Animals and treatment
Female four-six-week-old Sprague Dawley rats (n = 50) weighting 85-160 g ± 5 g and four- week-old Balb/c mice (n = 15) weighting 15 ± 2 g were purchased from shanghai Slac Laboratory Animal Co. Ltd (SPF grade, Experiment Animal Co., Ltd., Shanghai, China). The animals were raised and maintained at a pathogen-free sterile barrier facility (temperature, 22 ◦C; humidity, 50%-60%) under a 12-h light/dark cycle at Shanghai Pulmonary Hospital. All the animals were provided with standard rodent diet and water ad libitum. An acclimatization period of seven days was allowed before any initiation in experimental work.
The rats and mice were subjected to the passive smoking of five unfiltered cigarettes for three times daily (15–20 min for each time), 7 days a week for up to 18 weeks and 12 weeks, respectively. An automated cigarette-smoking apparatus (Model TE-10, Teague Enterprises, Davis, CA) was used for cigarette exposure as previously described . The control groups were exposed to fresh air. We simultaneously instilled 200 μl of LPS (lipopolysaccharide, 0.5 mg/L) into the tracheal of mice twice a week for up to four weeks to promote the development of COPD via inducing chronic inflammation. All animals were sequentially administered with 4.8 units/100 g body weight of porcine pancreatic elastase (Sigma-Aldrich Corp. St. Louis, USA) via instillation at week 17 and week 7 in rats and mice, respectively. The control groups were administered with saline. A few rats (n = 5) were sacrificed at week 12 for testifying the change of the pulmonary morphology and function. A few COPD rats (n = 20) were continuous nebulized salbutamol for three weeks to construct salbutamol-resistant COPD rats. We nebulized the RIS (20 µg) in salbutamol-resistant COPD rats for two weeks for exploring the long-term effect of RIS in vivo. All animals were euthanasia with carbon dioxide (CO2) at the end of point.
The RIS (HY-B0148, MCE, New Jersey, USA) was dissolved in PBS and salbutamol (HY-B1037, MCE, New Jersey, USA) was dissolved in ethanol, diluted in PBS with 1:1000 before using. The rats and mice were randomly divided into six groups. The useful data of respiratory parameters were measured from at least four different animals in each group. Group I is the normal rats and mice that measured respiratory parameters. Group II is control group of COPD rats and mice treated with PBS. Group III are the COPD rats and mice administered with RIS nebulization at different concentrations (0, 0.31, 0.625, 1.25, 2.5, and 5 mg/ml). Group IV is the COPD rats received salbutamol (0.125, 0.25, 0.5, 1 and 2 mg/ml) treatment. Group V are the salbutamol-resistant COPD rats treated with salbutamol nebulization at different concentrations (0.125, 0.25, 0.5, 1 and 2 mg/ml). Group VI are the salbutamol-resistant COPD rats treated with different concentration of RIS (0, 0.31, 0.625, 1.25, 2.5, and 5 mg/ml).
Non- invasive and invasive mechanics were used to monitor a few respiratory parameters representing lung functional ventilatory and bronchoconstriction of rats and mice. For rats, the lung function parameters mainly include specific airway resistance (sRAW), tidal volume (TV), peak expiratory flow (PEF), peak inspiratory flow (PIF), and mid-expiratory flow (EF50). For mice, the respiratory parameters mainly include TV, PEF, PIF, minute ventilation volume (MV), airway resistance (RI) and dynamic compliance (Cdyn). The different methods were utilized to assess these parameters in order to obtain more comprehensive results of RIS efficiency on COPD animals.
Non-invasive airway mechanics using the plethysmography method
The ventilatory and bronchoconstriction parameters, sRAW, TV, PEF, PIF, EF50 and MV of rats or mice were directly recorded with a software program of Buxco® Non-Invasive Airway Mechanics (NAM) (Data Sciences International [DSI], St. Paul, MN, USA) using the Plethysmography method without anesthesia. The rats or mice were restrained in a chambered special box which allows the independent measurement of nasal, mouth, thoracic and abdomen flows. The signal difference could be calculated to determine the specific airway resistance. The patented feature of NAM could detect the pulmonary function of animals under normal breath (Additional file 1: Fig. S1).
Invasive resistance and compliance system
The respiratory parameters, RI and Cdyn of mice were directly monitored by Buxco® FinePointe Resistance and Compliance system (RC) (Data Sciences International [DSI], St. Paul, MN, USA). The mice were anesthetized and instrumented with a detector in the trachea to directly measure RI and Cdyn after RIS treatment.
The above systems both have a small nebulizer that creates the mist out of the liquid agents. All the respiratory parameters of the animals were measured 5 min after the treatment of saline, RIS or salbutamol. The baseline of the parameters of animals was monitored before the treatment. The differences between the treated parameters and baseline parameters were documented as %change. Subsequently, the animals were sacrificed to collect bronchoalveolar lavage fluids (BALFs), and lung tissue samples for further experiments.
The primary culture of rats’ airway smooth muscle cells
Primary culture of airway smooth muscle cells (ASMCs) of rats were prepared based on the differential adhesion of different cells [15, 16]. Briefly, the rats were anesthetized, and the trachea smooth muscle was separated. The isolated airway smooth muscle tissue was cut into a 1mm3 pieces and then transferred to a 10 cm cell culture dish for 30 min. The primary cells were cultured with 10 ml of Ham's F-12 medium (Thermo Fisher Scientific, South San Francisco, CA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, South San Francisco, CA) in a 37 °C incubator. The cells obtained from airway smooth muscle might mix tracheal epithelial cells and fibroblasts besides airway smooth muscle cells (ASMCs). The tracheal epithelial cells and fibroblasts adhered to the cell dish within half an hour, while ASMCs generally required at least 1–4 h. Because of the different attachment times, ASMCs can be purified in the culture dish. When the cells achieved a confluence of 80%, the cells detached with 0.25% trypsinization to prepare a cell suspension which was then transferred into a new dish. After 1-h of cell culture, the upper cell suspension containing unattached ASMCs was aspirated to a new dish. ASMCs could be purred by repeating this process for four-five times.
The NR8383 alveolar macrophage cells were purchased from ATCC and cultured with Ham's F12 medium with 2 mM L-glutamine (Thermo Fisher Scientific, South San Francisco, CA) and 15% FBS under 37 °C and 5% CO2. The NR8383 cells received the treatment of 100 µm RIS for 48 h. The ASMCs received different concentrations RIS treatment for 24 h. The inflammation response was induced by LPS (100 ng/ml) (MCE, New Jersey, USA) stimulation for 30 min.
Inflammatory cell count in bronchoalveolar lavage fluids (BALFs)
BALFs collected from the pulmonary tissues as previously described . The lavage fluids were centrifuged for 15 min at 1600 × g. The supernatant was stored at − 80 °C, and the pelleted cells were resuspended in PBS for inflammatory cells analysis. 1 × 105 cells were subjected to cytospin centrifugation on glass slides, and fixed with 100% methanol for 20 min, following with Wright-Giemsa (Beyotime, China) staining. A differential cell count of BALFs’ cell pellets was performed under a light microscope at a magnification of 200 × according to morphological characteristics.
Enzyme-linked immunosorbent assay (ELISA)
The trachea was dissected in the anesthetized rats, followed with pre-cooled PBS (1 ml) injection. The PBS solution was kept in the pulmonary for 1 min and then gently extracted. A total of 3 ml of the lavage fluid was collected and centrifuged for 20 min at 1000 × g, and the supernatant was harvested. The cytokines (TNF-α, IL-1β, IL-8, and IL-6) of the supernatant were detected by ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer's protocols. The process was repeated three times.
The formalin-fixed, paraffin-embedded (FFPE) specimens of the lung tissue in rats and mice were cut into 5 μm sections, and deparaffinized by using xylene for 10 min twice at room temperature (RT). Heat-induced epitope retrieval was performed in 1 mM Tris buffer (1 mM EDTA, PH 9.0) for 20 min. And then the sections were incubated with primary antibody of anti‑CD68 (14-0688-82, Invitrogen, USA), diluted with 1:500 at RT for two hours. The secondary rat-anti-mouse antibody conjugated with HRP (18-4015-82, Invitrogen, USA) was added on the sections for one hour after washing the primary antibody. The protein level of CD68 was determined by staining with DAB (Diaminobenzidin) (Sigma Aldrich, Saint Louis, MO, USA) and confirmed by two pathologists (Wu and Hou) in Shanghai Pulmonary Hospital.
High-performance liquid chromatography (HPLC)
The molecular of RIS lacks the structure of chromophore, which we detected the concentration of RIS in lung tissue using precolumn derivatization method of HPLC [18, 19]. The amino group in RIS is used to combine with 9-fluoronylmethyl chloroformate (FMOC-Cl) to form a fluorescent group. Briefly, lung tissues (0.5 g) of rats were firstly grinded with 300 μl of standard solution. After precipitating with 200 μl of potassium dihydrogen phosphate solution (0.1 mol/L), 200 μl of calcium chloride solution (0.1 mol/L), and 400 μl of sodium hydroxide solution (1 mol/L), the precipitates were dissolved in 1 ml of sodium acetate buffer solution (0.2 mol/L, pH 4.5). The solution was then derivatized by Bond ELUT-DEA (ethylenediamine adsorption column), and ultimately RIS concentration was detected by a fluorescence detector of HPLC system (Shimadzu LCsolution, Janpan).
Data are expressed as mean ± SEM unless otherwise specified. One-way analysis of variance (ANOVA) using SPSS20.0 (version 20.0, SPSS, Inc., Chicago, IL, USA) software, and Graph Pad Prism 9.0 software (GraphPad, San Diego, CA, USA) was performed to compare the mean differences. A statistical difference was accepted at a p -value of < 0.05.
The details of immunofluorescence (IF), Hematoxylin-and-Eosin (H&E) staining, and western blot (WB) assay were documented in the Additional file 1: Methods.