Background and ObjectiveChronic obstructive pulmonary disease is characterized by airflow limitationthat is a kind of preventable and treatable diseases is not fully reversible airflowlimitation, showed progressive development of abnormal inflammation of cigarettesmoke and other harmful gases or harmful particles react with the lung related.COPD not only cause lung disease, but also involving the cardiovascular, skeletalmuscle, skeletal, digestive system and other systemic diseases[1]. Skeletal muscleatrophy in COPD patients reached20%to40%, not only increased mortality inpatients with COPD[2], but also seriously affect the prognosis of the disease. Recentstudies have shown that excessive apoptosis play an important role in skeletalmuscle atrophy[3-4], and accelerate the degradation of muscle fiber proteins is alsoimportant[5]. Apoptosis plays an important role in skeletal muscle atrophy, as afamily of cysteine proteases, Caspases molecular mechanism of apoptosis is animportant effector. Skeletal muscle apoptosis increased, resulting in reducing thenumber of muscle fibers, lead to weight loss of skeletal muscle atrophy in COPDpatients[6]. It was found that rats with muscle atrophy which caused by severe burnsand denervation were associated with increased apoptosis[7-8].Endoplasmic reticulum apoptosis pathway is one of the signal transductionpathway, Caspase-12is present in the endoplasmic reticulum, the endoplasmicreticulum stress-induced apoptosis plays an important role[9]. m-Calpain is a Ca2 +-dependent neutral cysteine protease present in mammalian cells and tissues, can beactivated after the activation of Caspase-12[10-11], and ultimately lead to a series ofcell apoptosis.. KalaiM proved Caspase-12expression mainly in the lungs andskeletal muscle[12]. Studies have shown that endoplasmic reticulum stress pathwayplays an important role in skeletal muscle atrophy in old rats[13]. Is the importantfactor Caspase-12of endoplasmic reticulum apoptosis involved in COPD rat skeletalmuscle(diaphragm and extensor digitorum longus) atrophy, and whether m-Calpainby activating Caspase-12involved in? Based on this theory, the design of thisexperimental study, with COPD rat model, by detectingapoptosis rate,the expressionof Caspase-12and m-Calpain protein and mRNA,and Caspase-12and m-Calpaincorrelation in diaphragm and extensor digitorum longus. Explore whetherCaspase-12and m-Calpain is involved in COPD rat diaphragm and extensordigitorum longus atrophy process, is m-Calpain involved through the activation ofCaspase-12?Materials and methodsAccording to a random number table,40healthy adult male Wistar rats weredivided into two groups, the model group and control group, each with20. Modelgroup modeling using the following method: experiments1st day to29th day andfrom31th day to60th day, the rats were placed in a sealed box exposure (homemadepassive inhalation) in the cigarette smoke twice a day, every30minutes, the timeinterval8hours for2months. PPE injected within the first30days of rat trachealmodel group,At the same time the control group with the same way to inject thesame amount of saline injection, more than no treatment. on60th day,10rats wererandomly selected pulmonary function testing in each group; All rats were sacrificedon day61th, the lung tissueã€diaphragm and bilateral extensor digitorum longus,afterweigh the diaphragm and the right of bilateral extensor digitorum longus,then havebeen determined as follows:1. The TUNEL method to detect the expression of apoptosis rate in diaphragmand extensor digitorum longus;2. Lung tissue, diaphragm and extensor digitorum longus for HE staining;3. The immunohistochemistry to detect the expression of Caspase-12and m-Calpain protein in the diaphragm and extensor digitorum longus;4. The polymerase chain reaction (RT-PCR) to detect the expression ofCaspase-12and m-Calpain mRNA in the diaphragm and extensor digitorum longus.Result1. Pulmonary function results showed: In the model group, PEFã€FEVO.3andFEVO.3/FVC%were significantly lower, suggesting the presence of small airwayresistance increased and obstructive ventilatory dysfunction phenomenon.2. TUNEL assay showed: diaphragm and extensor digitorum longus apoptosis rateincreased significantly in the COPD rat compared with the normal group (P <0.05),which prompted diaphragm and extensor digitorum longus exists apoptosis in modelgroup.3. HE staining of lung tissue biopsy, visible under an optical microscope, modelgroup in lung tissue inflammation and emphysema and chronic non-specificphenomenon, in line with the characteristics of pathological changes in COPD lungtissue.4. Diaphragm and extensor digitorum longus histopathology showed: In the modelof diaphragmatic rupture and extensor digitorum longus muscle fibers occurs,dissolution, necrosis and atrophy.5. The weight of diaphragm and extensor digitorum longus had negative correlatewith its apoptotic rates (r=-0.852, r=-0.844).6. Immunohistochemical results showed that compared with the control group, theexpression Caspase-12protein in diaphragm extensor digitorum longus of modelgroup was significantly increased, the differences were statistically significant (P<0.05). In model group, the expression of m-Calpain protein in diaphragm extensordigitorum longus were also enhanced significantly (P <0.05), a statisticallysignificant difference. And the protein of Caspase-12and m-Calpain in diaphragmand extensor digitorum longus were positively correlated (r=0.885, r=0.862).7. RT-PCR method test results showed that the expression of Caspase-12andm-Calpain mRNA in diaphragm and extensor digitorum longus of COPD model ratwere also increased significantly, the differences were statistically significant. Andthe mRNA of Caspase-12and m-Calpain in diaphragm and extensor digitorum longus of model group were correlated positively (r=0.787, r=0.774).ConclusionDiaphragm and extensor digitorum longus in the presence of apoptotic cells andmuscles atrophy in the model of COPD rat; There exist links beween apoptosis anddiaphragm and extensor digitorum longus atrophy; The expression of Caspase-12and m-Calpain protein in the diaphragm and extensor digitorum longus wereincreased, and the expression of Caspase-12and m-Calpain mRNA were alsoenhanced, then the expression of Caspase-12and m-Calpain protein and mRNAwere showed a positive correlation. Which imply Caspase-12and m-Calpain may beinvolved in the COPD rats diaphragm and extensor digitorum longus atrophy,m-Calpain by activating Caspase-12may participate in the process too. |