Establishment Of The COPD Mouse Model With Skeletal Muscle Dysfunction

ObjectiveChronic obstructive pulmonary disease(COPD)is a common respiratory disease in middle-aged and elderly people.In addition to the heterogeneous lung status,COPD patients often have significant extrapulmonary effects,in which skeletal muscle dysfunction(SMD)is associated with dyspnea and disease exacerbation,seriously affecting the activities of daily living in COPD patients.Among many pathogenic factors,smoke is the most important risk factor of COPD,directly damaging skeletal muscle.To explore the relevant mechanism of the occurrence and development of SMD in COPD and effective improvement methods,the establishment of the COPD animal model with SMD becomes a key step.However,the method has not yet been established at home and abroad.Therefore,in this study,a incremental cigarette smoke exposure protocol was used to establish a COPD mouse model,and to determine whether long-term cigarette smoke exposure could successfully establish a COPD model with SMD.MethodIn this study,forty male C57BL/6 mice aged 8 weeks were randomly divided into smoked model group and healthy control group,with 20 mice in each group.A25-week incremental cigarette smoke exposure program was carried out for the mice in the smoked model group to establish the COPD model with SMD.The mice in the control group were fed routinely without any intervention.During the modeling period,the body weight of the mice was measured once a month,and the grip strength was measured every two months.After 25 weeks,6 mice were randomly selected from each group,and the rest of the mice were fed normally to adjust the modeling scheme when the model failed and continue to establish the model.In this study,mice randomly selected from each group were tested for lung function first,and then the right gastrocnemius muscle of mice was completely removed for isolated muscle strength test.Then the lung tissue and the left gastrocnemius muscle were removed,and the pathological changes of the lung tissue and gastrocnemius were observed by HE staining.The average cross-sectional area of the alveoli,the average linear intercept of the alveoli,and the cross-sectional area of the gastrocnemius muscle fiber were calculated.The expression levels of protein degradation-related factors Atrogin-1 and Mu RF-1 in gastrocnemius muscle were detected by Western blot.Results(1)Compared with the control group,the characteristics of COPD after 25 weeks of cigarette smoke exposure in the smoked model group were obvious: chronic bronchial and emphysema-like changes were seen in the HE-stained sections of the mice in the smoked model group,namely a large number of inflammatory cell infiltration,airway structure destruction and tube wall thickening,alveolar structure disorder,rupture and fusion to form pulmonary bullae.Calculated by Image J software,the average cross-sectional area and average linear intercept of the alveoli in the smoked model group were significantly increased than those in the control group(p<0.05).The results of lung function tests showed that the lung function indicators such as MV,Cydn,FVC,and PEF in the smoked model group were significantly lower than those in the control group(p<0.05).TV and FEV50 were lower,but the differences between the groups did not reach statistical significance.(2)Compared with the control group,the characteristics of SMD in the smoked mice were obvious after 25 weeks of cigarette smoke exposure: compared with the mice in the control group,the monthly body weight of the mice in the smoked model group was significantly lower(p<0.01).The mass of the gastrocnemius muscle decreased,but it did not reach statistical significance.HE staining of the gastrocnemius muscle in smoked model group mice showed that the structure of muscle fiber was disordered,the arrangement was obviously sparse,and the muscle fiber gap increased.The muscle fiber cross-sectional area was significantly lower than that of the control group mice(p<0.05).Functionally,the grip strength of the mice in the smoked model group was significantly lower than that in the control group(p<0.05).The results of the isolated muscle strength test showed that compared with the control group,the isometric contraction force and muscle contraction force under35 Hz electrical stimulation in the smoked model group were significantly decreased(p<0.05).The force-frequency curve moved down significantly.The tetanic tension and muscle contraction force at 15 Hz,55Hz and 75 Hz were decreased,but the difference between groups did not reach statistical significance.(3)Compared with the control group,the expression levels of factors related to skeletal muscle protein degradation in the smoked model group were significantly changed after 25 weeks of cigarette smoke exposure: the results of Western blot showed that the protein expression levels of Atrogin-1 and Mu RF-1 were significantly up-regulated.The differences were statistically significant(p<0.05).ConclusionsThe 25-week incremental cigarette smoke exposure regimen resulted in significant characteristics of COPD in mice,as well as muscle fiber structure disorders,decreased weight and muscle strength,and increased muscle atrophy-related protein expression levels in mice.The results indicated that the25-week incremental cigarette smoke exposure regimen could successfully establish the model of COPD mice with SMD.