Tumor necrosis factor-like weak inducer of apoptosis regulates quadriceps muscle atrophy and fiber-type alteration in a rat model of chronic obstructive pulmonary disease

A total of 44 male Wistar rats (age, 8 weeks) weight, 180-220 g) were used in the present study. The rats were purchased from the Agricultural University of Hunan (Changsha, HuNan, China) and were housed in a pathogen-free, temperature- and humidity-controlled environment (70% humidity; 20 ± 2 °C) under a 12/12 h light/dark cycle in the Third Xiangya Hospital Experimental Animal Center of Central South University (Changsha, Hunan, China). Animals were tested periodically to ensure that they remained pathogen-free. For biochemical assays, the rats were randomly sorted into a control group (n = 16) and a COPD group (n = 28). Rats in the COPD group were exposed to cigarette smoke (CS) from commercial filter cigarettes (Leiothrix cigarettes, 8 mg of tar and 0.6 mg of nicotine per cigarette; Changsha Cigarette Factory, Changsha, China) for 90 d and on day 15 were exposed to porcine pancreatic elastase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) via tracheal dropping following tracheotomy, whereas the control group was injected with saline alone. Rats in the COPD group were exposed to the smoke emitted from 20 burning cigarettes per day, as described previously by Menegali et al. [32]. Briefly, animals were placed in a covered inhalation chamber (60 × 70 × 80 cm) and positioned under an exhaust hood. A cigarette was coupled to a plastic 60-ml syringe so as to draw in and expelled cigarette smoke into the exposure chamber. A total of 1 l smoke (20 puffs of 50 ml each) was aspirated from each cigarette and each puff was immediately injected into the inhalation chamber. When 1 L of smoke had been injected into the chamber, animals were maintained in this condition (3% smoke) for 6 min. The cover was then removed from the inhalation chamber and the exhaust hood switched on in order to remove the smoke within 60s. This process was immediately repeated. Animals were sacrificed in the day 90. Samples were isolated from all animals in each group for histological analysis. The right ventricle was perfused with sterile saline (0.9%) to remove blood from the lung. The right lung was fixed by infusion with 4% phosphate buffered formalin (pH 7.2) in 25 cm H₂O for 2 min at 4 °C through a tracheal catheter, after which it was removed and weighed. The right lungs were then fixed in 4% paraformaldehyde at room temperature for 24 h and embedded in paraffin. Serial sagittal sections (5 μm) were obtained for histological and morphometric analyses. Samples of left lung tissue and quadriceps muscle were stored at −70 °C for no more than a month for later experiment use. The study was approved by the Institutional Review Board of Central-South University and conformed to the guiding principles for research involving animals and human beings [33].

A small animal lung function instrument (PLY3211; Buxco Research Systems, Wilmington, NC, USA) provided by the School of Basic Medical Science of Central South University (Changsha, China), was used to measure pulmonary function in the rats. Briefly, at day 90 rats were weight and subsequently anaesthetized by an intraperitoneal injection of 10% chloral hydrate (3 ml/kg; Ming Bo Biological Technology Co., Ltd., Shanghai, China) and maintained under a deep plane of anesthesia. The trachea was opened with an inverted Y-shaped incision at the second and third cartilage ring and immediately intubated with a Y-type cannula. Inspiration and expiration volumes of the lungs were then measured. An outlet of the intra-tracheal Y-type cannula was connected to a pressure transducer linked to a pulmonary mechanics analyzer (PLY3211; Buxco Research Systems), and the other was used for administration of air into the lungs. A total of 6.0 ml air was administered into the trachea and the forced expiratory volume at 0.3 s (FEV0.3), forced vital capacity (FVC), peak expiratory flow (PEF) and ratio of FEV0.3/FVC were measured by the analyzer.

The evaluation criteria used to determine whether establishment of the COPD rat model was successful were based on measurements of weight change, hematoxylin and eosin (H&E) staining of lung tissue and lung function, as described previously [34].

To block endogenous peroxidase activity, 5 μm-thick deparaffinized sections were incubated with 1% H₂O₂ for 30 min at room temperature. To observe the morphological characteristics and detect protein expression in the quadriceps muscle, the quadriceps muscle tissues were reacted overnight at 4 °C with anti-TWEAK (ab37170; 1:400; Abcam, Cambridge, UK), anti-NF-κB (sc8008; 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PGC-1α (bs-1832R, 1:300, BIOSS, Beijing, China) and anti-MuRF1 (bs-2539R; 1:300; BIOSS, Beijing, China) antibodies. Samples were then washed with PBS and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (PV-8000; 1:200; ZSGB-BIO, Beijing, China) for 1 h at room temperature. Following the removal of non-reacted secondary antibodies by washing with PBS, samples were incubated with 3,39-diaminobenzidine (DAB; Sigma-Aldrich; Merck KGaA) in a DAB-4HCl-H₂O₂ solution to visualize immunolabeling. Some sections were also counterstained with hematoxylin and eosin and mounted with a coverslip with neutral resins. Immunohistochemical analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). A total of 4–5 images that were positive for protein expression were randomly selected and their integral luminosity values and average optical density were assessed using a light microscope.

The calcium-cobalt method developed by Padykula & Herman [35] was modified to improve the buffering capacity of the medium. Briefly, quadriceps muscle sections were fixed for 2 min in cacodylate-buffered 4% formaldehyde at pH 7.0. No fixation would have lead to sections floating off the coverslip while prolonged fixation would have affected enzyme reactivity [36, 37]. Sections were incubated for 20 min at 37 °C in a freshly-made medium consisting of 8 ml tris-(hydroxymethyl)-aminomethane (1.0 M), 4 ml calcium chloride (0.18 M) and 60 mg ATP disodium salt, which was made up to 30 ml in distilled water and adjusted to pH 9.5 with 0.1 N-HCl before being brought up to a final volume of 40 ml). Thus the final concentration of ATP was 2.4 mM. Following two washes in distilled water, sections were immersed in 2% cobalt chloride for 3 min, then washed again twice in distilled water and developed in dilute ammonium sulfide for 1 min at room temperature, and then were assessed using a light microscope and analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

To determine whether the COPD rat model was successfully established, the weight, lung function and lung histomorphology of rats were measured. All rats survived until the end of the 90-day experimental period. Relative to control rats, the body weights of COPD rats increased at a significantly slower rate (P < 0.05; Fig. 1a).

In measuring parameters of lung function, it was observed that the PEF, FEV0.3 and FEV0.3/FVC of COPD rats were significantly decreased in the COPD group relative to controls (31.40 ± 3.34 vs. 22.69 ± 3.88 ml/s, P < 0.05; 4.88 ± 0.49 vs. 3.60 ± 0.58 ml, P < 0.05 and 88.41 ± 4.17 vs. 61.18 ± 5.19%, P < 0.05, respectively; Fig. 1b).

Based on histological analysis, lung tissue samples obtained from control rats (Fig. 1c and d) exhibited thin alveolar septa, normal alveoli and no inflammatory cell infiltration, whereas those obtained from COPD rats exhibited damaged alveolar septa, alveolar enlargement and inflammatory cell infiltration (Fig. 1e and f). Collectively these data indicate that establishment of the COPD rat model was successful.

Relative to control rats, COPD rats exhibited significant reductions in the weight (2.8028 ± 0.0195 vs. 2.5131 ± 0.0147 g, P < 0.05; Fig. 2a), muscle fiber cross-sectional area (77.7860 ± 7.4126 × 10⁻⁴ vs. 66.1556 ± 8.3279 × 10⁻⁴ m², P < 0.05; Fig. 2b) and muscle fiber nuclear cell number (44.63 ± 6.16 vs. 41.23 ± 5.56; P < 0.05; Fig. 2b) of the quadriceps muscle. Histological analysis of quadriceps muscle samples obtained from control rats (Fig. 2c) identified neat and tight muscle fibers, narrow gaps between adjacent muscle fibers and an even distribution. By contrast, those obtained from COPD rats exhibited muscle fiber atrophy and a disordered fiber arrangement, a wide gap between adjacent muscle fibers, a reduction in nucleus number and an uneven size distribution (Fig. 2d). ATPase histochemistry (calcium-cobalt method) also indicated that the muscle fiber type of COPD model rats was altered (Fig. 2e–g). Relative to controls, the type I fiber content of COPD rats significantly decreased (30.9 ± 6.6 vs. 20.7 ± 7.6%, respectively, P < 0.05) and the type II fiber content significantly increased (79.3 ± 7.6% vs. 30.9 ± 6.6%, respectively, P < 0.05; Fig. 2g).

TWEAK drives different physiological effects, including cell proliferation, differentiation, angiogenesis, migration and apoptosis [28]. In the current study, it was observed that TWEAK localized to the membrane of quadriceps muscle cells (Fig. 3a and b). The presence of TWEAK was indicated by varying degrees of tan or brown granules or by the deposition of flakes, identified by analyzing pathological images of quadriceps muscle sections with Image-Pro Plus 6.0 software. Relative to control rats (Fig. 3a), it was observed that levels of TWEAK expression in the quadriceps muscle of COPD rats (Fig. 3b) were markedly increased. TWEAK expression was also measured by western blot analysis, whereby it was observed that levels of TWEAK in the COPD model group were significantly higher than that in the control group (0.8910 ± 0.0512 vs. 0.5803 ± 0.0733, respectively, P < 0.05; Fig. 3c). In addition, levels of TWEAK expression were inversely proportional to muscle weight and type I fiber proportion (P < 0.05; Fig. 3d and e), and were proportional to the ratio of type II fibers in quadriceps muscle (P < 0.05; Fig. 3f).

A previous study by the present authors demonstrated that the levels of NF-κB were increased in the peripheral skeletal muscle of COPD rats, where it potentially serves a key role in the pathogenesis of COPD (1). In the current study, immunohistochemical staining and western blot analysis were used to measure the levels of NF-κB and its potential downstream effectors MuRF1 and PGC-1α. Relative to controls, immunohistochemical staining identified altered expression in all three proteins in COPD rats (Fig. 4a). This was confirmed by western blotting, whereby significant increases in NF-κB and MuRF1 (both P < 0.05) and significant reductions in PGC-1α (P < 0.05) were detected (Fig. 4b–d).