2.1 PM2.5 sampling, extraction, and chemical analysis
The collection, extraction and component analysis of PM2.5 have been described in a previous study [7]. PM2.5 samples were collected on quartz filters (Tissuquartz,Pall, USA) using a high flow volume PM2.5 Sampler (Ecotech,Australia) at a flow rate of 1.13 m3/min, located on the top of a building in Xuhui District in Shanghai, China, from September 2017 to April 2018. The filters were cut into small fragments (1 cm × 1 cm), then immersed into ultrapure deionized water and eluted with an ultrasonic cleaner (KUDOS, Shanghai, China), followed by freeze-drying with a vacuum freeze dryer (Four-Ring Science Instrument Plant, Beijing, China). PM2.5 solid particulates were preserved at -80°C until required.
2.2 Cell culture, pharmacologic and genetic intervention
A549 cells (Shanghai Institutes for Biological Sciences, China Academy of Science, Shanghai) were cultured in F12K medium (Procell Life Science & Technology, Wuhan, China) with 10% fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin at 37°C in 5% CO2. According to the data from a preliminary study, we selected PM2.5 concentrations of 0, 8, 16, 32, 64, and 128mg/ml that induced oxidative stress in cells but did not lead to cell death.
A549 cells were treated with 32mg/ml PM2.5 to induce cell injury. Cells were pretreated with 10μM of Mdivi-1 (a DRP1 inhibitor) (Selleck, Shanghai, China) or 10μM BGP-15 (an OPA1 activator) (Selleck, Shanghai, China) for 2h and then were cultured with vehicle or PM2.5 for 48h (preliminary study results are shown in Supplementary Figure1). Human DRP1-knockdown plasmid and OPA1-overexpression plasmid sequences were commercially designed (Lncbio-technology, Xuhui, Shanghai, China). Lentivirus packaging was performed in 293T cells using Zorin virus packaging kit (Shanghai Zorin Biological Technology, Shanghai, China). After the lentivirus was prepared, the cells were stably transfected using Polybrene, screened using antibiotic puromycin dihydrochloride (Shanghai Zorin Biological Technology, Fengxian, Shanghai, China) resistance to obtain DRP1-knockdown (KD) and OPA1-overexpressing (OE) cells. These cells were also treated with 32mg/ml PM2.5 for 48h to induce injury.
2.3 Animal experiments
10-week-old male C57/BL6 mice, weight 22-25g, were purchased from SPF Biotechnology Co., Ltd (Shanghai, China). All the mice were housed in a specific pathogen-free facility where the circulating temperature is 22°C with 50-60% humidity, equal light-dark cycle, and with access to standard food and water ad libitum. All experimental studies involving animals were approved by the laboratory animal ethics committee of the institute. According to a preliminary study, mice were administered intraperitoneally with Mdivi-1 (20 mg/kg, dissolved in saline including 5% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG 300), 5% Tween 80) or BGP-15 (20mg/kg, dissolved in phosphate buffer solution(PBS)) one hour before intranasal instillation of 50μL of PM2.5 suspension (7.8mg/kg)(PM2.5 concentrations were selected according to the results of previous studies, and Mdivi-1 and BGP-15 concentrations were selected according to the results of the preliminary study in Supplimentary Figure 2 of the additional material) [7] or distilled water once a day for two consecutive days. The experimental procedures involving animals and their care were approved by the Laboratory Animal Ethics Committee of Shanghai Chest Hospital.
2.4 Cell viability assay
The effect of vehicle or PM2.5 on cell proliferation was measured by cell counting kit-8 (CCK8, Dojindo, Kumamoto, Japan). Cells were cultured in a 96-well plate with a density of 5×104cells/ml overnight at 37°C before exposure to vehicle or PM2.5. After 48h incubation, cells were washed twice with PBS and incubated with CCK8 working solution for 2h at 37°C following the manufacturer's protocol. The absorbance at 450nm was measured by microplate reader iMark (Molecular Devices, Sunnyvale, CA, USA).
2.5 Quantitative reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from A549 cells using TRIzol and then its concentration and purity were assessed. One μg total RNA was reverse transcribed into cDNA and quantitative real-time PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China) in an ABI ViiATM 7 System (Applied Biosystems, Foster City, CA, USA). The reaction conditions included 95°C for 30s, followed by 40 cycles of 95°C for 10s and 60°C for 30s, with a final cycle of 95°C for 15s, 60°C for 60s and 95°C for 15s. The primer sequences of the cytokines (IL-1b, IL-6, IL-18 and CXCL-8) and β-actin are shown in Table 1.
2.6 Measurement of intracellular and mitochondrial ROS in cells
Intracellular ROS generation was detected using 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells were seeded in 96-well black plates with a density of 5×104 /ml with 6 parallel wells in each group. Cells were stained with 10μM DCFH-DA at 37°C in the dark for 15 min. Then cells were washed with serum-free F12K for three times, the level of ROS was determined using a fluorescence plate reader (Molecular Devices) at 488/525nm. Mitochondrial ROS(mtROS) level was measured using MitoSOX Red (Invitrogen, Life Technologies, Carlsbad, CA, USA). Briefly, the cells were seeded in 96-well black plates with a density of 5×104 /ml with 6 parallel wells in each group. Cells were incubated with 5mmol/L MitoSOX Red probe for 10 min at 37°C. The cells were washed twice with PBS, and red fluorescence was determined at 510/580nm using a fluorescence plate reader.
2.7 Mitochondrial morphology in cells
Mitochondrial morphology was measured with Mito-Tracker Green FM assay (Invitrogen) according to the manufacturer’s instructions. Cells were seeded in confocal dishes with a density of 5×104cells/ml, and and incubated with 500μl of 200nM Mito-Tracker Green FM (Invitrogen) 30min at 37°C in the dark and washed twice with serum-free medium. 100ml of Hoechst 33342 (Beyotime Technology, Haimen, Jiangsu, China) was added and incubated for 30min at 37°C. The mitochondrial morphology was assessed using a confocal microscopy (Leica, Wetzlar, Germany). Cell area and mitochondrial area were measured by ImageJ software (https://imagej.nih.gov/ij/). Mitochondrial fragmentation was judged to havce occurred if >90% of mitochondria in the cytoplasm outside of the perinuclear compaction were punctate or circular, and mitochondrial perinuclear compaction was judged to be present if >90% of mitochondria accumulated in the perinuclear area.
2.8 Mitochondrial membrane potential (ΔΨm) in cells
Cells were seeded in 6-well plates (2×105 cells/well) and cultured with 1mL JC-1 staining working solution (Beijing Solarbio Science & Technology Co., Ltd.) for 20 min. The cells were later washed twice using PBS. Cells were photographed under an inverted fluorescence microscope. Red fluorescence was determined at 579/599nm and green fluorescence was determined at 490/516nm, using an inverted fluorescence microscope (Leica). The relative mitochondrial membrane potential level was evaluated by comparing the red fluorescence cells to green fluorescence cells in the groups.
2.9 Measurement of mitochondrial oxygen consumption rate (OCR) in cells
Cells were seeded on Seahorse XFe96 plates at a density of 10000 cells/well in complete F12K. The OCR was measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer’s instructions. The following inhibitors were added sequentially: 1.5μM oligomycin (an adenosine-triphosphate (ATP) uncoupler), 1μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an electron transfer chain (ETC) accelerator), 0.5μM antimycin A (a complex III inhibitor), and 0.5μM rotenone (a complex I inhibitor). OCR was calculated using the standard XFe96 Extracellular Flux Analyzer protocol.
2.10 Airway hyperresponsiveness (AHR) in mice
After anesthesia with an intraperitoneal injection of 0.2mL 1% pentobarbital, mice were tracheostomized and placed in a whole-body plethysmograph with inhalation of aerosolised acetylcholine (ACh) for the measurement of airway resistance and compliance (EMMS, Hants, UK). Lung resistance (RL) was recorded and presented as the percentage change from baseline RL of nebulizing PBS. The concentration of ACh required to increase lung resistance by 100% from baseline was calculated as PC100, and -logPC100 was taken as a measure of airway responsiveness.
2.11 Histological analysis and immunofluorescence in mice
The whole lung was removed, and the right lung was dissected and snap-frozen in liquid nitrogen for later analysis.The left lung was inflated with 4% paraformaldehyde under 25cm of water pressure and then embedded in paraffin. Paraffin blocks were sectioned to expose the maximum surface area of the lung tissue in the plane of the bronchial tree. Four μm sections were cut and stained with hematoxylin and eosin (H&E). The extent of lung inflammation was evaluated in the H&E-stained lung sections as described previously [21] using the following scale: 0 = no inflammatory response, 1 = mild inflammation with foci of inflammatory cells in the bronchial or vascular wall and in alveolar septa, 2 = moderate inflammation with patchy inflammation or localized inflammation in walls of the bronchi or blood vessels and alveolar septa and less than 1/3 of the lung cross-sectional area is involved, and 3 = severe inflammation with diffuse inflammatory cells in walls of the bronchi or blood vessels and alveoli septa; between one-third and two-thirds of the lung area are involved.
The localization and expression of pulmonary surfactant-associated protein C (SFTPC) was examined by immunofluorescence staining. Lung sections were incubated with anti-SFTPC primary antibody (1:200, Proteintech, Wuhan, Hubei, China) at 4°C overnight, and the secondary antibody (Goat anti-rabbit IgG (Alexa Fluor 488) ,1:500, Abcam, Cambridge, MA, USA) at room temperature in the dark for 60 min. Nuclear staining was performed with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime) staining solution, and imaging was obtained.
2.12 Western Blot analysis in cells and mice
Total protein from A549 cells and mouse lung tissues were respectively homogenized with a RIPA lysis buffer (Beyotime), and protein concentrations were quantified by a Bicinchoninic Acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). 30μg of protein per lane was separated through 10-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: DRP1 (1:1000, Cell Signaling Technology, Danvers, MA,USA), MFF (1:1000, Cell Signaling Technology), MFN2 (1:1000, Cell Signaling Technology), OPA1 (1:1000, Cell Signaling Technology), phosphorylated (phospho) DRP1 (p-DRP1)(1:1000, Cell Signaling Technology), PINK1 (1:1000, Abcam), PARK2 (1:1000, Abcam), SQSTM1/p62 (1:1000, Abcam), LC3 (1:500, Abcam), MLKL (1:1000, Affinity Biosciences, Liyang, Jiangsu, China), RIPK1 (1:1000, Cell Signaling Technology), RIPK3 (1:1000, Cell Signaling Technology), overnight at 4°C. Membranes were then incubated with a horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell Signaling Technology,USA) and then visualized by chemiluminescent detection.
2.13 Statistical analysis
Data were presented as mean ± SD. GraphPad Prism 8 was used to compare multiple groups by One-way ANOVA with Bonferroni’s post hoc test (for equal variance) or Dunnett’s T3 post hoc test (for unequal variance).Two-way ANOVA was performed for comparisons of %change in lung resistance between individual groups. P<0.05 was considered significant.