| Objective:Amyotrophic lateral sclerosis (ALS) is a chronic progressiveneurodegenerative disease, selectively damage the upper and lower motorneurons, which leads to chronic progressive muscle weakness and atrophy,finally succumb to respiratory failure, but there is no effective treatment.5%-10%of the ALS has family history, called familial amyotrophic lateralsclerosis (FALS),90%is sporadic amyotrophic lateral sclerosis (SALS).Theyare similar in clinical and pathological performance. Studies found that20%of the FALS is related to encoding a gene mutations of Cu/Zn superoxidedismutase (Cu/Zn SOD1).And there are several hypotheses in thepathogenesis of SALS, including glutamate excitotoxicity, autoimmunemechanisms, oxidative stress, neurofilament abnormalities, mitochondrialdysfunction and so on, these factors influence each other, and all of them arerelated to oxidative stress. So oxidative stress is the focal point in treatmentinvestigative of amyotrophic lateral sclerosis.Studies have found the pathogenesis of ALS is related to oxidative stress,and the relationship between oxidative stress and ALS is also a researchhotspot in recent years. The antioxidant defense system including enzymesand non-enzymes. ARE (antioxidant response element) is located in the5'-flanking region of many genes essential for both detoxification andantioxidant proteins, which is the essential sequence to induced activation.The key molecules that antioxidants play a neuroprotective effect is Nrf2protein. The activation of Nrf2/ARE signaling pathway can induce theincreases of a series of endogenous cytoprotective genes in a variety of organs,including the antioxidant proteins, antioxidant enzymes, anti-inflammatoryand detoxification protein. The increase in the expression of these antioxidant enzymes and antioxidant proteins in nerve can act against oxidative damagecaused by hydrogen peroxide, glutamate and dopamine. HO-1is an importantantioxidant protein. It decomposes hemoglobin to produce nitric oxide, freeiron and biliverdin. Biliverdin and its metabolite generated bilirubin arephysiological free radical scavenger, which can protect neuron againstoxidative stress. Therefore the activation of Nrf2/ARE signaling pathway isexpected to become a new therapeutic target in neurodegenerative disease.L-3-n-butylphthalide (L-NBP) is a new type of anti-cerebral ischemicdrug developed in recent years. It was initially extracted from seeds of theChinese celery, Apium graveolens Linn. DL-3-n-butylphthalide (DL-NBP)was approved by the State Food and Drug Administration of China for clinicaluse in ischemic stroke patients in2002. Studies have demonstrated thatDL-NBP is neuroprotective by acting through multiple-targets, includingreducing oxidative stress injury, increase cerebral blood flow of the ischemicregion, inhibiting the inflammatory response, and reconstructing the cerebralmicrocirculation of the ischemic area, improving the mitochondrial functionand anti-apoptotic and so on. Therefore, Butylphthalide may affect theincidence of the SOD1-G93A mice to a certain extent.In present study, western blot and immunohistochemistry were used toassess the expression levels of Nrf2and HO-1of lumbar anterior horn motorneurons of Tg (SOD1-G93A) mice treated with DL-NBP, aim to find theneuroprotective mechanism of DL-NBP in ALS.Methods:1The establishment of experimental animal modelsTransgenic human SOD1-G93A mice and their non-transgenic littermateswere generated by breeding male hemizygous carriers(B6SJL-Tg(SOD1-G93A)1Gur/J) to female B6SJL/F1hybrids, both of whichwere purchased from the Jackson Laboratory (Bar Harbor, ME, USA). DNAwas obtained from the tails of transgenic progeny, and the presence of theSOD1-G93A transgene was confirmed by polymerase chain reaction (PCR).2Animal and treatment 48hSOD1-G93A positive mice were randomly divided into low, medium,high doses Butylphthalide group and the vehicle control, the negativelittermates hSOD1-G93A mice were used as negative control group, with12mice in each group, half male and half female. DL-NBP and corn oil wereadministered by oral gavage to the SOD1-G93A mice once per day. From theage of6weeks, respectively, in accordance with60mg/kg,120mg/kg and180mg/kg dose of DL-NBP to the three groups, the vehicle control was giventhe same volume (10ml/kg) corn oil gavage once a day.3Onset and lifetimeReferenced1-5score methed invented by Vercelli et al.4point wasdecided to be the time of onset, and1piont was the time of death.5: healthy without any motor dysfunction,4:when hung up the hind legs stretched abnormally or tremors,3: obvious paralysis and destabilized gait,2: the hind limbs were fully paralyzed, animals could only crawl on theforelimbs,1:fully developed paralysis of the hind limbs, animals are not able tostraighten up after turning them on the back for20s.4ImmunohistochemistyMice were anesthetized with10%chloral hydrate and perfusedtranscardially with4%paraformaldehyde for20min.The lumbar enlargementswere quickly dissected and fixed in4%pareformaldehyde for48hour. Thetissues were then cut into30μm sections using a vibratome(LeicaVT1000S).By immunohistochemisty we observed the expression anddistribution of Nrf2and HO-1in the spinal anterior horn neurons.5ImmunoblottingMice were anesthetized with10%chloral hydrate intraperitonealinjection, then extracted the lumbar spinal cord of mice quickly and put intoliquid nitrogen freezing, storage at-80℃. Extracted total protein to detect theexpression of Nrf2and HO-1protein in lumbar spinal cord byWestern-blotting. 6Statistical analysisSPSS version13.0for Windows (SPSS) was used for all statisticalanalyses. The data are depicted as mean values and standard errors of themean (SEM). Kaplane-Meier survival analysis was used for diease onset andsurvival comparisons. Biochemical and pathological results were analyzedusing ANOVA and the rank test. P <0.05was considered as statisticallysignificant.Results:1.Onset and lifetimeLow (60mg/kg), medium (120mg/kg) and high dose (180mg/kg)treatment group delayed onset by11,6, and13days respectively,statistically significant compared with the vehicle control. Low, medium andhigh dose treatment group extended the lifespan of the SOD1-G93Atransgenic mice by6,2, and12days respectively, only the high dose groupeffective in extending the lifespan, the low dose and middle dose had nostatistical significance compared with the vehicle control.2.Western blot and immunohistochemicalWestern blot and immunohistochemical analysis showed that comparedwith the vehicle control Butylphthalide (180mg/kg) can increase theexpression of Nrf2and HO-1in the SOD1-G93A transgenic mouse spinalanterior horn motor neurons.Conclusions:Butylphthalide can postpone the onset of SOD1-G93A transgenic miceand prolong survival, neuron protective effect may be achieved by improvingthe antioxidant capacity. Butylphthalide may become an effective treatmentfor amyotrophic toilet sclerosis drug in future... |
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