PM2.5 sampling and toxic air preparation
PM2.5 was continuously collected from several traffic arteries, Shenyang, China (December 2012 to February, 2013) using Mini Vol PM portable sampler (Air Metrics, USA) at a flow rate of 6–8 L/min. PM2.5 samples were prepared according to the method of Ma et al. Briefly, the dust collection was calculated after drying the filter membrane. The sampling filter membranes were stored at -20°C or cut into 1 cm ╳ l cm size. PM2.5 samples were extracted from sampled filter strips by immersing them in normal saline and then sonicating them for 30 min in a normal saline bath sonicator (KH5200-220V, 50HS). The samples of the underlying particle were collected, dried by vacuum freeze, and stored at -80°C prior to use. To determine and adjust the concentration of PM2.5 preparations, 100 µl aliquots of PM2.5 were placed on filters and air-dried. The samples and filters were weighed on a microbalance. PM2.5 was prepared in normal saline at 10 mg/ml and sonicated for 1 min prior to use.
Carbon monoxide (CO), nitrogen dioxide (NO2) and sulfur dioxide (SO2) were purchased from Dalian Special Gas Industry Co., LTD (Dalian, China). Three kinds of different concentration, including low, median and high, were set up. When used for experiments, the different concentration gas would be released into the toxicant exposure cabinet under the control of gas flowmeter.
Animals
In this study, 84 Wistar rats (42 female rats and 42 male rats) were randomly selected from the Animal Captivity Park of the Pharmaceutical Department of Shenyang Pharmaceutical University, China.Animal feeding environment: temperature is 22 ± 1℃, humidity is 40–60%. Rats were randomly divided into 6 experimental groups (n = 6) and corresponding control groups (n = 6) with different exposure time (1, 7 and 30 days, respectively) and exposure concentration. with 7 Wistar rats in each group. All animal experiments are approved by the Animal Care and Experimental Ethics Committee of the National Institute of The Environment, China.
Exposure Of Animals To Pm2.5 And Mixed Toxic Gas
Rats in the experimental group were intranasally injected with 1 mL of physiological saline containing PM2.5 for 5–10 minutes. The PM2.5 content in Group 1, Group 2, and Group 3 were 0.04mg/m3, 0.4mg/m3, and 4mg/m3, respectively. After 24 hours, put it in the poison exposure cabinet. The mixed toxic gas containing CO, NO2 and SO2 enters the tank under the control of a liquid gas dynamic exposure control device (Tianjin Development Zone Industry and Trade Co., Ltd., Tianjin, China). The concentrations of CO, NO2, and SO2 in the mixed toxic gases in Group 1, Group 2, and Group 3 were 0.04, 0.15, and 0.12 mg/m3; 0.4, 1.5, and 1.2 mg/m3; and were 4, 15, and 12 mg/m3, respectively. The rats in the control group were gavaged with 1ml saline to release normal fresh air to the control group.
Identification Of Bacteria From Posterior Pharyngeal Wall
Wistar rats were sacrificed after dusting on day 1, day 7 and day 30, and trachea and lung tissues were collected, and the repaired pieces were placed in 2.5% glutaraldehyde fixative for use. The trachea and lung tissue were fixed in 2.5% glutaraldehyde at 4°C for 2 hours, and then fixed in 1% OsO4 at 4°C for 2 hours. Then dehydrated with graded alcohol, washed with acetone, and embedded in epoxy resin.
On days 1, 7, and 30, ether anesthetized the secretions of the posterior pharyngeal wall into 0.5 mL of saline. Absorb 50 bacteria and place them on a blood plate to cultivate bacteria. All plates were incubated in a 5% CO2 incubator (Thermo Scientific Forma, MA, USA). API (API system, Merieux, France) and atb test paper were used to separate and identify different colonies.
Scanning And Transmission Electron Microscopy
A three-dimensional scanning (KYKY-1000B, Shanghai, China) scanning electron microscope (SEM) was used to characterize the tissue. The size and morphology of tissue samples were observed by transmission electron microscopy (H-500, Hitachi, Tokyo, Japan).
Cytokine Detection In Balf And Serum
After the rats were sacrificed, the secretions from the posterior wall of the pharynx were taken, and bronchoalveolar lavage was performed with saline three times on days 1, 7, 30. Centrifuge the bronchoalveolar lavage fluid (BALF), collect the supernatant, and detect interleukin 4 (IL-4), interleukin 6 (IL-6), interferon (INF-TNF-), tumor necrosis factor (TNF- TNF-) levels. At the same time, 2–5 mL of blood was punctured after heart exposure to prepare serum and detect cytokines. Cytokines in BALF and serum were detected by enzyme-linked immunosorbent assay. CLINIBIO-128, ASYS Hitech Gmbh, Austria) according to the manufacturer's instructions.
Cytokine Detection In Lung Tissues
Detection of cytokine levels in lung tissue. Total RNA was extracted using RNeasy Mini kit (Qiagen Sciences) according to the manufacturer's protocol. First-strand cDNA synthesis kit (Invitrogen, California, USA) was used to synthesize first-strand cDNA from total RNA. Real-time PCR was performed in iCycler (MxPro3500, Stanagene, La Jolla, CA) using SYBR green supermix (TaKaRa Biotechnology, Dalian, China) and PCR primers. See Table 1 for real-time PCR primer sequences. The amplification conditions are: 95 ℃ 30 s, 95 ℃ 60 s, 60 ℃ 31 s, 40 cycles. GAPDH mRNA was used to normalize differences in amplification efficiency.
Table 1
Sequences of primers used in real time PCR.
Gene | Primer | Sequence |
IL-4 | sense | 5’-TTTGAACCAGGTCACAGA-3’ |
| antisense | 5’-GACCGCTGACACCTCTAC-3’ |
IL-6 | sense | 5’-GAGAGCATTGGAAGTTGGGC-3’ |
| antisense | 5’-CTTCCAGCCAGTTGCCTTCT-3’ |
IFN-γ | sense | 5’-ACGCCGCGTCTTGGTT-3’ |
| antisense | 5’-GTGCGATTCGATGACACTTAT-3’ |
TNF-α | sense | 5’-CATCTGCTGGTACCACCAGTT-3’ |
| antisense | 5’-TGAGCACAGAAAGCATGATC-3’ |
GAPDH | sense | 5’-ACCACAGTCCATGCCATCAC-3’ |
| antisense | 5’-TCCACCACCCTGTTGCTGTA-3’ |
IL-4: interleukin 4; IL-6: interleukin 6; TNF-α: tumor necrosis factor α; IFN-γ: Interferon γ |
Statistical analysis
All values were expressed as mean ± SD. Data was analyzed using statistical software (SPSS 22.0, Chicago, USA) with a two-way ANOVA (PM2.5, ozone as factors). The Tukey’s multiple comparison procedure to elucidate the differences between the treated groups and the control ones. The systematic description of the statistically significant effects determined from two-way ANOVA and post hoc comparisons in studies. For the purpose of the study, we have adhered to three kinds of comparisons, which are clearly defined in the figure captions under the figures. The level of p < 0.05 was defined as statistical significance.