRH-MAG4, Huironghe, China) was used to spray bleomycin into the lungs of mice through the glottis and tracheal. After bleomycin inhalation, the mice were kept upright, allowing bleomycin to enter as much of their lung tissue as possible by the gravity. On day 7, 1 × 106 cells (BCs or MSCs) were resuspended with 50 μL PBS and then transplanted into lung tissue of anesthetized mice through endotracheal intubation. The cells of CoQ10 + BCs group were incubated with 10 mM CoQ10 for 24 h before transplantation and resuspended into 50 μL PBS containing 10 mM CoQ10, and the other steps were the same. The body weight of mice was recorded on day 7, 14 and 21 respectively. Lung tissues were extracted on day 21.
Histological examination of lung tissue
The lungs were perfused with 4% paraformaldehyde overnight at 4 °C and performed standard paraffin embedding protocols containing dehydrated with gradient ethanol and embedded with wax. Slicer (Leica, Germany) was used to cut the lung tissues into slices with a thickness of 5–7 μm. The slices were placed on a slide coated by poly-lysine and then stored at room temperature until further use. H&E staining and Masson staining were performed according to the standard protocols. The degree of pulmonary fibers was evaluated through Ashcroft scoring standard. The blue part of Masson staining was quantified by Image J (collagen fibers).
Measurement of lung hydroxyproline
To evaluate the extent of pulmonary fibrosis, the total collagen contents were measured using a hydroxyproline assay kit (abcam, Cat.ab222941) according to the manufacturer’s instructions. Briefly, the left lobes of lungs were completely dried in the oven and the weights of dry lobes were measured. The samples were then hydrolyzed at 95 °C in each tube containing 6N HCl for 2 days. The hydrolysates were centrifuged and then supernatants in each tube were transferred to fresh tubes. 5 μL of each sample or standard was mixed with 5 μL of citrate-acetate buffer into 96-wells plate in triplicate. 100 μL of Chloramine-T solution was added into each well and the mixture was incubated for 20 min. Then, 100 μL of Ehrlich’s solution was added into each well and the samples were incubated at 65 °C for 20 min. Absorbance was measured at 550 nm and the amount (μg/mg) of hydroxyproline was calculated by comparison to the standard curve.
Immunofluorescence staining of pulmonary α-SMA, SPC, and human mitochondrial protein
The sections were reconstituted in citric acid buffer (Boster Biological Technology Co., ltd, California, USA) at 120 °C. The nonspecific antigens were blocked with 1% BSA for 20 min. Sections were incubated with primary antibody of α-SMA (SAB, Cat. 40482, 1:200 dilution,), or with primary antibody of SPC (Abcam, Cat. ab211326, 1:200) and anti-human mitochondria antibody (CHEMICON, Cat. MAB1273, 1:200) overnight at 4 °C, and then washed in PBS 3 times. Then sections were then incubated with the suitable secondary antibodies in dilution of 1:400 at room temperature for 1 h. Sections were washed in PBS 3 times and then incubated with DAPI (Beijing Solarbio Co., Ltd., Beijing, China) for 5 min, followed by washed in PBS 5 times. Slides were stored in a dark place at 4 °C, and staining of pulmonary α-SMA, SPC or human mitochondrial protein were recorded with a fluorescence microscope.
Statistical analysis
The data were analyzed by Graphpad Prism 8.0 software (La Jolla, CA, USA). The survival curve was drawn using the Kaplan Meier method. All data were expressed as mean ± standard deviation (SD). T-test was used for analysis between two independent sample groups and single-factor analysis was used for comparison between multiple groups of data. P < 0.05 was considered statistically significant.