| OBJECTIVES1 To establish a new COPD rat model which is more close to the clinical pathogenesis and evaluate its characteristics and long-term stability, and provide research base for the in-depth research for COPD.2 To explore the mechanism of Bufeijianpi Granules on diaphragmatic muscle dysfunction in rat with COPD, based on the COPD model prepared by the combination of cigarette smoke inhalation (CSI) and repeated bacterial infections (RBI).METHODS1 Rats were randomly divided into Control group, CSI group, RBI group and the combination of CSI and RBI (CCR) group,40 animals each group. Stable COPD models were prepared by simple CSI or RBI or CCR respectively, and respiratory function and pathological changes including bronchial wall thickness, bronchial lumen stenosis, alveolar amount in unit area, alveolar size, small pulmonary vessels thickness were detected monthly, and deposition of typeâ… ,â…¢andâ…£collagen in lung tissue at the eighth week end.2 Rats were randomized into control group (CG), model group (MG), Bufeijianpi high-doses group (HDG), medium-doses group (MDG), low-doses group (LDG) and aminophylline group (AG), rats with stable COPD were prepared by CCR mentioned in STUDY ONE. The drug interventions of Bufeijianpi Granules and aminophylline were conducted after the first two months, lasting three months. Pulmonary function and pathological changes (including bronchial wall thickness, bronchial lumen stenosis, alveolar amount in unit area, alveolar size, small pulmonary vessels thickness, diaphragmatic histology and the ultrastructure, phrenic nerve discharge, respiratory movement, in vitro diaphragmatic tension and endurance test, enzymatic activity of adenosine triphosphate (ATP) dehydrogenase and succinic acid dehydrogenase (SDH) in the diaphragmatic muscle, reactive oxygen species (ROS) and calcium ion (Ca2+) concentration in the diaphragmatic muscle cells.RESULTS1 Compared to the Control group, TV, PEF and EF50 in rats treated with CSI or RBI or CCR rats reduced significantly (P<0.05 or P<0.01) after the first eight weeks, TV and EF50 in rats treated with CCR decline significantly than that in rat treated with CSI or RBI (P<0.05 or P<0.01). The histological changes in the lungs treated with CSI or RBI or CCR could be observed obviously at the eighth week end, and showed the trend aggravated gradually in the following months. Pathological changes in rats treated with CCR was more apparent than that in CSI or RBI, including bronchial wall thickening, bronchial luminal stenosis, unit area alveolar numbers decrease, alveolar size enlargement, and small pulmonary blood vessel wall incrassation (P<0.05 or P<0.01). Typeâ… ,â…¢andâ…£collagen deposition increased significantly in rats treated with CCR (P<0.05 or P<0.01) compared to the controls, but only type III collagen increased significantly than rat treated with CSI or RBI (P<0.01). RVHI in COPD rats decreased obviously compared to the Control rats (P<0.05 or P<0.01).2 TV, PEF, EF50 in MG, HDG, MDG, LDG and AG rats decreased obviously (P<0.05 or P<0.01) compared to CG rats before medicine interention. High- or medium-doses Bufeijianpi Granules improved TV, PEF and EF50 significantly (P<0.05 or P<0.01). Carbon dioxide partial pressure (PCO2) increases significantly (P<0.01) and oxygen partial pressure (PO2) reduced (P<0.01) in arterial blood in MG rats, PCO2 reduced and PO2 improved distinctly in rats treated with Bufeijianpi Granules, especially in rats treated with high-or medium-doses of Bufeijianpi Granules (P<0.05 or P<0.01), with close dose dependence (P<0.05 or P<0.01). Lung histological impaired obviously in MG rats, and Bufeijianpi Granules could obviously improve the damages in the lungs, specially in that treated with high-or medium-dose. Histology and ultrastructure of diaphragmatic muscle in MG rats changed significantly, and high- or medium-dose Bufeijianpi Granules could repair the damages superiorly than aminophylline. RVHI reduced significantly in MG rats (P<0.01), Bufeijianpi Granules could significantly improve RVHI, but that treated with high- or medium-dosage showed more significant (P<0.05 or P<0.01) and better than that treated with aminophylline (P<0.05 or P< 0.01). Nervus phrenicus discharge duration and interphase extends significantly (P<0.05 or P<0.01) in MG rats, and discharge area and range decreases (P<0.01), high- or medium dose Bufeijianpi Granules could shorten the duration and interphase of the nervus phrenicus discharge, increase area and amplitude of the discharge (P<0.05 or P<0.01), which is better than AG (P<0.05 or P<0.01). Inspiratory time (Ti) and expiratory time (Te) prolonged (P< 0.01) in MG rats, respiratory rate (RR) reduced (P< 0.01), high- or medium-dose Bufeijianpi Granules can significantly shorten Ti and Te, improve RR (P<0.05 or P<0.01), and showed superiority than in AG. Respiratory excursion (RE) and area (RA) decreased obviously (P<0.01) in MG rats, high- or medium-dose Bufeijianpi Granules can significantly improve RE and RA (P<0.05 or P<0.01) with a colse dose-response relationship (P<0.01), and were better than AG (P<0.05 or P<0.01). In vitro diaphragmatic muscle tension and endurance reduced significantly (P< 0.01) in MG rats, and significantly improved (P<0.05 or P<0.01) in rats treated with high- or medium-dose Bufeijianpi Granules, which showed better efficacy than AG (P<0.05 or P<0.01). Rate of type I and IIA fiber in diaphragmatic muscle increased significantly (P<0.01) in MG rats, while that of typeâ…¡B fiber decreased (P<0.01), high- or medium-dose Bufeijianpi Granules can significantly improve the fiber ratio (P<0.05 or P<0.01), with a close dose-response (P<0.05 or P<0.01), and were better than AG (P<0.01). Enzymatic activity of ATP dehydrogenase reduced significantly (P<0.01) and SDH increased (P<0.01) in the diaphragmatic muscle in MG rats, high- or medium-dose Bufeijianpi Granules can significantly improve the activity of the enzymes (P<0.05 or P<0.01), and were better than AG (P<0.01). ROS levels in the diaphragmatic muscle cells increased significantly (P<0.01) in MG rats, high- or medium-dose Bufeijianpi Granules can significantly reduce the ROS levels (P<0.01), and they were better than AG (P<0.05 or P<0.01). Ca2+ concentration in diaphragmatic muscle cell significantly reduced (P<0.01) in MG rats, high- or medium-dose Bufeijianpi Granules can significantly improve Ca2+ concentration (P<0.05 or P<0.01), with a close dose-response realtionship (P<0.05 or P<0.01), and had better eutherapeutic than AG (P<0.01). Correlation analysis showed that there were significant positive correlation and related closely (correlation coefficient r>0.5) between the diaphragmatic muscle fatigue index (FI) and Te (r=0.701, P=0.000), ROS (r=0.700, P=0.000); ROS and nervus phrenicus discharge interphase (r=0.527, P=0.000), Te (r=0.660,P=0.000); RE and RA (r=0.743, P=0.000), Pt (r=0.731, P=0.000), PO (r=0.795, P=0.000), ATP (r=0.670, P=0.000), Ca2+ concentration (r=0.772, P=0.000); RA and Pt (r=0.599, P=0.000), PO (r=0.656, P=0.000), Ca2+ concentration (r=0.645, P= 0.000); Pt and PO (r=0.882,P=0.000), ATP (r=0.730, P=0.000), Ca2+ concentration (r=0.813, P=0.000); PO and ATP (r=0.790, P=0.000), Ca2+ concentration (r=0.880, P=0.000), ATP and Ca2+ concentration (r=0.810, P=0.000).CONCLUSIONS1 Smoking combining with repeated bacterial infections can not only duplicate a COPD modle in a relatively short 8 weeks, and the pulmonary function and histological damages progressed in a steady development in the following 32 weeks, showing more good advantages and stability. This model will provide a basic tool and research foundation for forward research for COPD.2 Mechanism of diaphragm (respiratory) muscle fatigue may be relevant to extension of nervus phrenicus discharge interphase, reduction of diaphragmatic muscle ATP enzyme activity and Ca+ concentrations, excessive generation of SDH and ROS, and etc..3 Bufeijianpi Granules can improve diaphragm function in COPD rats, its mechanism might involve improving nervus phrenicus discharge, increasing diaphragmatic ATP enzyme activity and Ca2+ concentration, restraining the generation of ROS and etc..
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