Animals
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. 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. 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.
PM exposure system and characterization of the test atmosphere
Fig. 1 depicts the design of the study. Rats were exposed to PM as described previously [6]. 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.
xposure to BMF. We used China fir sawdust (40 g/per exposure) 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-h periods, 5 days per week. The test atmosphere was measured during the first hour.
Exposure to MVE. 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 min to produce sufficient MVE. Rats were exposed for two 2-h periods, 5 days per week. The test atmosphere was measured during the first 2 h.
Ambient BMF and MVE samples were collected throughout the duration of exposure, and concentrations of organic carbon, elemental carbon, polycyclic aromatic hydrocarbons, and metals were measured at the Guangzhou Institute of Chemistry, Chinese Academy of Sciences (Guangzhou, China) according to a previous method [12, 13].
Sample preparation
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. Colon contents were collected under sterile conditions, snap-frozen in liquid nitrogen, and stored at −80°C for microbial and SCFA analysis. Bronchoalveolar lavage fluid was collected as previously reported [14].
Lung morphometric analysis
As described previously [6], 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 were quantified by the mean linear intercept [15].
Enzyme-linked immunosorbent 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.
Microbiota analyses
DNA was isolated from colon contents 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 [16]. 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′ [17]. 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 [18]. 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 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.
Statistical analysis
Data are reported as mean ± standard deviation (SD). Comparisons were performed using ANOVA and p values were corrected with the least significant difference (LSD) 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 were assessed using Spearman’s rank correlation. p < 0.05 was considered significant.