Materials
Ethics approval for the study was obtained from the Fourth Military Medical University. Sprague-Dawley(SD) rats (2 male, 120g±20g and 102 female, 200g±20g) were provided by the Experiment Animal Center of the Fourth Military Medical University(Xi’an, China) . 200g/L PQ solution dichloride was obtained from Tianjin Shipule Agriculture Pesticides Technology & Development Inc(Tianjin, China). MP was obtained from Pfizer Inc (New York, USA) . Fluorescein isothiocyanate(FITC)-conjugated anti-mouse CD34, CD45, CD44 and CD166 antibodies were obtained from Biolegend(San Francisco, USA). The nucleoprotein extraction kit was obtained from Promega (Madison, USA), the NF-кBp65 polyclonal antibody from Santa Cruz (California, USA). The rat Interleukin-1β(IL-1β) and Tumor necrosis factor-α(TNF-α) and Interleukin-6(IL-6) and Interleukin-10(IL-10) ELISA kits from Sigma (St.Louis,USA). Malonaldehyde (MDA) and superoxide dismutase (SOD) kits were obtained from the Nanjing jiancheng Bioengineering Institute(Nanjing, China).
Isolation, culture and identification of BMSC
Male rats were sacrificed by cervical dislocation and the femur and tibia were harvested. The bone marrow cells were gently resuspended into single cells. BMSCs were isolated by Percoll gradient centrifugation, and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)-F12 supplemented with 10% fetal bovine serum Cells were seeded in 25 cm2 culture flasks at 1 × 105 cells/cm2 and maintained at 37°C with 5% CO2 in a humidified incubator. Culture medium was exchanged after 48 h and then every 2–3 days. Cells were cultured to 80–90% confluence and then digested with 0.25% trypsin containing 0.02% EDTA for 1 min. Then, cells were centrifuged, resuspended and passaged at a 1:3 ratio. Third passage cells were tested using flow cytometry for cell surface expression of CD34, CD45, CD44 and CD166 [9].
Grouping
Female SD rats were randomly divided into five groups: 1) PQ group (n = 24): PQ solution was intraperitoneally injected at a dose of 20 mg/kg. 2). BMSC group (n = 24): BMSCs (0.5 mL, 1 × 107 cells) were injected via the tail vein at 6 h after PQ injection. 3). MP group (n = 24): MP (20 mg/kg) was administered via the tail vein once a day for 2 consecutive days at 2 h after PQ injection. 4). BMSC + MP group (n = 24): BMSCs and MP were administered as described above. 5) Normal control group (n = 6): rats were intraperitoneally injected with 0.9% saline.
Macro observation
Animals were observed for general activity, feeding and survival for 14 days after PQ poisoning.
Wet/dry weight ratio of lung tissue
At 1, 3, 7 and 14 days post-injection, the right lung was isolated and the trachea ligated. After blotted off the blood and other contaminants, the wet weight was measured. Then the lung was dried in a 70°C oven for 72 h and the dry weight was measured. The lung wet/dry weight ratio was calculated.
Determination of cytokine levels
Plasma was collected on days 1,3,7 and 14 post-injection and the concentrations of TNF-α, IL-1β, IL-6, IL-10, MDA and SOD measured by respective ELISAs kits.
Nucleoprotein extraction and Measurement of NF-кBp65 expression
On days 1, 3, 7 and 14 post-injection, the left-sided lung tissues were isolated, and rinsed with cold phosphate buffer solution (PBS). Nucleoprotein lytic/extraction buffer solution A (2 ml) was added and the tissues incubated on ice for 30 min. The tissue was then homogenized, mixed with 500μl of 10% Nonidet P-40 and centrifuged. The precipitate was rinsed twice with cold PBS and the suspension was discarded. Nucleoprotein extraction/lytic solution B (2 mL) was added to the precipitate on ice for 30 min and the solution centrifuged again to harvest the nucleoproteins. Nucleoproteins (10μg) were measured by western blotting. The images were scanned using a gray-scale scanner and the averaged gray values were used to determine the NF-кBp65 expression level.
Statistical analysis
All data were statistically analyzed by SPSS 11.5 software and expressed as mean±standard deviation. The 2-group comparison were performed with t-test. P<0.05 was considered statistically significant.
