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. As shown in Fig. S1, 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 [49, 69, 70]. The components of AL diet and CR diet are shown in Table S1. The food intake and the water consumption were monitored daily (Fig. S2) and the body weights were recorded weekly (Fig. 1A). 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 the 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. The characteristics of the real-ambient PM exposure system has been described in our previous study [8]. As shown in Fig. S3, this system permitted circulating the ambient air into the chambers in absent of 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. Both AF control and PM exposure system were equipped with an auto-temperature control module to keep a constant temperature of the input air. The meteorological condition inside the chambers was closely monitored to keep a relatively constant temperature, humidity, ventilation frequency, air-flow rate, and noise (Table S2). We measured PM concentration in the chambers using an Aerosol Detector DUSTTRAKTM II and analyzed the particle size spectrum using an 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. 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).
Blood collection and tissue preparation
At the end of PM exposure, mice were anaesthetized with 100 mg/kg sodium pentobarbital. Mouse blood was collected into the EDTA-coated tubes (BD Biosciences, San Jose, California). 100 μL blood was subjected for comet assay. The plasma was isolated from blood by centrifugation at 450×g for 10 min at room temperature and stored at -80 ℃ before use. After the blood were collected, lung tissues and liver tissues were rapidly harvested and weighed. Then the tissues were divided into two parts for fixation in 4% paraformaldehyde in PBS and snap freeze in liquid nitrogen and stored at −80 °C, respectively.
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 Biotech Inc., Nantong, 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, Coulter, CA, 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 0.1μM 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 [71].
TUNEL staining
The lung sections (5 μM) were stained via terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining (Beyotime Biotech Inc., Nantong, China) to examine the apoptosis of cells in mouse lung according to the manufacturer’s instructions. Murine lung sections without any stimuli were incubated with 0.01 U/ul DNase I for 10 min at room temperature (RT) and were regarded as a positive control. For quantification of apoptotic cells, 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) [72]. Briefly, the frozen sections (5 μm) were incubated with 10 μM DHE at 37℃ for 30 min. The human bronchial epithelial cells (16HBE) treated with 100 μM H2O2 for 30 min were regarded as a positive control. The slides were viewed under a fluorescence microscope (Leica, Germany). The intensity of DHE was analyzed by ImageJ software.
Immunohistochemistry of F4/80
F4/80, also called as EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1), is a heavily glycosylated G-protein-coupled receptor and is a well-known biomarker for mouse macrophages [73]. F4/80 was detected by immunohistochemistry (IHC). Briefly, 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 30min at 37℃, the slides were incubated with primary antibodies against F4/80 (#30325; Cell Signaling Technology, MA, 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 Biotech Inc., Nantong, China) for 10 min. The nuclei were counterstained with hematoxylin for 10 s. The monocytes (THP-1) treated with 100 ng/ml phorbol myristate acetate for 48 h were regarded as a positive control. Positive staining cells were counted in 20 random fields per section in each group by light microscopy (Leica, Germany) and the average number of positive cells per section was calculated.
Immunofluorescence of γ-H2AX
The phosphorylation of the histone 2AX at ser139 (γH2AX), which is a marker of DNA double-strand breaks (DSB) [74], is assessed by immunofluorescence. In brief, lung sections (5μm) were mounted onto the slides and subjected for deparaffinization, rehydration, antigen retrieval and inactivation of endogenous peroxidase as described above. After blocking with 2% BSA for 30 min at 37℃, the slides were incubated with primary antibodies against γ-H2AX (#ab11174; Abcam, Cambridge, UK) followed by incubation for Alexafluor-488 conjugated secondary antibody (Thermo Fisher ScientificTM, Waltham, MA, USA) and 4’,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. Digital images were taken by a laser scanning confocal microscope (Leica, Germany). 16HBE cells treated with 5 μg/ml etopside for 12 h were regarded as a positive control. Positive staining cells were counted in 20 random fields per section in each group and the average number of positive cells per section was calculated.
Cytokine analysis
Cytokines in BALF and plasma, 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). The z-score transformation was utilized to calculate the relative level of cytokines according the following equation: Z= ( is the mean value, SD is standard deviation).
Cell culture
The neuroblastoma cells (Neuro-2A), monocytes (THP-1), liver hepatocellular carcinoma cell (HepG2), human embryonic kidney cells (HEK), colon carcinoma cells (HCT-116) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The human bronchial epithelial cells (16HBE) was a gift form Dr. D. C. Gruenert (University of California, San Francisco) [75]. HepG2, 16HBE, HEK, and HCT116 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). Neuro-2A and THP-1 were cultured in minimum Eagle’s medium (MEM, Gibco, USA) and Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, USA), respectively, supplemented with 10% FBS. Cells were incubated at 37 ℃ in a humidified chamber with 5% CO2.
Ex vivo assays
Colorimetric cell viability assay (MTS; Promega Corporation, Madison, WI, USA) was used to determinate the effect of the plasma on cell viability of multiple cell lines, including 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 for 48 h. At the end, 20 μL of MTS reagent were added to each well and incubated for 2 h. The absorbance at 490 nm was determined. The cell that not treated with the plasma from the mouse blood were regarded as a negative control. The cell viability was presented as fold changes relative to the cell viability of the negative control.
Immunoblotting
Total cellular protein of liver tissues 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 (#9662), cleaved caspase 3 (#9664), CYP1A2 (#14719) (Cell Signaling Technology, MA, USA), CYP1A1 (#ab3568), CYP1B1 (#ab33586), GSTT1 (#ab199337), GSTM1 (#ab113432), UGT1A1 (#ab194697) (Abcam, Cambridge, UK), and β-actin (#60008; 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). The frozen tissues of lung and liver (approximately 5 mg) were subjected to RNA isolation using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA samples were applied for 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 by PCR. 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 (Beijing Genomics Institute, Wuhan, China). To process sequences, the reads were filtered to obtained using SOAnuke (https://github.com/BGI-flexlab/SOAPnuke) [76]. The clean reads were aligned to a reference genome using Bowtie2 (http://bowtie-bio.sourceforge.net/Bowtie2/index.shtml) [77]. Then, the FPKM method was used to calculate the unigene expression level by RSEM (http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html) [78, 79]. Differentially expressed genes (DEGs) were identified with the R package DEGseq [80]. DEGs were restricted, with the fold-change greater than 1.5 times, P value lesser than 0.05 as the thresholds, by performing pairwise comparisons of the gene expression profiles of different diets with or without PM exposure (“CR-AF vs. AL-AF”, and “CR-PM vs. AL-PM”). The pheatmap R package was used to draw a heatmap of DEGs. To annotate the biological of the DEGs, the canonical pathway analysis and the disease and function analysis were conducted by Ingenuity Pathway Analysis (IPA) software (Qiagen, Germany). Significant differences were defined as P value was less than 0.01 and the absolute value of the z score was greater than 2.
Quantitative polymerase chain reaction (qPCR)
We conducted reverse transcription with an Advantage RT-for-PCR Kit (Takara, Tokyo, Japan), and performed quantitative realtime PCR (qRT-PCR) with a SYBR Green PCR Master Mix (Toyobo, Tokyo, Japan). β-Actin is served as an internal control. The 2–ΔΔCt method was used to calculate the relative expression of mRNAs. The primers used for qPCR are shown in Table S3.
Examination of urinary OH-PAHs
Mouse urine collected by housing animals in the metabolic cages (Type Y-3101, Yuyan Instruments Co. Ltd., Shanghai, China) for 24 h at the end of PM exposure [81]. The urine was centrifuged at 1000×g for 10 min at 4 ℃. The supernatant was filtered through 0.22 μM filter (Pall Corporation, USA). 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.