Effects Of TNF-α On Proteolysis Of Skeletal Muscle In Rats With COPD And Its Mechanism

Background: Chronic obstructive pulmonary disease (COPD) is a disease and progressive development characterized by an irreversible airflow obstruction. It is a common disease in the respiratory and a major cause of morbidity and mortality throughout the world. It was found, in the 1980s, that weight loss is a common symptom in COPD. Recent studies showed that about 25%-65% patients with COPD apparently suffered a gradual weight loss in the course of the disease. Nutritional status is not only a reflection the severity of disease, but also an independent predictor of survival: patients with low body weight died sooner, even after controlling of the established factors associated with mortality (i.e. age, smoking history, FEV₁, and supplemental oxygen requirement). Weight loss is an independent predictor of mortality. The measurement of body weight alone, however, does not fully present the extent of nutritional depletion in COPD; the evaluation of body composition is necessary. A normal-weight COPD patient with reduced fat-free or lean mass is still at the risk. Malnutrition in COPD, especially the loss of muscle mass, has become a hot spot in the research of COPD. Currently, foreign and domestic researches on malnutrition in COPD patients mainly involve three aspects: 1. the mechanism of malnutrition in COPD patients; 2. effects of malnutrition on COPD patients; 3. interference to COPD patients with malnutrition. The purpose of all the researches is to take measures to improve the nutritional status of patients and to prevent the occurrence of malnutrition.Malnutrition is common in COPD, but its mechanisms remain obscure. It is confirmed that the factors which induce the malnutrition in COPD include energy imbalance, hypoxia, disuse atrophy, cytokine-mediated inflammation, and the frequent use of corticosteroids. But treatments including nutritional supplementation, oxygen therapy, exercise training turned out to be ineffective. This indicated that energy imbalance, hypoxia and disuse atrophy resulted but partly in the malnutrition in COPD. Recently some researchers have found elevated levels of tumor necrosis factor alpha in patients with stable COPD and weight loss compared with those without weight loss or non-COPD control subjects. They consider systemic inflammation mediated through inflammatory cytokines may play an important role in COPD-associated weight loss.Tumor necrosis factor alpha (TNF-α) is a reassured cytokine highly relevant to the malnutrition of COPD. Several studies have demonstrated that TNF-αis the major cytokines responsible for malignant and inflammatory-mediated cachexia, malnutrition patients with stable COPD had higher serum level of TNF-αthan that of the non-malnutrition patients, indicating a close relation between TNF-αactivation and concerning its mechanism of malnutrition in COPD. However, no reports have been published that TNF-αeffects the proteolysis of skeletal muscle of the patients with COPD.Part 1: Change of serum levels of TNF-αin COPD model rats and its relation with nutritional status; Change of levels in TNF-αin Homogenates of skeletal muscle of COPD model rats and its relation with proteolysis of skeletal muscle.Objective : To investigate the impact of TNF-αon the nutritional status and proteolysis of skeletal muscle.Methods: Ninety healthy male adult Wistar rats were randomly divided into two groups: normal control group (n=20) and model group (n=70). The COPD rat model were established by intratracheal instillation of PPE (20u/100g.Bw) and being exposed to cigarette smoke for 8 weeks, 30 minutes per day. The control group was not exposed to smoke but was in intratracheal instilled of the same amount of Saline on the same day. Malnutrition was defined when the weight of the rats in the model group was lower than 90% of the mean body weight of the control group. When the incidence of malnutrition reached 29% in the model group, 10 rats were randomly chosen in the malnutrition group, the rats were re-divided into 4 groups: the control group, the non-malnutrition group, the malnutrition group, the TNF-αMcAb treatment group. The TNF-αMcAb treatment group were recieved 4 days'therapy of intravenous injection of TNF-αMcAb 0.1 mg/Kg.Bw, the rest were given the same amount of Saline. Another 10 days later, all of the 90 rats were euthanized.1) We measured the concentrations of TNF-αin the serum and the homogenates of skeletal muscle by enzyme-linked immunosorbent assay (ELISA).2) We measured the concentrations of glucose, albumin, triglyceride in the plasm by automatic biochemisty analyzer.3) We measured the contents of 3-methylhistidine, tyrosine in homogenates of skeletal muscle by High Performance Liquid Chromatography (HPLC).Results:1) The levels of serum TNF-αof the malnutrition group were significantly higher than those of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01).The levels of serum TNF-αof the non-malnutrition group was significantly higher than those of the control group (P<0.01).2) The levels of glucose, triglyceride in the plasm of the malnutrition group were significantly higher than those of the control group (P<0.01, P<0.01), the non-malnutrition group (P<0.01, P<0.01), the TNF-αMcAb treatment group (P<0.01, P<0.01); The levels of albumin in the plasm of the non-malnutrition group were significantly lower than those of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01).3) The weight of diaphragmatic muscle of the malnutrition group were significantly decreased compared with that of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01).The weight of diaphragmatic muscle of the non-malnutrition group was significantly increased compared with that of the control group (P<0.01). The weight of extensor digitorum longus of the malnutrition group were significantly decreased compared with that of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01).The weight of extensor digitorum longus of the non-malnutrition group was significantly increased compared with that of the control group (P<0.01).4) The contents of 3-methylhistidine, tyrosine in homogenates of diaphragmatic muscle and extensor digitorum longus of the malnutrition group were significantly higher than those of the control group (P<0.01, P<0.01, P<0.01, P<0.01), the non-malnutrition group (P<0.01, P<0.01, P<0.01, P<0.01), the TNF-αMcAb treatment group (P<0.01, P<0.01, P<0.01, P<0.01); The contents of 3-methylhistidine, tyrosine in homogenates of diaphragmatic muscle and extensor digitorum longus of the non-malnutrition group were significantly higher than those of the control group (P<0.01, P<0.01, P<0.01, P<0.01).5) There were no significant differences in the contents of glucose, albumin, triglyceride in the plasm between the TNF-αMcAb treatment group and the non-malnutrition group (P>0.05, P>0.05, P>0.05), There were no significant differences in the weight of diaphragmatic muscle and extensor digitorum longus between the TNF-αMcAb treatment group and the non-malnutrition group (P>0.05, P>0.05), There were no significant differences in the contents of 3-methylhistidine, tyrosine in homogenates of diaphragmatic muscle and extensor digitorum longus between the TNF-αMcAb treatment group and the non-malnutrition group (P>0.05, P>0.05, P>0.05, P>0.05).Conclusion:1) TNF-αis one of the factors to cause the malnutrition-in COPD model rats.2) An increase of proteolysis of skeletal muscle was found in COPD model rats, especially in the malnutritional rats.3) TNF-αis one of the causes of the increase of proteolysis of skeletal muscle.Part 2: Effect of TNF-αon the increase of proteolysis of skeletal muscle of COPD model rats.Objective: To investigate the effect of TNF-αon the increase of proteolysis of skeletal muscle of COPD model rats.Methods:The COPD rat model were established by the same method as described in part one. Eighty Wistar rats were divided into 3 groups: control group, non-malnutrition group and malnutrition group .4 rats were randomly chosen in the malnutrition group and recieved 4 days'therapy of intravenous injection of TNF-αMcAb 0.1 mg/Kg.Bw. Another 10 days later, all the 80 rats were euthanized. We semi-quantitatively analyzed the expression of TNF-αmRNA, ubiquitin mRNA, proteasome 20S C2 subunit mRNA in the skeletal muscle.Results:1) The expression of TNF-αmRNA in the malnutrition group was significantly increased compared with that of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01).The expression of TNF-αmRNA of the non-malnutrition group was significantly increased compared with that of the control group (P<0.01).2) The expression of ubiquitin mRNA of the malnutrition group was significantly decreased compared with that of the control group (P<0.01), the non-malnutrition group (P<0.01), the TNF-αMcAb treatment group (P<0.01). The expression of ubiquitin mRNA of the non-malnutrition group was significantly decreased compared with that of the control group (P<0.01).3) The expression of proteasome c2 subunit mRNA of the malnutrition group was significantly decreased compared with that of the control group (P<0.01), the non-malnutrition group (P<0.01), TNF-αMcAb treatment group (P<0.01). The expression of proteasome c2 subunit mRNA of the non-malnutrition group was significantly decreased compared with that of the control group (P<0.01).4) There was no significant difference in the expression of TNF-αmRNA between the TNF-αMcAb treatment group and the non-malnutrition group (P>0.05), the expression of ubiquitin mRNA, proteasome c2 subunit mRNA of TNF-αMcAb treatment group was significantly decreased compared with that of the non-malnutrition group (P<0.01),Conclusion:1) Ubiquitin-proteasome pathway (UPP) is a major pathway in the proteolysis of skeletal muscle of COPD model rats.2) TNF-αis one of the important factors in enhancing proteolysis in skeletal muscle of COPD model rats, and the mechanism is related to the enhanced activity of the ubiquitin system at gene level.