The Effect Of NBP On The Ferritin Expression In Spinal Of The HSOD1^G93A Transgenic Mice

Objective: Amyotrophic lateral sclerosis （ALS） is a chroniclydegenerative disease of the nervous system that progressively and selectivelyinvolves the motor neurons of the brain and spinal cord. The clinicalmanifestations are progressive muscle weakness, muscle atrophy and the signof tractus pyramidalis, and finally injurys the respiratory muscle. The timefrom onset to death is generally3to5years. Its concrete pathogenesis is notclear, but it is generally thought that glutamate toxicity, oxidative stress,cytoskeleton abnormality, shaft slurry transportation disorder and so on manykinds of causes are related. At present, in addition to riluzole can delay theprogression of disease,the effective treatment is not existed.Oxidative stress is considered as the finally path of a variety of neurallydegenerative disease, and is more and more attentioned in the pathogenesis ofALS. The reasons that the central nervous system is sensitive to active oxygeninclude that,1) the low content of natural antioxidant-glutathione in theneurons2) the higher composition of unsaturated fatty acids in membranes3)the increasing demand of oxygen because of the high metabolism of brain.Iron is a necessary trace element for human body, it has manyphysiological functions, but too much iron aggregated in the body can lead tooxidative stress and cause damage to DNA, lipid, protein by Fenton reaction.The steady state of iron depends on a variety of metabolism related protein,while Ferritin plays an importantly regulatory role, it can combine with freeferrous iron ion; thus, prevent Fenton reaction and the occurrence of oxidativestress. Researches have proved that iron metabolic abnormality is in theamyotrophic lateral sclerosis patients, the ferritin in their serum is increased,also, the iron ion chelating agent can deplye the degeneration of motor neuronand progression of disease in SODl^G93Atransgenic mouse, and then prolonglife. Dl-3n–butylphthalide（NBP） is the first new drugs with independentintellectual property rights in the field of cardiovascular of our country. It ispicked up from celery seed, with relative molecular mass for190.24, and thatit has a variety of neural protection function, can be directly through the blood-brain barrier, reconstruct microcirculation of ischemia area, protect themitochondria from damage, and improv the energy metabolism, etc. Studieshave confirmed that it can obviously improve the symptoms of patients withvascular dementia, at the same time, increase the SOD activity in serum andreduce the content of MDA, and against oxidative stress.In previous studies, we found that Dl-3n–butylphthalide（NBP） canprolong the life of SODl^G93Atransgenic mice. This experiment observes theeffect of Dl-3n–butylphthalide（NBP） on the onset time and survival period,also, the Ferritin expression in the spinal of familial amyotrophic lateralsclerosis transgenic model SODl^G93A, aiming to explore the influnce of NBPon SODl^G93Atransgenic mice and its protective mechanism.Methods:1. Animal group and drug interventionThrough the PCR technological appraisal, we selected SODl^G93Agenepositive mice72and randomly divided them into Dl-3n–butylphthalide（NBP） group （early onset and end-stage）,placebo group （early onset andend-stage）and blank control group（early onset and end-stage）, each group was12, male and female were equal in each group. At the age of42days,we givedthe treatment group Dl-3n–butylphthalide （180mg/kg）, at the same time,the placebo group was given the corn oil （10mg/kg） using the method oflavage, once a day. The materials were drawn respectively on the early-stage（110days or so） and end-stage.2. The decision of infection and survival periodWhen the SODl^G93Atransgenic mouse had the signs that their hind legswere paralyzed, we determined them to be onset, the time from birth to beonset was recorded as the onset time. We placed the SODl^G93A transgenicmouse to be lie on their back on a flat plate, if they can’t turn over to be normal position or weight lost more than20%, we regarded them as death, thetime from birth to death was recorded as a living maturity.3. ImmunohistochemistryWe anesthesiaed the mice using10%chloral hydrate, and then fixed themby heart perfusion with4%polyformaldehyde about20-30minutes. Thelumbar segment spinal cord of mice was fixed in paraformaldehyde about48hours, we cut the specimen into the thickness of25um using Oscillationslicing machine, and then observed the expression of Ferritin in the motorneurons of anterior horn of spinal cord of the SODl^G93Atransgenic mouseusing the way of immunohistochemistry. We conformed the neurons thatcontour dyeing were clear and diameter were more than25um as the alphamotor neurons.4. Western blotThe mice was anesthesiaedwith10%chloral hydrate, we left the freshspecimen into liquid nitrogen, the section of spinal cord was preserved in–80℃, and then, we used western blot method to observe the quantitativeexpression of total Ferritin in spinal.5. Statistical methodsAll data was applicated with the software of SPSS13.0.The survival timewas analysised using Kaplane-Meier （K-M） survival curve. Measurementdata was expressed in the forms of x±s. All the pathology results andbiochemical indicators were analysised by the way of ANOVA. P <0.05hadstatistical significance.Results:1. The onset time and survival periodThe onset time of Dl-3n–butylphthalide （NBP） group was118.0±2.5,placebo group was109.5±2.2,and blank control group was107.1±1.0. Compared with placebo group（109.5±2.2）, the onset of Dl-3n–butylphthalide （NBP） group（118.0±2.5） was delayed by9days, P=0.011, P <0.05, it had statistical significance. Compared with blank control group（107.1±1.0）, the onset of Dl-3n–butylphthalide （NBP） group（118.0±2.5） was delayed by11days, P=0.000, P <0.05, it had statistical significance. But theonset time between placebo group（109.5±2.2）and blank control group（107.1±1.0）had no statistical significance, P=0.209, P>0.05.The survival period of Dl-3n–butylphthalide （NBP） group was138.6±2.3, placebo group was128.1.±2.8,and blank control group was127.4±2.3Compared with placebo group（128.1.±2.8）, the survival period of Dl-3n–butylphthalide （NBP） group（138.6±2.3）was delayed by10days, P=0.034, P <0.05, it had statistical significance. Compared with blank control group（127.4±2.3）, the survival period of Dl-3n–butylphthalide （NBP） group（138.6±2.3）was delayed by11days, P=0.000, P <0.05, it had statistical significance. Butthe survival period between placebo group（128.1.±2.8）and blank controlgroup（127.4±2.3）had no statistical significance, P=0.555, P>0.05.2. ImmunohistochemistryAt the early onset time, the comparation of Ferritin positive neuronnumbers in Dl-3n–butylphthalide （NBP） group （23.83±1.75）, placebogroup（17.67±3.39）, blank control group （13.50±1.73） had statistical difference,P=0.000. The cytoplasm and membrane around of the motor neurons inplacebo group and blank control group were both colored evenly, yet, themotor neurons of Dl-3n–butylphthalide （NBP） group were only coloredsurrounding membrane, the staining of nucleus and cytoplasm is not obvious.At the end-stage, the comparation of Ferritin positive neuron numbers inDl-3n–butylphthalide （NBP） group （8.25±1.22）, placebo group （5.33±0.78）,blank control group had statistical difference, P=0.000.Compared with Dl-3n–butylphthalide （NBP） group, the numbers of motor neuron that expressedFerritin in placebo group and blank control group were decreased, they werecolored in both cytoplasm and nucleus. We can see the proliferation of glialcells.3. Western blotAt the early onset, the relative abundance of Ferritin expression inlumbar myeloid in Dl-3n–butylphthalide （NBP） group （0.81±0.23）, placebogroup （1.57±0.43）and negative control group （0.35±0.13）had statistical difference, P=0.006, P <0.05. The comparation of relative abundance betweenNBP group and placebo group had statistical difference, P=0.039, P <0.05.The comparation of that between NBP group and negative control group hadstatistical difference, P=0.042, P <0.05, and that between placebo group andnegative control group also had statistical difference, P=0.006, P <0.05.At the end-stage, the relative abundance of Ferritin expression in lumbarmyeloid in Dl-3n–butylphthalide （NBP） group（1.45±0.45）, placebo group（1.73±0.36）and negative control group （0.78±0.36）had statistical difference, P=0.023, P <0.05. The comparation of relative abundance between NBP groupand placebo group had no statistical difference, P=0.449, P>0.05. Thecomparation of that between NBP group and negative control group hadstatistical difference, P=0.047, P <0.05, and that between placebo group andnegative control group also had statistical difference, P=0.010, P <0.05.Conclusion: Dl-3n–butylphthalide （NBP） can reduce the Ferritinexpression in spinal cord of ALS mice and delay the onset time; also, prolongthe survival time of ALS mice.