| Objective: Amyotrophic lateral sclerosis (ALS) is a fatal neurodege- nerative disorder affecting specifically the motor system. The death of spinal motor neurons leads to limb weakness with muscle atrophy and to a progressive involvement of respiratory muscles leading to death within 3 to 5 years after the onset. Several mechanisms have been put forward to explain motor neuron degeneration, including oxidative stress, glutamate excitotoxicity, and apoptosis, suggesting that the disease is multifactorial in origin. However, the precise molecular mechanisms responsible for the selective loss of motor neurons remain obscure. Some of the therapeutic approaches that have used to the clinical arena cannot effectively control the progression of disease.A growing body of evidence involves skeletal muscle mitochondrial dysfunction in ALS: morphological abnormalities of mitochondria (swelling) are very early events in ALS .mitochondrial dysfunction can cause interaction of damage process, oxidatie stress, mitochondrial energy metabolization obstacle, intracellular calcium overload and excitability amino acids release excessive can cause including ALS happening or worse. This pathogenesis is increasingly concerned.Imbalances in the generation of reactive oxygen species(ROS) and cellular antioxidant defenses lead to oxidative stress, which results in oxidative damage. oxidative stress-related mitochondrial involvement as a key determinant of motor neuron degeneration. Uncoupling proteins (UCPs) are members of the growing family of mitochondrial carrier proteins. The founding member of UCP family is UCP1, formerly known as thermogenin.UCP1 is almost exclusively expressed in the brown adipose tissue (BAT) and is responsible, through the diversion of energy from ATP synthesis to thermogenesis, for the thermogenic function of this tissue, especially in rodents. UCP2, -3, -4, and -5 (also known as BMCP1) were further cloned by homology with UCP1, and their thermogenic activity in vivo is still controversial. Recently, there is reported that UCP3 are linked to oxidative stress resistance, mitochondrial function, and ultimately cell survival, these three functions being defective in ALS. So we speculate that UCP3 express will change in progression of ALS.The study was aimed to investigate the protective effects to mitochondrial by observing the express of UCP3 and the change of ROS level .We are in the point of antioxidantactivities and participating in metabolic disorder to understand the mechanism in amyotrophic lateral sclerosis disease progression.Methods: We used transgenic male mice with the missense mutation (G93A) in the SOD1 gene that were 30,60,75,90, 100-120,130-150 days old. Each group includes three mice. Nontransgenic littermates served as controls. All mice were normal female mice. We use 0.9%hydration aldehydes (350mg/kg body weight) intraperitoneal injection anesthesia.Gastrocnemius muscles were dissected either from transgenic mice or nontransgenic littermates. Tissues were immediately frozen in liquid nitrogen and stored at -80°C until use. We used Western blot to detect the protein expression of UCP3 in all phases of transgenic mice and used RT-PCR to detect the mRNA expression of UCP3 in all phases of transgenic mice, and used assay Kit to detect the muscle homogenate level of MDA,CAT and SOD. The data were analyzed with SPSS 13.0.Results:1 Results of the UCP3 protein expression in skeletal mucle:1.1 With the progression of disease, G93A transgenic mice UCP3 protein expression of skeletal muscle show a rising trend.Western blot showed that there were no significant differences in G93A mice UCP3 protein expression in groups of 30d,60d,75d,90d. The expression of UCP3 in G93A mice increased in onset group compared to those in 90d group (P<0.05). The expression of UCP3 in G93A mice increased in ending group compared to those in 90d group(P<0.05). There were no significant differences in G93A mice UCP3 protein expression between the groups of onset and ending.1.2 In our study, western blot showed that the protein of UCP3 expressed in both the skeletal muscle of G93A mice and control mice, Western blot analysis documented the expression of UCP3 in G93A mice increased in onset group compared to those in control group (P<0.05) and increased greatly in ending group compared to those in control group (P<0.01).1.3 While with aging, western blot analysis documented that there were no significant differences UCP3 protein expression of control mice in each group.2 Results of the UCP3 mRNA expression in skeletal mucle:2.1 With the progression of disease, G93A transgenic mice UCP3 mRNA expression of skeletal muscle show a rising trend. The expression of UCP3 in G93A mice increased in 60d group compared to those in 30d group (P<0.05). The expression of ucp3 in G93A mice increased in 90d group compared to those in 60d group (P<0.05). The expression of ucp3 in G93A mice increased in ending group compared to those in 90d group(P<0.05). There were no significant differences in G93A mice UCP3 mRNA expression between the groups of onset and ending.2.2 In our study, RT-PCR showed that the mRNA of UCP3 expressed in both the skeletal muscle of G93A mice and control mice, RT-PCR analysis documented the expression of UCP3 in G93A mice increased in 90d and onset group compared to those in 90d group and control group. (P<0.05) and increased greatly in ending group compared to those in control group (P<0.01).2.3 While with aging, RT-PCR analysis documented that there were no significant differences UCP3 mRNA expression of control mice in each group.3 Results of MDA in skeletal muscle:3.1 With the progression of disease,The level of MDA in G93A mice were no significant differences in groups of 30d,60d,75d,90d. The level of MDA in G93A mice increased in onset group compared to those in 90d group (P<0.05). The level of MDA in G93A mice increased in ending group compared to those in 90d group (P<0.05). The level of MDA in G93A mice were no significant differences between the groups of onset and ending.3.2 Our study documented the level of MDA in G93A mice increased greatly in onset group compared to those in control group. (P<0.01) and increased in ending group compared to those in control group (P<0.05).3.3 While with aging, The level of MDA in control mice were no significant differences between each groups.4 Results of CAT in skeletal muscle:4.1 With the progression of disease,The level of CAT in G93A mice show a trend of increasing before decreasing .When it come to 90d ,it become the lowest . There is a statistically significant difference (P<0.05).4.2 Our study documented the level of CAT in G93A mice decreased greatly in 90d group compared to those in control group (P<0.05).4.3 While with aging, the level of CAT in control mice were no significant differences between each groups.5 Results of SOD in skeletal muscle:5.1 Our study documented that there were no different in the level of SOD and Cu-Zn SOD between G93A mice and control group.5.2 With the progression of disease, while with the progression of disease, the level of SOD and Cu-Zn SOD in G93A mice were no significant differences between each groups.5.3While with aging, the level of SOD and Cu-Zn SOD in control mice were no significant differences between each groups.Conclusions: Through the experimental study in ALS mouse, the level of ROS were detected increasing consistent with this, we observed the abnomal increase expression of UCP3 mRNA and protein expression in skeletal muscle of G93A transgenic mice.And before onset the expression of UCP3 mRNA were increasing. It is confirmed that ucp3 can anti-oxidation and participate in the body metabolic disorder in ALS. We provide a high value evidence in early diagnosis and a marker of disease monitoring from muscle level, and it may be a therapy targets for ALS. |
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