2.1 Preparation of MP extracts
MP, ATCC15531 strain (American Type Culture Collection, Rockville, MD) was cultured in modified Hayflick medium (GZBIOTEST Co., Ltd, Guangdong, China) containing PPLO broth, horse serum, 25% yeast extract, which was added with penicillin G (1000 u/ml), thallium acetate (0.025%), glucose (0.5%) and phenol red (0.002%), pH = 7.6. MP was cultured at 37 °C under 5% CO2 for 7 days.
2.2 Qingfei Tongluo formular water extracts
Qingfei Tongluo formular and was purchased from Longhua hospital affiliated to Shanghai University of traditional Chinese medicine. All medical materials were decocted by boiling in distilled water to obtain QT water extracts, and water extracts was adjusted to 3.7 g/ml.
2.3 Animals
BALB/c mice (3-week-old, 15 ± 1 g) were purchased from Shanghai sippr bk laboratory animals Co. Ltd. (Shanghai, China) and fed with feedstuff and water in a specific pathogen free (SPF) environment. All mice were with ether anesthesia and divided into five groups as control, model, AZM, QT (46.25 mg/g) and QT (92.5 mg/g) randomly. Mice in control group were treated with 100 µl normal saline by nasal drip [5]. Model, AZM, QT (46.25 mg/g) and QT (92.5 mg/g) groups were given with nasal drops containing 100 µl MPFH (1 × 107 ccu/ml) for 2 days. Control and model groups were given by gavage once a day with 0.25 ml/20 g normal saline. AZM, QT (46.25 mg/g) and QT (92.5 mg/g) groups were given by gavage with 46.25 mg/g AZM (Pfizer, New York, USA, 1164096), 46.25 mg/g QT water extracts and 92.5 mg/g QT water extracts 3 days, respectively. Ethical approval for the study was provided by the independent ethics committee, Shanghai University of traditional Chinese medicine.
2.4 LC-MS analysis of QT
An Agilent 1100 HPLC system, equipped with a quaternary pump, an autosampler, a degasser, an automatic thermostatic column compartment, a DAD and an LC/MSD Trap XCT ESI mass spectrometer (Agilent Technologies, MA, USA), was used for the separation. The separation was performed on a GS-120-5-C18-BIO chromatographic column (5 µm, 250 ⋅ 4.6 mm i.d.) with the column temperature set at 35°C. A linear gradient elution of A (0.1% formic acid water) and B (acetonitrile) was used with the gradient procedure as follows: 0 min, B 5%, to 60 min B 40% (v/v). The flow rate was 1.0 ml/min and the injection volume was 10 µL. DAD was on and the target wavelength was simultaneously set at 210 nm. The split ratio to the mass spectrometer was 1:3. The acquisition parameters for negative ion mode were: collision gas, ultra high-purity helium (He), nebulizer gas (N2), 35 psi, drying gas (N2), 10 l/min, drying temperature, 350 °C, HV, 3500 V, mass scan range, m/z 100–2200, target mass, 500 m/z, compound stability, 100%, trap drive level, 100%. All the data were analysis by Chemstation software.
2.5 Inflammatory cells
Leukocyte recruitment to alveoli was determined in the broncho alveolar lavage fluid (BALF). Briefly, animals were sacrificed under ether anesthesia and trachea was exposed and intubated with a catheter, and then repeated 1 ml injections of PBS were made until a total of 3 ml of BALF was recovered. BALF was centrifuged at 3,400 × g for 10 min, and supernatant was frozen at -80 °C until analysis of inflammatory mediators. Cells in the pellet were resuspended in PBS for quantification of leukocytes with a haemacytometer, and cell populations were enumerated from Diff-Quik Stain kit (Thermo Fisher Scientific Inc.).
2.6 Histological examination
Inferior lobe of right lung isolated on the 3th day was fixed with 4% paraformaldehyde, embedded with paraffin and cut into slices for HE staining. Morphometric analysis was performed by optical microscope (LEICA DMLB, Germany).
2.7 ELISA
Cytokine (IL-6, IL-10, TNF-α and IL-13) was measured by ELISA assay. Briefly, lung homogenates were lysed in lysis buffer pH 7.4 which is consisted of 300 mM NaCl/L, 15 mM TRIS/L, 2 mM MgCl2/L, 2 mM Triton X-100/L, 20 ng pepstatin A/mL, 20 ng leupeptin/mL, and 20 ng aprotinin/mL. Then lung homogenates were centrifuged at 1500 × g for 15 min at 4 °C; the supernatant was frozen at -20 °C, until cytokine measurement by ELISA as per manufacturer’s protocol (Ray Biotech).
2.8 Western blot
Lung tissues were harvest and washed twice with PBS and lysed in ice-cold radio immunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China) with freshly added 0.01% protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and incubated on ice for 30 min. Tissue lysis was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant (20–30 µg of protein) was run on 10% SDS-PAGE gel and transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Bredford, USA). The blots were blocked with 5% skim milk, followed by incubation with primary antibodies. Antibodies against COX-2, TLR2 and TLR4 were purchased from Abcam. Antibodies against NF-κB, β-actin and H3 were purchased from Santa. Blots were then incubated with goat anti-mouse secondary antibody (Beyotime, Shanghai, China) or goat anti-rabbit secondary antibody (Beyotime, Shanghai, China) and visualized using enhanced chemiluminescence (ECL, Millipore).
2.9 Statistical analysis
The GraphPad Prism 5.0 software system was employed for statistical analysis. Data are expressed as the mean ± standard error. Student‘s t test was used to compared the differences between two groups, while one-way analysis of variance was used when more than two groups were compared. P < 0.05 was taken as statistical significance.