Preparation of Ardisia japonica
Ardisia japonica (500 g), purchased from Anguo TCM market (Hebei, China) was added into 500 mL of water and soaked for 20 min. Thereafter, the mixture was boiled and then another 500 mL of water was added. Then, the medicine was decocted into 100 ml using slow fire. Finally, a decoction (100 g/100 mL) was prepared and kept at 4 ℃ for use.
Animals and COPD model
120 healthy male SD rats (120 ± 20 g, 6 weeks) were purchased from Tengxin Biotechnology Co., Ltd. in Chongqing [Certificate No. SCXK (Chongqing) 2012-0005] and housed in a SPF condition with a standard 12-h light/12-h dark cycle and ad libitum access to food and water. The average humidity was 40%-60%, and the average temperature was 20-25 ℃. The experiments were approved by the Ethics Committee of Guiyang College of Traditional Chinese Medicine.
The COPD model was established as previously described (26). Briefly, SD rats were put in a smoking box (40 x 50 x 60 cm). In each smoking box, 10 rats were placed and exposed to 8 cigarettes each time and the smoking process lasted about 30 min. The passive smoking was delivered for twice each day for consecutive 28 days. On the first and the 14st days, lipopolysaccharide (LPS) (0.2 mL, 200 μg/200 μL, SIGMA) was administrated through intratracheal injection in the anesthetized rats (10% chloral hydrate, 0.37 mL/100 g). After anesthesia, the rats were fixed on the table in supine position. Longitudinal incision along the midline neck (about 1 cm) was prepared to deliver LPS. The rats were erected to keep the LPS flowing along the wall of the trachea to the alveoli. The wound was sutured layer by layer, and penicillin powder was applied to the incision. Eight days after modeling, animals were anesthetized with 5% isoflurane and airway tissues were fixed in 4% paraformaldehyde (PFA). The lung injury was confirmed by H&E staining.
The experiments were divided into normal group (Group A), COPD group (Group B), COPD + low dose of AJ group (0.75 mL/kg, Group C); COPD + medium dose of AJ group (1.5 mL/kg, group D); COPD + high dose of AJ group (3 mL/kg, group E); COPD + dexamethasone group (Group F). AJ was delivered through intragastric administration once daily for 30 days. In COPD + dexamethasone group, the rats were treated with 1 mg/kg dexamethasone (intramuscular injection, once daily) for consecutive 30 days. After that, inferior lobes of the right lung tissues were collected for molecular biochemical experiments or fixed in 4% for pathological staining (Fig.1).
H&E staining
Inferior lobes of the right lung tissues were fixed in 4% paraformaldehyde for ~1 week at 4˚C. Thereafter, the tissues were dehydrated, embedded, and sliced. The paraffin section is dewaxed and hydrated. The tissues were rinsed for several hours, thereafter dehydrated, embedded, and sliced. The paraffin section is dewaxed and hydrated. The sections were stained with hematoxylin for 5 min and with eosin for 3 min. The images were taken by a light microscopy.
Preparation of airway fibroblasts
Under the anesthesia with isoflurane (5%), the rats were decapitated and thoracotomized in aseptic conditions. Fresh airway tissue of COPD rats was washed repeatedly with PBS in aseptic worktable for 5 times. Sterile scalpel was used to collect the tissue around the airway. The tissue was cut into 2 mm x 2 mm with sterilized ophthalmic scissors and pasted into the culture plate, and placed in a 5% CO2 incubator 37˚C for 4 h. The cells completely adhered to the wall and the newly prepared DMEM (Gibco) + 20% FBS (Hyclone) medium was added to the culture plate, and the cells were further cultured in a 37˚C, 5% CO2 incubator. The growth of the cells was observed and then treated routinely. The cultured dishes were fixed in 4% paraformaldehyde for 15 min, and permeated with 0.5% Triton X-100 (PBS) for 20 min at room temperature. 5% BSA was dripped into the dish and sealed at 37 ˚C for 30 minutes. A sufficient amount of diluted vimentin (1:250, ab92547, Abcam) was dripped into the dish and incubated at 37 ˚C for 3 h. The diluted fluorescent antibody Cy3 (1:200) was added and incubated at 37˚C for 30 min. DAPI (4',6-diamidino-2-phenylindole) was used to stain the nuclei. The images were observed under fluorescence microscope.
The collected airway fibroblasts were divided into nine groups as listed in Fig.1A: Normal, lithium chloride (Licl), 4-Ethyl-5,6-Dihydro-5-methyl-[1,3] dioxolo[4,5-j] phenanthridine (HLY78), TPA (cat. no. 425KO21, Solarbio) and epidermal growth factor (EGF) (L1450411, Cyagen) groups. The cells were treated with 10 mM Licl, 10 μM HLY78, 10 nmol/L TPA and 50 ng/ml EGF for 24 h, respectively.
ELISA
Matrix metalloprotein (MMP)-9, platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) levels were detected by ELISA following the instructions of the assay kits (QIYI Biotech, Shanghai, China). The reagents in the kit were kept at room temperature for 30 minutes. A standard curve was established to calculate the levels of target proteins. The airway tissues were homogenated and centrifuged at 11,000 g for 10 min at 4 ℃. The supernatants were used in the experiments. All standard samples and test samples required 3 duplicates. A blank control was set without sample and enzyme reagent. Absorbance was detected at the wavelength of 450 nm by a Microplate Reader (RT-6100, Rayto).
Inferior lobes of the right lung tissues were fixed in 4% paraformaldehyde for ~1 week at 4˚C. Thereafter, the tissues were dehydrated, embedded, and sliced. The paraffin section is dewaxed and hydrated. Immunostaining of histological sections was performed using monoclonal antibodies against RhoA (1:200, ab187027, Abcam, USA), Wnt5a (1:200, ab174963, Abcam, USA) and β-catenin (1:200, ab32572, Abcam, USA). Endogenous peroxidase activity was blocked with 3% (v/v) H2O2 for 5 minutes at room temperature. Subsequently, tissues were incubated with primary antibodies overnight at 4 ˚C, followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 30 min at room temperature and visualized with 3,3’-diaminobenzidine chromogen for 3 minutes at room temperature. The images were taken under a light microscopy.
Western blotting
Homogenates from inferior lobes of the right lung tissues were obtained from each group and lysed. The protein level was detected by Bicinchoninic Acid Kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%) and transferred onto nitrocellulones membranes. The membranes were blocked with Tris-buffered saline (TBS), containing 0.1 % Tween-20 (TBST) and 5 % fat-free milk for 2 h at room temperature and then incubated (overnight, at 4 ℃) with the rabbit antibodies against RhoA (1:200, ab187027, Abcam, USA), Wnt5a (1:200, ab174963, Abcam, USA) and β-catenin (1:200, ab32572, Abcam, USA) and Actin (1:500, Zsbio, Beijing, China) followed by a 2-h incubation with a peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000, Zsbio, Beijing, China) at room temperature. Chemiluminescent substrate detection reagent was applied to show the staining.
Data analysis
The data were expressed by mean ± standard deviation and statistically analyzed by SPSS 19 (SPSS, Inc., Chicago, IL, USA). One-way ANOVA with Newman-Keuls as the post-hoc test was applied to determine statistical significance. A value of P<0.05 was considered to be significant.