Male Sprague-Dawley rats (7–9 weeks in age) were purchased from Guangzhou University of Chinese Medicine (Guangzhou, China). The Animal Medical Center of Guangzhou Medical University reviewed and approved all experiments (identification number: GY2019-009). A total of 54 rats were randomly divided into three groups: control, BMF, and MVE (n = 6 per group) for three exposure durations (4, 12, and 24 weeks). All rats were kept in a specific pathogen-free room and were housed three to a cage. Except the time of the PM exposure, the animal facility conditions of the BMF and MVE groups are the same as those of the control group. The animal facility maintained temperature and relative humidity at 23 ± 2°C and 40%–70%, respectively. Lighting was artificial with a sequence of 12 h light (06:00–18:00) and 12 h dark. Commercially available rodent food pellets and water were provided ad libitum. Rats were weighed every 2 weeks throughout the study. Corncob bedding and cage are replaced every 3 days. The rats were observed for any sign of illness a minimum of twice daily.
PM exposure system and characterization of the test atmosphere
Fig. 1A depicts the design of the study. Rats were exposed to PM as described previously . Briefly, all animals were exposed in whole-body inhalation chambers for 4 h/day, 5 days/week either 4, 12, or 24 weeks. PM mass concentrations, particle size distributions, and gas concentrations were monitored each day of exposure. DustTrak II aerosol monitors (model 8530, TSI, Shoreview, MN, USA) were used to monitored PM mass concentrations and particle size distributions. Testo 340 portable flue gas analyzers (Testo, Lenzkirch, Germany) were used to monitored gas concentrations (O2, carbon monoxide, nitrogen oxides, and sulfur dioxide) in the exposure rooms. The control group was exposed to clean air.
Exposure to BMF. As Chinese fir (Cunninghamia lanceolata) is the major indigenous tree species that occupies approximately 25% of plantations in subtropical areas of southern China , we used Chinese fir sawdust (40 g/per exposure), as a representative, to produce BMF smoke, which was sent into the animal exposure room through a piston pump (5 L/min). Rats were exposed to BMF smoke for four 1-hour periods, 5 days per week. The test atmosphere was measured during the first hour.
Exposure to MVE. Previous studies indicated that exposure to higher traffic-related air pollutants was strongly associated with increased COPD prevalence . Therefore, the gasoline-powered motorcycle was used as a source of MVE to stimulate a real-world pollution. MVE was produced by a Wuyang model WY48QT-2, 1.6-Kw, 125-cm3, one-cylinder, four-cycle, gasoline-powered motorcycle (Guangzhou, China). Premium low-sulfur gasoline (<150 ppm; Petro Inc., El Paso, TX, USA) was used to produce MVE. Prior to the exposure session, the motorcycle engine was operated in an idle state for 2 minutes to produce sufficient MVE. Rats were exposed for two 2-hour periods, 5 days per week. The test atmosphere was measured during the first 2-hour interval.
Ambient BMF and MVE samples were collected throughout the duration of exposure to determinate the mass concentration and composition of atmospheric aerosol. Concentrations of organic carbon, elemental carbon, polycyclic aromatic hydrocarbons, and metals were further measured at the Guangzhou Institute of Chemistry, Chinese Academy of Sciences (Guangzhou, China) according to previous studies [12, 13].
Measurement of lung function
Spirometry data were obtained as previously described using a Forced Pulmonary Maneuver System (Buxco Research Systems, Wilmington, NC, USA) . Rat were sedated with 3% pentobarbital (1mL/Kg), tracheostomized and intubated, then placed supine in the body chamber and connected to the system. According to the procedures, the FRC (functional residual capacity), FEV20 (forced expiratory volume in 20 seconds), FEV100 (forced expiratory volume in 100 seconds) and PEF (peak expiratory flow) were measure. At least three acceptable maneuvers for each test of every mice were conducted to obtain a reliable mean spirometry data.
Rats were sacrificed by CO2 after 4, 12, and 24 weeks of exposure (on days 29, 85, and 169, respectively). Blood samples were collected from the heart and centrifuged at 1,700 × g for 15 min at 4°C. Serum was stored at −80°C. Proximal colon contents were harvested using sterile instruments for each individual animal and site. Fresh proximal colon contents samples were snap-frozen in liquid nitrogen then stored at −80°C for microbial and SCFA analysis.
Bronchoalveolar lavage fluid differential cell count
Bronchoalveolar lavage fluid (BALF) was collected as previously reported . Cells were isolated by centrifugation at 300 × g for 10 min at 4°C and stained with Diff-Quik stain (Baso Diagnostics, Zhuhai, China). Differential cell counts were assessed from 400 cells counted on each slide.
Lung morphometric analysis
As described previously , lung tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin using standard methods. Sectioning and staining were performed by the Pathology Center of the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). All slides were scanned and analyzed using an image analyzer platform (Leica, Wetzlar, Germany). Alveolar enlargement and destruction, and the bronchial wall thickness was calculated as describe previous .
Serum Levels of Lipopolysaccharide and total BALF protein assay
Serum levels of LPS were measured using a commercial chromogenic end-point TAL kit (Xiamen Bioendo Technology Co., Ltd., Xiamen, China). All procedures were performed according to the manufacturer’s instructions. The total protein in the BALF determined by Bicinchoninic Acid (BCA) method using a commercial BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The concentration of endotoxin from DMSO-extract of particulate matter were performed at the Kingmed Diagnostics Center (Guangzhou, China). All procedures were performed according to the Pharmacopoeia of the China (2015 edition) volume IV.
DNA was isolated from colon contents and BALF samples using a Qiagen QIAamp® DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. 16S rRNA gene amplification, in vitro transcription, and labeling and hybridization were performed following the Illumina 16S Metagenomic Sequencing Library Preparation guide . We used a MiSeq rRNA amplicon sequencing protocol to PCR-amplify the V3–V4 variable regions (amplicon size expected: approximately 460 bp). 16S amplicon PCR forward primer was 5′-(TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG)-3′ and 16S amplicon PCR reverse primer was 5′-(GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C)-3′ . All samples were paired-end sequenced on an Illumina MiSeq PE250 platform (San Diego, CA, USA) by the RiboBio Genome Center (Guangzhou, China). 16S rRNA gene sequence analysis, including raw sequence filtering and taxonomic classification, was performed as described previously . The bar diagrams of alpha diversity indices and relative abundance were drawn using GraphPad Prism 8 software (La Jolla, CA, USA).
Quantification of SCFAs in colon contents
Seven SCFAs (acetic, propionic, butyric, isobutyric, valeric, isovaleric, and caproic acids) in the proximal colon were measured by high-performance gas chromatography-mass spectrometry (Agilent 6890N; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s recommendations. Briefly, a total of 100 mg of the colon contents were homogenized by ultrasonication in 600-μL reactions containing 100 μL of phosphoric acid (15%), 100 μL of 4-methylvaleric acid (250 μg/mL), and 400 μL of diethyl ether. The mixtures were vortexed and centrifuged until a clear supernatant was obtained. The sample was subjected to a high-performance liquid chromatography column (Agilent Technologies) for analysis of SCFAs.
Data are reported as mean ± standard deviation (SD). Comparisons were performed using ANOVA and p values were corrected for multiple testing with the Bonferroni method. Statistical analysis was performed using SPSS version 24 (IBM SPSS, Armonk, NY, USA). Correlations between serum LPS levels and the pulmonary mean linear intercept (MLI) were assessed using Spearman’s rank correlation. p < 0.05 was considered significant.