|
Objective: amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS) is a fatal neurodegenerative disease that affects the body's motor system, manifested as muscle atrophy and weakness, and eventually progressed to respiratory muscles, life-threatening, usually after the onset of 3 -5 years of death. Motor neuron degeneration has been shown by a number of mechanisms explanations, including oxidative stress, glutamate excitotoxicity and apoptosis. This indicates that ALS is a multifactorial origin of the disease. However, the selective loss of motor neurons exact mechanism remains unclear, there is still no specific treatment. ALS pathogenesis, early in the disease appears abnormal skeletal muscle mitochondria, dysfunction and abnormal pathological changes in energy metabolism, mitochondrial dysfunction can lead to a series of interactions injury, oxidative stress, mitochondrial energy metabolism, intracellular calcium overload, excessive release of excitatory amino acids, which can lead to the onset or worsening of ALS. The pathogenesis is gaining attention. Machine ROS (reactive oxygen species, ROS) generation and scavenging of balance disorders, leading to a large accumulation of ROS in the body, resulting in oxidative stress. Oxidative stress-induced mitochondrial damage in motor neuron degeneration process has played an important role. Uncoupling protein (uncoupling proteins, UCPs) located in the mitochondrial inner membrane, mitochondrial anion carrier protein family members. Currently UCPs has five members, namely UCP1-UCP5, which UCP2, 3 antioxidant function has increasingly attracted attention. In the pathogenesis of ALS, oxidative stress involved in them, so we speculate, UCP3 expression in various stages of ALS onset will change. SOD1-G93A transgenic mouse study is an ideal animal model of ALS, the study by observing the SOD1-G93A transgenic mouse skeletal muscle uncoupling protein 3 expression and mitochondrial changes in the level of oxygen free radicals, in-depth study of uncoupling protein 3 and oxidative stress, mitochondrial function for UCP3 clear whether protective effects against oxidative stress and involved in metabolic disorders as a starting point to explore its amyotrophic lateral sclerosis and progression of the disease in rats. Methods: A total of 36 mice, both females. Were divided into two groups: experimental group was internationally recognized SOD1-G93A transgenic mice (SOD1-G93A transgenic mice), 18; same litter control group, 18. The course is divided into groups according to 30 days, 60 days, 75 days, 90 days, duration of symptoms (onset ,100-120 days), end-stage (ending ,130-150 days), n = 3. With 10% chloral hydrate (350mg/Kg body weight) were anesthetized by intraperitoneal injection of fresh frozen in liquid nitrogen quickly take skeletal muscle and stored at -80 ℃ refrigerator. Using the Western blot were used to detect various disease stages of mouse skeletal muscle uncoupling protein-3 protein expression. By RT-PCR detection of various disease stages of mouse skeletal muscle uncoupling protein-3mRNA change in expression levels. Muscle tissue specimens for lipid peroxidation product malondialdehyde (MDA), catalase (CTA) and superoxide dismutase 1 (SOD1) determination. Spss13.0 and use the data for analysis. Results: 1 SOD1-G93A transgenic mouse skeletal muscle UCP3 Protein Expression 1.1 With disease progression, SOD1-G93A transgenic mouse skeletal muscle UCP3 protein expression showed increasing trend, which 30d, 60d, 75d, 90d between the four groups no significant increase, the difference was not statistically significant; symptomatic group was significantly higher than 90d (P lt; 0.05); end-stage were significantly higher than 90d (P lt; 0.05); symptoms of end-stage compared with no statistically significance. 1.2 SOD1-G93A transgenic mouse skeletal muscle UCP3 protein expression compared to the control group with the same nest, duration of symptoms increased, the difference was statistically significant (P lt; 0.05), end-stage significantly increased, the difference was statistically significant (P lt; 0.01). 1.3 littermate controls UCP3 protein levels did not change with increasing age. 2 SOD1-G93A transgenic mouse skeletal muscle UCP3 mRNA expression 2.1 Dynamic with disease progression, SOD1-G93A transgenic mouse skeletal muscle UCP3 mRNA expression showed increasing trend, which compared 60d 30d UCP3 mRNA expression increased significantly (P lt; 0.05); 90d 60d UCP3 mRNA expression compared, the difference was statistically significant (P lt; 0.05); end-stage were significantly higher than the 90d, the difference was statistically significant (P lt; 0.05); symptoms of Compared with end-stage was not statistically significant. 2.2 SOD1-G93A transgenic mouse skeletal muscle UCP3 mRNA expression compared to the control group with the same nest, 90 days, duration of symptoms increased, the difference was statistically significant (P lt; 0.05), end-stage significantly increased, the difference was statistically significance (P lt; 0.01). 2.3 littermate controls mRNA expression did not change with increasing age. 3 SOD1-G93A transgenic mouse skeletal muscle MDA assay 3.1 with disease progression, SOD1-G93A transgenic mouse skeletal muscle homogenate MDA content showed increasing trend, which 30d, 60d, 75d, 90d no significant change between the four groups the difference was not statistically significant; symptomatic group was significantly higher than 90d (P lt; 0.01); end-stage were significantly higher than 90d (P lt; 0.05); symptoms of end-stage compared with no statistical significance. 3.2 SOD1-G93A transgenic mouse skeletal muscle homogenate MDA in the control group compared with the same nest, the symptom was significantly increased, the difference was statistically significant (P lt; 0.01), end-stage increased, the difference was statistically significant ( P lt; 0.05). 3.3 MDA littermate control group did not change expression levels increased with age. 4 SOD1-G93A transgenic mouse skeletal muscle CAT assay 4.1 with disease progression, SOD1-G93A transgenic mouse skeletal muscle CAT levels decreased first and then increased, 90d is the lowest, the difference was statistically significant (P lt; 0.05 ). 4.2 SOD1-G93A transgenic mouse skeletal muscle CAT content compared to controls with the same nest, 90d significantly lower, the difference was statistically significant (P lt; 0.05). 4.3 littermates CAT expression level of the control group did not change with increasing age. 5 SOD1-G93A transgenic mouse skeletal muscle SOD assay 5.1 with disease progression, SOD1-G93A transgenic mouse skeletal muscle homogenates total SOD content and type SOD (Cu-Zn SOD) showed no significant change, does not have a statistically significant difference . 5.2 SOD1-G93A transgenic mouse skeletal muscle homogenates total SOD content and type SOD (Cu-Zn SOD) levels compared with littermate control group, no significant difference was not statistically different. 5.3 littermate control group SOD content and type SOD (Cu-Zn SOD) showed no significant change was not statistically different. Conclusion: Through the SOD1-G93A transgenic mice skeletal muscle UCP3 expression levels and the determination of oxygen free radicals, the experimental results show that as the disease advances SOD1-G93A transgenic mice increased ROS generation in skeletal muscle, and compared with the control group was significantly up litter high, with the corresponding UCP3 mRNA and protein expression levels, and presymptomatic UCP3 mRNA appeared an increasing trend. This study confirms the UCP3 able to participate in the body against oxidative stress and metabolic disorders in the development and progression of ALS disease play a role in clinical practice from the level of the muscle to provide higher value for early diagnosis and monitoring of disease markers, and to provide an ALS possible therapeutic targets.
|