Animal models
Six-week-old C57BL/6 mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China.). Mice were housed in cages under a 12-hour dark/light cycle with free access to food and water. After 2-week acclimation period, mice were divided into two weight-matched groups and fed with different diets, including ad libitum diet (AL, n = 40) and caloric restriction diet (CR, n = 40) for a period of 4 weeks, respectively. CR was progressive, initiated at 10% restriction during the first week, 25% during the second week, and to 40% for the remainder experimental period, according to the method described previously [45]. The components of AL diet and CR diet were given in Table S1. The food intake and the body weight were recorded daily (Table S2, Fig. 1A-1B). All animal procedures were conducted in accordance with the guidelines of the Animal Care and Protection Committee of Sun Yat-sen University and Hebei Medical University.
Real-ambient PM Exposure
Mice were kept in isolated ventilated cages (IVC) and exposed to PM in a real-ambient PM exposure system located at heart of city Shijiazhuang, China, where the annual average concentration of PM2.5 was ranked in the top 5 Chinese cities over the past decade [8]. This system permitted circulating the ambient air into the chambers absence concentrating the ambient PM. The air channels of the control chambers are equipped with a three-layer HEPA filter (air filter, AF), which provides an excellent barrier to block fine ambient PM (PM1 = 0). The concentrations of fine PM in the exposure chamber and ambient air were monitored with Aerodynamic Particle sizer Spectrometer 3321 (TSI Incorporated, Shoreview, MN, USA). Two groups of male mice (n = 20/group, 5 mice/cage) fed with AL or CR diet were exposed to PM for 24 h per day, 7 days per week for 4 weeks, from Jan 4th to Feb 1st, 2018. The other two groups of male mice (n = 20/group, 5 mice/cage) were kept in the control AF chambers (PM1 = 0). At the end of the experiments, mice were sacrificed, and the biological samples were collected for further analyses as described below.
PM Collection, Extraction And Components Analysis
In the course of PM exposure (4 weeks), the ambient PM2.5 was collected onto Teflon filters daily at a flow rate of 1.05 m3/min using a High-Volume Air Samples (Thermo Fischer Scientific, Waltham, MA, USA) nearby the PM exposure system. The filters were combined for chemical analysis. Organic components were extracted by Soxhlet extraction for quantification of polycyclic aromatic hydrocarbons (PAHs), nitro- and alkyl- derivatives of PAHs, polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-dioxins (PCDD). Water-soluble fractions were extracted by sonication for analyses of metals and anion species with inductively coupled plasma-mass spectrometry (ICP-MS; ELEMENT2; Thermo Finnegan, San Jose, CA, USA).
BALF Analysis
Bronchoalveolar lavage fluid (BALF) was collected as described previously [8]. The cells and supernatant were separated by centrifugation of BALF at 400 × g for 7 min at 4℃. Total protein, lactate dehydrogenase (LDH) and albumin contents in BALF supernatant were determined by BCA Protein Assay Kit (Beyotime biotechnology, China), LDH release assay kit (Promega, Corporation, Madison, WI, USA) and Albumin Assay kit (Nanjing Jiancheng Bioengineering Institute, China), respectively. The number of cells was determined with a cell counter (Beckman, USA). 1 × 104 cells were spread on microscope slides, fixed with 96% ethanol and stained with May-Grünwald-Giemsa. The number of macrophages and polymorphonuclear neutrophils (PMNs) were counted under microscope (Leica, Germany).
Histopathological Analysis
The lung tissues were removed, washed with 0.1 µM phosphate buffered saline (PBS, pH7.4), fixed in 4% formalin for 24 h at room temperature, dehydrated by graded ethanol, and embedded in paraffin. Tissue sections (5 µm) were made and stained with hematoxylin and eosin. The histological examination was performed under a light microscope. The histopathological analysis of lung injury was conducted quantitatively as described previously [65].
TUNEL Staining
Apoptosis of cells in mouse lung was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining (Beyotime, China) according to the manufacturer’s instructions. For quantification of apoptotic cells, five sections and 20 random fields per section in each group were counted, and the average number of apoptosis per section was calculated.
Detection Of ROS In Mouse Lung
Reactive oxygen species (ROS) levels in the lung tissues were detected with the fluorescent probe DHE (dihydroethidium; Sigma, USA). Briefly, the frozen sections were incubated with 10 µM DHE at 37℃ for 30 min. The slides were viewed under a fluorescence microscope (Leica, Germany). The intensity of DHE was analyzed by ImageJ software.
Immunohistochemistry
Lung sections (5 µm) were mounted onto the slides, deparaffinized and rehydrated, and heated in 0.1 M citrate buffer (pH 5.8) for antigen retrieval. To inactive endogenous peroxidase, we incubated slides with 3% H2O2 at RT for 15 min. After blocking with 2% BSA for 30 min at 37℃, the slides were incubated with primary antibodies against γ-H2AX (Abcam, USA), p53 (CST, USA) and F4/80 (CST, USA) overnight at 4 °C. After incubation with the corresponding secondary antibody for an additional 1 h at room temperature in dark, the slides incubated in 10 µg/ml 3, 3N-diaminobenzidine tertrahydrochloride (DAB; Beyotime, China) for 10 min. The nuclei were counterstained with hematoxylin for 10 s. The fluorescent intensity was analyzed using ImageJ software.
Cytokine Analysis
Plasma cytokines, including interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12p70), and transforming growth factor beta (TGF-β1) were measured with an ELISA assay kit (R&D Systems, MN, USA).
Ex vivo assays
Colorimetric cell viability assay (MTS; Promega Corporation, Madison, WI, USA) was used to determinate the effect of the plasma on cytotoxicity of multiple cell lines, Neuro-2A, THP1, HepG2, HEK, HCT116, 16HBE. We seeded the cells in a 96-well plate at a density of 3 × 103 and cultured them in a medium containing 10% of fetal bovine serum (FBS) and 1:100 plasma isolated from the mouse blood. Forty-eight h after seeding, 20 µL of MTS reagent were added to each well and incubated for 2 h. The absorbance at 490 nm was determined and the cytotoxicity was presented as fold changes relative to the cell viability of the negative control.
Immunoblotting
Total cellular protein was extracted by RIPA lysis buffer (150 M of NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris (pH 7.4)) containing protease inhibitors. 20 µg soluble proteins were separated by 8 ~ 12% SDS-PAGE and were transferred onto a nitrocellulose membrane (Pall Corporation, NY, USA). After being blocked with 5% fat free milk, the membranes were incubated with primary antibodies against caspase3, cleaved caspase 3, CYP1A2 (Cell Signaling Technology, MA, USA), CYP1A1, CYP1B1, GSTT1, GSTM1, UGT1A1 (Abcam, UK), and β-actin (Proteintech Group, AL, USA). Immunolabeling was visualized with HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Santa Cruz Biotechnology, CA, USA). The density of the specific bands was quantified using ImageJ software.
RNA Sequencing
For each group, we randomly selected 3 mice for conducting RNA sequencing of mouse lung or liver tissues, 12 mice in total from 4 groups of mice (AL-fed or CR-fed with or without PM exposure). Total RNA samples of mouse lung and liver tissues were extracted using TRIZOL reagents and subjected to RNA sequencing (Beijing Genomics Institute in Shenzhen, China). Briefly, Oligo dT magnetic beads were employed to trap mRNAs with poly A tails, and the mRNAs were fragmented and reversely transcribed to double-stranded cDNA (dscDNA) by random primers. The cDNAs were ligated adaptors and subjected to amplification. The PCR products were then denatured, and single stranded PCR products were cyclized by splint oligos with DNA ligase to construct cDNA library. The sequencing was performed with BGISEQ-500 platform, generating 23.87 M reads per sample. The average mapping ratio with reference genome was 94.60% and the average mapping ratio with gene was 81.87%. 17,519 genes were detected in this experiment.
We set fold-change greater than 1.5 times, P value lesser than 0.05 as the standard to define differentially expressed genes (DEGs). These DEGs were analyzed by Ingenuity Pathway Analysis (IPA) software (Qiagen, Germany). The increased and/or decreased activity of pathways and functions of the DEGs were defined via IPA. Significant differences were defined as P value was less than 0.01 and the absolute value of the z score was greater than 2.
Examination Of Urinary OH-PAHs
Concentration of seven hydroxylated metabolites of PAHs (OH-PAHs), including 1-OHNap, 2-OHNap, 1-OHPhe, 4-OHPhe, 9-OHPhe, 2-OHFlu, 1-OHPyr, in mouse urine were analyzed with a LC-20A high performance liquid chromatography system (HPLC; Shimadzu, Japan) coupled with a Q-Trap 5500 mass spectrometer (MS/MS; AB SCIEX, USA). Five isotopically labeled chemicals were used as internal standards: d8-2-OHNap d9-2-OHFlu, 13C6-4-OHPhe, 13C6-1-OHPyr. The concentration was calculated by extrapolating the peak area of the sample from standard sets. Urinary OH-PAHs concentrations were adjusted with the content of urinary creatinine (µg/g Cre).
Statistical analysis
Data are shown as the mean ± S.D. All statistical analysis was performed with SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t-test was applied to analyze the difference between two groups and one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was used for comparisons between multiple experimental groups. Differences were considered significant at P < 0.05.