2.1 Establishment of the COPD rat model
Seven-week-old SPF male Sprague-Dawley (SD) rats (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed under conditions of 12-hour light/dark cycle, 25-26℃, and 50-60% humidity at the animal facility, Beijing Chaoyang Hospital. Rats were given ad libitum access to food and water. All animal procedures approved by the Animal Care and Ethics Committee of Beijing Chaoyang Hospital were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Beijing Chaoyang Hospital, Capital Medical University. After a 1-week adaptation, the rats were randomly divided into three CS-exposed groups and the respective control groups according to the duration of CS exposure: a 3-month CS group (S3, n = 10) with a matched no-smoking control group (C3, n = 14); a 6-month CS group (S6, n = 9) with a matched control group (C6, n = 15); and a 6-month CS Group followed by 3 months cessation (S6N3, n = 9) with a matched control group (C9, n = 7).
The CS group rats were exposed to CS with Baisha Filter cigarettes (tobacco type; tar, 10 mg; nicotine content, 1.0 mg; and carbon monoxide, 13 mg) for 1 h twice a day. The SIBATA SG-300 oro-nasal cigarette suction system (Shibata Scientific Technology Ltd., Tokyo, Japan) and a CS exposure chamber were used. The exposure lasted for 3 min/cigarette, and 20 cigarettes were used for each exposure procedure, with a maintained level of total particulate matter of 993.6 ± 125.7 mg/m3. The control rats were housed under the same conditions without CS exposure. The COPD model was established by evaluating the pulmonary functional changes and lung tissue histological changes for further experiments. The rats were excluded if the rats died prematurely, if the rats did not meet the following experimental schedules and criteria, or if the examples failed to meet quality control standards described below.
2.2 Pulmonary function test and lung histopathological analyses
Pulmonary function was assessed usinganAniRes2005 animal lung function analysis system (Beijing Beilanbo Technology). The forced expiratory volume in 0.3 s (FEV0.3), forced vital capacity (FVC), and the ratio of FEV0.3/FVC were calculated.
Following the pulmonary functional assay, the rats were sacrificed by exsanguination from the abdominal aorta. The lungs were inflated with 4% paraformaldehyde solution through the endotracheal intubation at a constant pressure of 25 cmH2O for 10 min. Then, the right lower lobe was removed from rats after ligating the bronchi, fixed in 4% paraformaldehyde solution for 48 h, and embedded in paraffin. These paraffin-embedded tissues were cut into 5-μm sections and stained with hematoxylin and eosin (H&E, Scy Tek Laboratories).
Emphysema, reflecting pulmonary injury, was assessed by measuring the mean linear intercept (MLI) and the mean alveolar number (MAN), as previously described . Ten fields (100×) for the MLI and twenty fields (200×) for the MAN (without the large trachea and blood vessels) were randomly selected and a cross was drawn through the center of each field. The number of alveolar intervals (Ni) lying on the cross, the total length (L) of the cross, the area of the field (S), and the number of alveoli (Na) in each field was determined (MLI = L/Ni and MAN = Na/S) using Adobe Photoshop CS5.
Semi-quantitative histological assessment of H&E sections was performed blindly using an ordinal scoring system designed to distinguish the degree of lung inflammation . Severity scores ranging from 0 to 4 were used, and severity was assessed by noting the most advanced grade present within the specific sample irrespective of its horizontal extent. The extent was defined as the horizontal distribution of pathology, where a score of 0, 1, 2, or 3 meant that none of the lung was involved, ≤ 1/3 involvement, 1/2 involvement, or ≥ 2/3 involvement, respectively. The overall score was defined as a combined assessment of severity and extent (overall score = severity × extent).
2.3 Contractile function of the diaphragm in vitro
Diaphragmatic contractile properties were measured using the RM6240 biological signal acquisition and processing system (Chengdu Instrument Factory, Sichuan, China). The diaphragm was quickly removed and immediately immersed in Kreb’s solution containing the following (mmol/l): 118NaCl, 4.7KCl, 1.25CaCl2, 1.2MgSO4, 1.2KH2PO4, 25NaHCO3, and 11Glucose; pH value, 7.3–7.4. A rectangular muscle bundle, ~5mm wide, parallel to the long axis of the fibers, was cut in the upper region of the right hemi-diaphragm with the central tendon and rib reserved. The diaphragm bundle was placed vertically into the glass bath filled with Kreb’s solution at 37°C and perfused with a mixture of 95% O2 and 5% CO2.
The rib end of the diaphragm was tied to an “L”-shaped metal bar at the bottom of the bath and the other end was connected to the tension transducer. Two silver stimulating electrodes separated by a distance of 1cm were inserted into the bundle. After a 30-min thermo-equilibration period, the bundle was extended to its optimal length (Lo, the length with peak twitch force) and stimulations were delivered through an electronic stimulator. Subsequently, the threshold voltage at Lo was determined, defined as the minimum stimulus intensity that caused the maximum contraction of the diaphragm bundle. The voltage was then increased by 20% to ensure supramaximal stimulation and this voltage was used during the subsequent experiments.
Twitch characteristics: Two twitches were recorded at Lo to determine the twitch tension (Pt), the maximum tension rise rate (+dT/dtmax), and the maximum tension fall rate (-dT/dtmax). The average value for the two twitches was used for further analysis.
Maximal tetanic tension (Po): The bundle was stimulated twice tetanically at 160 Hz with a 0.2ms wave width for 250 ms to obtain a clear plateau in force generation. The Po was recorded as the plateau tension and the average value for the two stimulations was used.
Force-frequency curve: The bundle was stimulated at the following frequencies: 10, 20, 40, 60, 80, 100, and 150 Hz. Each stimulus was separated by a 2-min interval. The curve was plotted with stimulation frequency on the X axis and tension on the Y axis.
Fatigue index (FI):The FI was assessed with a low-frequency protocol in accordance with the modified Burke method . The muscle bundle was fatigued by using 330-ms stimulations repeated at 40 Hz and applied every second for 2 min. The FI was calculated as the force at the end of the 2-min stimulation divided by the maximum force during the initial stimulation.
Standardization of values: The values of Pt and Po were corrected using the cross-sectional area (CSA) of the muscle bundle. The CSA (cm2) was calculated by dividing the weight by the specific density (1.056g/cm3) and muscle length.
2.4 Electron microscopy
Samples of the diaphragm were processed for electron microscopy according to the standard methods at the Electron Microscope Laboratory of Peking University People’s Hospital. Briefly, each diaphragm sample was immediately fixed with 3% glutaraldehyde solution and 1% osmic acid solution, and then dehydrated with different concentrations of ethanol. The samples were embedded in epoxy resin. Ultrathin sections were obtained along the long axis of muscle fibers and stained using uranyl acetate and lead citrate. The ultrastructure of the diaphragm cells, such as the muscle fiber arrangement, “Z-line,” and mitochondrial morphology, was observed and photographed in three copper nets for each sample using an FEI Tecnai Spirit Transmission Electron Microscope at constant calibrated magnifications of 6,000× and 1,1500×. Ten randomly selected fields from each copper net were analyzed (6,000×). The mitochondrial number density (NA) and mitochondrial volume density (Vv) were established and calculated by Image Scope image analysis software. Ten horizontal and 10 vertical lines were cross-drawn equidistant through the space. NA (/μm2) = mitochondrial number of reference space/whole space area; Vv (%) = number of points that hit mitochondria/number of points that hit reference space .
2.5 Quantitative Real-Time polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, USA). First-strand cDNA synthesis was performed using PrimeScript RT Master Mix (TaKaRa Biotech, Dalian, China). The mRNA levels of genes of interest, such as PTEN induced putative kinase 1 (Pink1), BCL2 interacting protein 3 (Bnip3), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), optic atrophy 1 (Opa1) and dynamin-related protein 1 (Drp1), were measured by qPCR using SYBR Green Master Mix (TaKaRa Biotech, Dalian, China). The total amount of mRNA was normalized to Actin levels. The primers needed are listed in Table 1.
2.6 Western Blot
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was run on 8 or 10% gradient gels with equal amounts 50ug protein loaded. Relative amounts of Pink1, Bnip3, Mfn1, Opa1 and Tubulin were assessed using the following primary antibodies: (1) Pink1 (product no. ab23707, Abcam, Cambridge，MA); (2) Bnip3 (product no. ab109326, Abcam, Cambridge, MA); (3) Mfn1 (product no.ab104274, Abcam, Cambridge, MA); (4) Opa1 (product no. ab157457, Abcam, Cambridge, MA); (5) Tubulin (product no. ab108342, Abcam, Cambridge, MA). Secondary antibodies (Zhongshan Golden Bridge, Beijing, China) were applied at room temperature for 2 hours. Protein bands were visualized using ECL reagent. Image J software was used for subsequent quantification and statistical analysis.
2.7 Statistical Analysis
For normally distributed continuous variables, the data are presented as the mean±SD, unless stated otherwise. To compare continuous variables, the Kolmogorov–Smirnov test was used to test the normality of the data. Statistical comparisons between two groups were performed via two-tailed Student’s t-test for normally distributed continuous variables or the Mann–Whitney U test for non-normally distributed continuous variables. Statistical comparisons of three or more groups were performed using One-way ANOVA (Least significant difference post hoc test) for normally distributed variables, and one-way Kruskal–Wallis tests (Dunn’s post hoc test) for non-normally distributed variables. Statistical significance was set at p<0.05. The data were analyzed with SPSS 17.0 software.