The Study Of Nrf2/ARE Pathway In ALS Transgenic Mouse Model

Objectives: In 1993, Rosen DR found that some of the familial amyotrophic lateral sclerosis have mutation of gene coding copper / zinc superoxide dismutase (Cu / Zn SOD) of the SOD1. The discovery provides us with a clear reason for motor neuron death. So far, 20 percent of fALS and 4% Sporadic amyotrophic lateral sclerosis (sALS) have the SOD1 gene mutation and more than 100 kinds of mutation have been identified. Among them, glycine (Gly) mutation for alanine (Ala) located site 93 in the gene is the most common. Transgenic mice transfected with mutant SOD1 gene have the same clinical manifestations with ALS patients,which has been internationally recognized as the ideal model for ALS research.In ALS patients and transgenic animal models, there are proof of oxidative damage of some biological macromolecules such as proteins, lipids and nucleic acid. These suggests that oxidative stress involve in the pathogenesis of the disease. whether the body's own antioxidant system plays a protective role in the disease or play a more protective role in the progress of the disease is not sure. Some researchers reported that the activation of Nrf2/ARE signaling pathway can induce an endogenous increase including the antioxidant enzymes, antioxidant proteins, anti-inflammatory and detoxification proteins, all of these can play a protective role. This study will investigate the expression of antioxidant enzymes Nrf2 and HO-1, NQO1 in the spinal cord of B6SJL-TgN (SOD1G93A) 1Gur transgenic mice (Carry mutant SOD1 gene) during the different periods of life and help us to understand the oxidation resistance ability and the Nrf2/ARE pathway in ALS transgenic mouse.Methods:1 the propagation of transgenic miceAll animals were kept in constant temperature (25-27℃), hang wet and sterile conditions (Specific pathogen free SPF) environment and fed with the sterilization of SPF rodents feed particles. To maintain B6SJL-TgN (SOD1G93A) 1 Gur transgenic mice under-gene mutation, B6SJL SOD1G93A / + hemizygous males and B6SJLF1 / J hemizygous females were mated , the genetic offspring mice were identified with a mutant gene determination .2 the experimental grouptheSOD1G93Amice were divided into three groups Symptomless-stage (80 days), early symptoms (120 days), end-stage. wild-type mice and hSOD1 were the control, hSOD1mice were used to exclude the influence of human SOD.3 Nrf-2 protein level determinationplasma proteins were extracted from Spinal cord tissue and measured by Synergy-HT-Meibiaoyi OD numerical measu- rements, protein concentration , protein of 100 on the sample volume. Take SDS-PAGE electrophoresis, 100 V 4℃to film two hours, 5% skim milk closed one hour; an anti-incubation, 4℃, overnight; TPBS concussion rinsing 5 min×5; fluorescence two-incubated at room temperature for 2 h; TPBS concussion Rinse 5 min×5; Odyssey Infrared Imaging System Analysis. 4 HO-1, NQO1 gene expression level determinationSpinal cord of 6 mg was isolated for total RNA extraction. OD values were measured by Synergy HT-by-Meibiaoyi measurement. 1.8-2.0 of OD260/OD280 ratio were selected for reverse transcription of RNA and cDNA synthesis. HO-1, NQO1, nrf-2 gene were amplified and took electrophoresis in 1.5% agarose gel (containing Goldview dye). Theresults were printed by Automatic GBOX-HR gel imaging systems and analyzed by the gel image analysis Analysis system.Results: Compared with the two control groups, the Nrf-2 protein level and antioxidase HO-1, the NQO1 gene expression of spinal cord from the ALS transgene mouse in Symptomless-stage were not changed; while the Nrf-2 protein level decreased and antioxidase HO-1, the NQO1 gene expression increased during the symptom-stage and end-stage.Conclusion: The Experiment was studied in amyotrophic lateral sclerosis transgenic mouse model-ALS SOD1G93A mice. During the early stage and end-stage of diseases, there are activation of Nrf-2 and increase expression of downstream antioxidant enzymes including HO-1 and NQO1 in the spinal cord of transgenic mice. However, the protective effect of these enzymes needs further study.