| Objective: Amyotrophic lateral sclerosis (ALS) is a chronic, progressiveneurodegenerative disease, characterized by muscle weakness and atrophythroughout the body due to the degeneration of both the upper and lowermotor neurons. The disease has a long history of more than100years since itsdiscovery. Although a large number of researches have been done, itspathogenesis is still unclear, and no effective treatment is available. Thepatient’s life quality is poor. Most die3to5years after onset of symptoms.Currently, there are many hypotheses about the pathogenesis of ALS, such astoxicity of excitatory amino acids, oxidative stress, immunologicalmechanisms, mitochondrial dysfunction and abnormal protein aggregation, etc.However, the fundamental reason of the selective motor neuron loss remainsunknown, and there is still no specific treatment.SIRT protein (sirtuins) also known as longevity protein, originally foundin yeast, is a kind of NAD+dependent deacetylation protease, which is highlyconserved from bacteria to humans. It is divided into seven subtypes, namelySIRT1-SIRT7. Many researches are focused on SIRT1, which is highlyhomologic with SIR2gene of yeast. It can make the histone acetylation,regulate the activity of many proteases, and can also influence the expressionof protein from the gene expression level, involved in regulating metabolism,energy restriction, prolong life, energetics, inflammation, immune function,apoptosis and tumorigenesis, cell proliferation and aging, autophagy, skeletalmuscle cell function, lipometabolism, endothelial function and myocardialprotection, circadian rhythm and many other physiological functions. In recentyears, researches on Sirtuins in various aging diseases such as type2diabetes,cancer, cardiovascular diseases, as well as metabolic disorders and itsassociated complications, hereditary diseases and neurodegenerative diseases are increasing. In the studies of prion disease model, SIRT1, as a protectiveprotein, can maintain cell survival by protecting mitochondria. In Parkinson’sdisease (PD) model, SIRT1may fight against oxidative stress damage.Meanwhile, in alzheimer’s disease (AD), studies indicate that SIRT1mayreduce the formation of β-amyloid protein precursor (APP) and the depositionofβ-amyloid protein.Resveratrol（Res）is a polyphenol compounds and is widely used indifferent kinds of experiments as a kind of protective protein, which isconfirmed to activate SIRT1. Res mainly extracts from the rootstocks of thepolygonaceae plants. Resveratrol has anti-inflammatory, anti-cancer,anti-oxidation, cardiovascular disease resistance, and many other roles. What’smore, there are more and more researches focused on its beneficial roles inneurodegenerative disease.However, does SIRT1expression change in ALS, the same as a kind ofneurodegenerative disease? Some scholars claim that SIRT1in ALS increases,but there are also some others declare that SIRT1reduces. However, in ALS,its function and changes of expression have not yet been finalized. We want tostudy the dynamic changes of SIRT1expression and the effects of resveratrolon SIRT1in the motor cortex and lumbar spinal cord of SOD1-G93A with thedisease progression, what we are also interested in.SOD1-G93A transgenic mice are one of ideal models of ALS. Thepresent study was designed to observe the dynamic variation of SIRT1inmotor cortex and lumbar spinal cord of SOD1-G93A transgenic mice with theprogress of the disease, as well as the change of SIRT1after given resveratrol,an activator of SIRT1, in the hope of deepening the research on ALS further,and exploring whether SIRT1is feasible to be a therapeutic target of ALS, aswell as the roles of resveratrol in ALS.Methods: SOD1-G93A transgenic mice were used as the experimentalanimals. According to the course, the mice were divided into3stages, namelypreclinical stage (60days) group, onset stage group and end-stage group. Eachgroup contained3mice. The wild type (WT) mice served as control group. Resveratrol (Sigma，101237259)0.375g was dissolved in7.5ml anhydrousethanol, then the above liquor was added water to50ml, so we got7.5mg/mlsolution. Meanwhile, we prepared13.5%ethanol solution as a solvent control.70-day （preclinical stage） old SOD1-G93A transgenic mice served astreatment groups. The treatment groups are divided into blank group, vehiclegroup, and resveratrol group. Each group continued to be divided into twosubgroups according to the time of administration, and each group contains6mice. Mice in the resveratrol group and vehicle group were administrateddaily with the resveratrol solution of30mg/kg/d and an equal volume ofvehicle through gastric perfusion respectively from70days to onset stage(Continuous dosing20-30days) and end stage (Continuous dosing50-60days), then got samples. The blank group was without any treatment. After10%hydration aldehydes (350mg/kg body weight) intraperitoneal injectionanesthesia, decapitated, extracted the lumbar spinal cord and motor cortex ofmice immediately, and they were frozen in liquid nitrogen, then stored at-80℃; fixated tissues via heart perfusion by4%paraformaldehyde, dissectedthe lumbar spinal cord and motor cortex of mice and fixated them in4%paraformaldehyde or2.5%glutaraldehyde. By using Western blot techniqueand immunohistochemical technique, the study was designed to observeSIRT1expression in motor cortex and lumbar spinal cord of SOD1-G93Atransgenic mice, and the changes of SIRT1after given resveratrol. Applyspss13.0to analyze the data.Results:1The dynamic variation of SIRT1in motor cortex and lumbar spinalcord of SOD1-G93A transgenic mice with ALS progession: Through Westernblot technique to detect expression of SIRT1in motor cortex and lumbarspinal cord of SOD1-G93A transgenic mice, the results show that expressionof SIRT1in onset stage group and end-stage group increases than the controlgroup, while60days group has no significant change with the control group.Immunohistochemistry shows that the immunoactivity of SIRT1in the onsetstage group and the end-stage group increases, and motor neurons of lumbar spinal cord in end-stage group obviously decrease.2The changes of SIRT1after given resveratrol in motor cortex andlumbar spinal cord of SOD1-G93A transgenic mice: in the onset stage group,SIRT1expression of the resveratrol group decreases than the vehicle group,but it has no obvious change compared with the blank group. And anincreased expression of SIRT1was observered in the vehicle group than in theblank group. While in the end-stage group, it has no significant change amongeach treatment groups. Immunohistochemistry shows, in the onset stage groupof SOD1-G93A transgenic mice, the immunoactivity of SIRT1in motorcortex and lumbar spinal cord after given resveratrol decreases than thevehicle group; but increases than the blank group. Besides, theimmunoactivity of SIRT1in the end-stage group has no significant changeamong each treatment group.Conclusions: With ALS progresses, the expression of SIRT1in motorcortex and lumbar spinal cord of SOD1-G93A transgenic mice increases. Inthe onset stage group, the expression of SIRT1in the resveratrol groupdecreases than in the vehicle group, but has no significant differencecompared with the blank group. While the expression of SIRT1in the vehiclegroup increases obviously than both the resveratrol group and the blank group.In the end-stage group, it has no significant change among each treatmentgroups. The result shows that resveratrol does not have the effect on activatingSIRT1, even plays a role in reducing SIRT1. Not like our expectation,resveratrol does not have a protective effect. Maybe the protection role ofresveratrol in ALS model is limited. The exact mechanism of resveratrolhasn’t come to a conclusion yet is still not clear, and needs to be studiedfurther. |
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