The Effect Of Resveratrol On Bcl-2and Bax Expression In Lumbar Spinal Cord And Cortex Of SOD1G93A Transgenic Mice

Objective: Amyotrophic lateral sclerosis (ALS) is a kind of progressiveneurodegenerative disease, and mainly involves the upper and lower motorneuron (mainly including spinal cord, brain stem, motor cortex), characterizedby progressive limb weakness and muscle atrophy, while sensory system andsphincter dysfunction hardly occur. With the progress of the disease, mostpatients die of respiratory failure3-5years after onset of symptoms. There isno effective treatment of ALS so far. ALS is divided into two types: sporadicamyotrophic lateral sclerosis (sALS) and familial amyotrophic lateral sclerosis(fALS). sALS accounts for about90%to95%of all diagnosed patients, andfALS accounts for about5%to10%, of which approximately20%has beenconfirmed to be related with mutation of copper-zinc superoxide dismutase(SOD1) gene. Until now, the aetiology and pathogenesis of ALS remainunknown. A variety of mechanisms may participate in its pathogenesis,including heredity, oxidative stress, glutamate excitotoxicity, mitochondrialdysfunction, abnormal protein aggregation and degradation disorders,overload of Ca2+,axonal transport obstacles, cell apoptosis, etc. At present, thecolleague of our laboratory detailing the changes and roles of mitochondrialdysfunction, including mitochondrial fission, fusion and mitochondrialautophagy in the pathogenesis of ALS. No matter mitochondrial fission,fusion, autophagy or various other mechanisms may lead to the occurrenceand development of ALS by inducing cell apoptosis pathway.Apoptosis is a necessary physiological process to maintain homeostasis,growth and development. The cells in our body metabolize all the time inprocess of growth and development. While generating new cells, mutated oraging cells can be cleared through apoptosis mechanism in order to maintainnormal homeostasis and function. Apoptosis pathway is mainly divided into three types: death receptor apoptotic pathway, mitochondrial apoptoticpathway and endoplasmic reticulum apoptotic pathway. The mitochondrialapoptotic pathway is a hot spot in current researches. The main site of theBcl-2family locates in the membrane of mitochondria and plays an importantrole in the mitochondrial pathway of apoptosis. The Bcl-2protein family isdivided into two categories, antiapoptotic protein such as Bcl-2andpro-apoptotic protein such as Bax. When cells are stimulated by abnormalsignals, pro-apoptotic proteins undergo a conformational modification andtranslocate from cytoplasm to mitochondrial outer membrane, thus leading tothe formation of permeability transition pore and subsequent cytochrome Crelease, finally cause cell apoptosis. Studies have confirmed that in animalmodels of Alzheimer’s disease (AD), Parkinson’s disease (PD) and ALS,Baxand Bcl-2expression is altered in the progress of diseases. In ALS animalmodels, mitochondrial apoptosis pathway is affected by inhibiting Bax geneexpression,which not only reduce the loss of motor neurons, but also improvethe axonal function and extended lifespan. Resveratrol is a class ofpolyphenolic compounds of diphenylethylene family that have beneficialeffects on cardiovascular disease, cancer, aging and neurodegenerativediseases. In animal models of prion disease, resveratrol can reduce thetranslocation of pro-apoptotic protein Bax to the mitochondrial outermembrane, preventing the release of cytochrome C and protect neurons. As aneurodegenerative disease, how does the Bcl-2and Bax expression change inthe ALS pathogenesis? What kind of effect do the resveratrol have on theexpression of Bcl-2and Bax? It remains to be further confirmed.At present, the SOD1-G93A transgenic mice are one of the most idealALS models. This study was designed to observe the dynamic changes ofBcl-2and Bax in the lumbar spinal cord and motor cortex of SOD1G93A miceat different stages of the disease，as well as the changes of Bcl-2and Baxexpression in the lumbar spinal cord and motor cortex of SOD1G93A miceafter resveratrol treatment. To further explore the role of cell apoptosis in thepathogenesis of ALS, and finally, assess the viability of the resveratrol treatment application for ALS.Methods: SOD1-G93A transgenic mice were used as the experimentalanimals. The wild type mice served as control group. There were four groups:control group, pre-symptoms stage(60-day-old) group, onset stage group andending stage group. Each group included six mice.70d SOD1-G93Atransgenic mice were selected as the treatment group,they were divided intoresveratrol group(ALS-Res),vehicle group(ALS-Veh), the control group(ALS).Each group according to different delivery time can be divided into twosubgroups, six mice in each group. Resveratrol group were given the mixtureof resveratrol and ethanol (30mg/kg/d Res), vehicle group received an equalvolume of ethanol, the control group did not receive any treatment.Continuous intragastric infusion was exerted to onset stage(20-30days ofcontinuous dosing) and ending stage (continuous administration of50-60days). After10%hydration aldehydes (350mg/kg body weight) intraperitonealanesthesia was injected, mice were decapitated, extracted the lumbar spinalcord and motor cortex immediately, and they were frozen in liquid nitrogenand stored at-80℃, tissueswere fixated via heart perfusion by4%paraformaldehyde, dissected the lumbar spinal cord and motor cortex of miceand fixated them in4%paraformaldehyde or2.5%glutaraldehyde. Westernblot, Confocal microscopy and immunocytochemistry were used to detect theprotein expression of Bcl-2and Bax in the tissue of lumbar spinal cord andmotor cortex of mice. Data were analysed by spss13.0.Results:1. The expression of Bcl-2and Bax in the lumbar spinal cord and motorcortex of SOD1-G93A mice is measured at different stages of the disease.(1)Bcl-2, Bax expression in the motor neurons of lumbar spinal cord andmotor cortex: Immunohistochemical staining result shows the lower expre-ssion of Bcl-2and higher expression of Bax at the onset stage and endingstage of disease compared with WT mice. There is no significant difference ofBcl-2and bax expression between the60-day-old group and control group.Confocal microscopy shows the lower expression of Bcl-2and higher expression of Bax in the onset and ending stage of disease group comparedwith WT group. There is no significant difference of Bcl-2and bax expressionbetween the60-day-old group and control group.(2) The protein levels of Bcl-2and Bax in the lumbar spinal cord of ALSmice: using western blot, we find the protein levels of Bcl-2decrease at theending stage group compared with WT group. There is no significantdifference between theonset stage group,60-day-old group and control group.The protein levels of Bax increase at the onset stage and ending stage groupscompared with WT group. There is no obvious difference between the60-day-old group and control group.2. Bcl-2and Bax expression in different periods of SOD1-G93A micelumbar spinal cord and motor cortex after resveratrol treatment.(1)Onset stage: Immunohistochemical staining result shows that afterapplication of resveratrol intervention in SOD1-G93A mice the expression ofBcl-2was increased, and Bax expression was reduced in lumbar spinal cordmotor neurons and the motor cortex, compared with the control group; Inresveratrol group Bcl-2and Bax expression levels did not change comparedwith the vehicle group, Confocal detect co-localization of Bcl-2, Bax andneuronal markers SMI32: In SOD1-G93A mice lumbar spinal cord motorneurons, Bcl-2expression levels increased in resveratrol group and vehiclegroup compared with the control group, while the expression of Bax reduced.Compared with the vehicle group, Bcl-2and Bax in the motor neurons of theresveratrol group showed no significant change. Western blot detection:Compared with the control group, resveratrol group and vehicle group Bcl-2levels increased, while Bax was reduced, the difference was statisticallysignificant; compared with the vehicle group, the resveratrol group Bcl-2showed no significant changes, the difference was not statistically significant.(2)Ending stage:Immunohistochemical staining result shows that afterapplication of resveratrol intervention in SOD1-G93A mice the expression ofBcl-2was increased, and Bax expression was reduced in the motor cortex,compared with the control group; In resveratrol group Bcl-2and Bax expression levels did not change compared with the vehicle group, Confocaldetect co-localization of Bcl-2, Bax and neuronal markers SMI32: InSOD1-G93A mice lumbar spinal cord motor neurons, Bcl-2expression levelsincreased in resveratrol group and vehicle group compared with the controlgroup, while the expression of Bax reduced. Compared with the vehicle group,Bcl-2and Bax in the motor neurons of the resveratrol group showed nosignificant change. Western blot indicate that there is no significant changes ofBcl-2and Bax in resveratrol group, vehicle group and the control group.Conclusion:1.With the progression of ALS, the anti-apoptotic protein levels of Bcl-2was reduced in the central nervous system motor neuron of SOD1-G93Atransgenic mice,and pro-apoptotic protein level of Bax was elevated,whichsuggest that apoptosis mediated by mitochondrial pathway has beenupregulated with the disease progression.2.The effect of resveratrol in the treatment of ALS by intervention ofmitochondrial apoptosis pathway at onset stage and ending stage is limited.