The Effects Of NBP On AR Expression In The Mice Model Of ALS

Objective: Amyotrophic lateral sclerosis （ALS） is a degenerativedisease of nervous system that selectively injurys upper and lower motorneurons, reflecting progressive muscle weaknessand atrophy, more than90%of ALS cases are sporadic and about10%are familial. The mechanism ofmotor neuron degeneration in ALS remains unkonwn oxidative、mitochondriadisfunction、excitotoxicity、neuroinflammatory、calcium overload、abnormalprotein aggregation may be involved in, and so far, there is no effective cure.Sex hormones is the synthesis of steroid hormones by the gonads of theanimal body, as well as the placenta, adrenal cortical mesh with organization，promote sexual organs mature, the role of secondary sexual characteristics anddevelopment, and maintenance functions. Female animals ovary secretes twomajor sex hormones, estrogen and progesterone, the most active is estradiol,which we know is the most important estrogen shows a lot of different role innerve tissue such as neurotrophic and neuroprotective effect.estrone and estriolare the metabolic conversion products of that. The male animals testis secretethe androgen testosterone-based. Androgen can act on the androgen receptoror aromatase into estrogen play a protective role. This conversion has provenin the central nervous system and sexual differentiation of the brain. Ofdegenerative diseases of the nervous system is affected by many factors suchas environment, genetics, gender differences, gender differences are more andmore attention.DL-3n-butyphthalide （NBP） is extract from seeds of the Chinese celery, itwas effective in treating ischemic cerebrovascular disease. Many studies havedemonstrated that NBP possesses neuroprotective by improving mitochondrialfunction, inhibiting calcium overload, reducing neuronal apoptosis andinflammation. Recent research has showed that NBP has also curative effecton the neurodegenerative disease. In the present sytudy, we apply NBP to interfere familial amyotrophiclateral sclerosis mice model （SOD1G93Atransgenic mice）, and observe thechanges of behaviors of SOD1G93Atransgenic mice by lifespain, investigatethe spinal cord anteral horn motor neurons numbers by toluidine bluestaining and AR expression in the spinal cord anterior horn by IHC in thefinal stage, aim to study the effects of NBP on SOD1G93Atransgenic mice.Methods:1Animal and treatmentThe48hSOD1^G93Agene positive mice were screened and genotyped.And then were randomly divided into NBP groups and placebo group, thehSOD1^G93Agene negative mice were selectived as littermates. Each grouphas12mice with equal numbers of males and females. At the age of6weeks,L-NBP was administered by oral gavage daily at a dose of180mg/kg/dayrespectively, placebo group received coequeal （10ml/kg/day） corn oil alone.2Life span AssessmentThe animals were considered as sacrificed when they were unable to rightthemselves within20s after placed on either side, this day was recorded asdeathtime, The life span of the mice was the time from born to death.3PathologyAfter10%chloral hydrate anesthesia, animal tissues were fixated viaheart perfusion by4%paraformaldehyde for20min. The lumbar enlargementswere fixated in4%paraformaldehyde for48hour. Then they were dehydratedby gradient ethanol, transparentized by xylene, embedded by paraffin andmade into conventional histological sections futher （5μm thick）.5μm thickparaffin sections were used for hematoxylin-eosin staining to observe thespinal anterior horn neurons survival condition.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 into25μm using a vibratome （LeicaVT1000S）. By immunohistochemisty we observe AR expression and distribution of the spinalanterior horn neurons. Each mouse to take three slices, each slice collect atleast five fields,200magnification observation, counting the edge of theventral spinal cord is located in the central canal of the spinal cord anglekernel Ren staining clear diameter>25micron nerve cells. Take unilateralanterior horn motor neuron counts the average statistics.5Statistical analysisThe data representation are the Mean±SEM, all experiment data weretreated with SPSS13.0statistical software, Kaplane-Meier survival analysiswas used for survival comparisons, and repeated measures ANOVA test wasused for assessment of rotation test comparisons. Biochemical andpathological results were analyzed using ANOVA or Nonparametric test. p <0.05was considered as statistically significant.Results:1Life spanThe life span of the NBP-treated group was135.25±2.111days,placebogroup was124.833±1.492days, respectively compared with the hSOD1^G93Agene blank group122.579±1.373days. NBP-treated group significantlyextended the lifespan of SOD1G93A transgenic mice, P=0.000.MalehSOD1^G93A survival of transgenic mice （139.00±2.573） compared femalehSOD1^G93A survival of transgenic mice （130.00±1.924days）（P=0.000）,with a statistically significant difference in the NBP group.2hematoxylin-eosin staininghematoxylin-eosin staining results display, in final stage, NBP-treatmentgroup reserved more neurons than placebo group,（9.47±2.466vs4.67±1.175）,compared with placebo group, P=0.000. NBP-treatment group reserved moreneurons than blank group,（9.47±2.466vs4.47±1.125）, compared with placebogroup, P=0.000.Male hSOD1^G93A transgenic mice more neurons than femalehSOD1^G93A transgenic mice was,（11.57±0.976vs7.63±1.685）, comparedwith female hSOD1^G93A transgenic mice in the NBP group, P=0.00, witha significant difference. 3ImmunohistochemistyThe immunohistochemistry results showed that the number of androgenreceptor-positive neurons in NBP-treatment group was5.87±1.598, theplacebo group was2.93±1.1, compared with the placebo group, P=0.00,statistically significant. The number of androgen receptor-positive neurons inmale NBP-treatment group was6.75±1.282, female transgenic mice was4.86±1.345, compared with the female transgenic mice （P=0.015）, with astatistically significant difference. An intense cytoplasmic immunostaining ofmotor neurons for AR in NBP-treated group, and very low levels ofcytoplasmic AR immunoreactivity was detected in spinal cord sections ofplacebo group.And in male NBP-treatment group the expression of theandrogen receptor higher than female transgenic mice.Conclusions: DL-3n-butyphthalide （NBP） can extend the survival ofhSOD1^G93A transgenic mice, transgenic mice and male hSOD1^G93A haslonger survival than the female hSOD1^G93A transgenic mice.DL-3n-butyphthalide （NBP） may play a protective role through the androgenreceptor, and the gender differences of the survival in NBP-treatment groupmay be associated with the expression of androgen receptors in the anteriorhorn motor neurons.