Study On The Therapeutic And Preventive Effect Of DL-3-n-butylphthalide In A Transgenic Mouse Model Of Amyotrophic Lateral Sclerosis The Expression Of Superoxide Dismutase-1 In Peripheral Venous Blood Of Healthy People

Part 1:Study on the therapeutic and preventive effect of DL-3-n-butylphthalide in a transgenic mouse model of amyotrophic lateral sclerosisBackgroundAmyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a lethal neurodegenerative disease characterized by progressive muscular atrophy, paralysis and bulbar symptoms resulting from the progressive and selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem as well as spinal cord. Clinically, ALS usually onset at the age of 30-60 years. The patient of ALS eventually dies of respiratory paralysis or infection. The averages survival time after onset is about 3-5 years. Approximately 90% of ALS cases are sporadic, while 10% are familial. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) have been linked to 15-20% of familial ALS and about 5% of sporadic cases.Transgenic mice or rats over-expressing a mutant form of the SOD1 gene develop an ALS-like phenotype. The pathogenesis of ALS remains unclear. Although the mechanism underlying neuronal cell death in ALS remains poorly understood, exaggerated neuroinflammatory process seems to play an important role in the pathogenesis of neuronal degeneration in both familial and sporadic ALS Possible mechanisms may include excitatory amino acid toxicity, oxidative stress, mitochondrial dysfunction, abnormal neurofilament phosphorylation, apoptosis and inflammatory response cascade.Nevertheless, there is no cure for this lethal disease. Riluzole is the only therapeutic drug that has been approved by the Food and Drug Administration for ALS treatment; however, it only extends the life span of an ALS patient a few months. Thus, there is an urgent need for an efficacious therapeutic treatment for ALS patients. L-3-n-butylphthalide (L-NBP) is a compound that was initially extracted from seeds of the Chinese celery, Apium graveolens Linn. DL-3-n-butylphthalide (DL-NBP) was then synthesized in the laboratory and approved by the State Food and Drug Administration of China for clinical use in ischemic stroke patients in 2002. Studies have demonstrated that DL-NBP is neuroprotective by acting through multiple-targets. DL-NBP significantly reduces oxidative damage, improves mitochondrial function, reduces neuronal apoptosis and inhibits inflammation. Furthermore, DL-NBP attenuates P-amyloid-induced cell death in neuronal cell cultures, blocksβ-amyloid-induced microgliosis, and improves cognitive performance in an animal model of Alzheimer's disease. However, DL-NBP has not been tested as a therapeutic treatment of ALS.ObjectiveThis present study was aimed to investigate the effect of DL-NBP in the transgenic mouse model of ALS(ALS-SOD1G93A), study the efficacy of DL-NBP administration on disease onset and progression, to study if DL-NBP can reduce the glial activation,neuroinflammation and apoptosis, and to investigate the possible mechanism how DL-NBP exert neuroprotective effect.MethodsHemizygous breeding pairs of SOD1G93A transgenic mice on a B6SJL background (B6SJL-Tg (SOD1-G93A-1Gur/J) were obtained from Jackson Laboratory.The mouse line was maintained as hemizygotes by breeding Tg(SOD1-G93A)+/- males with C57BL6XSJL hybrid females. The offspring were genotyped by PCR according to the vendor's protocol. T Beginning at 8 weeks of age, disease progression of each group were monitored weekly, including the mental state,weight,suspending tail test,Rotarod test and hanging wire test. The onset of symptoms is defined when tremors and shaking of limbs is present, upon suspending the mouse briefly in the air by its tail. Symptoms typically occur at postnatal day 90 (approximately) and extend to symmetrical paralysis of the hindlimbs followed by complete paralysis at the terminal stage. Because of ethical considerations, transgenic animals were euthanized when they were unable to right themselves within 20s, and this time point was recorded as the time of death.Electromyography (EMG) was measured using a Nicolet Viking IV. EMG recorder according the manufacturer's protocol. Briefly, mice were deeply anesthetized with 10% chloral hydrate. Compound muscle action potential (CMAP) and a modified statistical method of motor unit number estimation (MUNE) was measured on the gastrocnemius muscle. Mice were anesthetized and transcardially perfused with 0.9% NaCl. The spinal cords (L4-5) were removed and postfixed. A total of 200 serial transverse sections (10μm thick) were cut on a cryostat and mounted on gelatin-coated slides for Nissl staining and immunohistochemistry. The spinal cords (C1-L3) were quickly removed and preserved in liquid nitrogen for preparation of tissue lysates for Western blot analysis. The number of the motor neuron in the spinal cord anterior horn was counted. To examine the glial activation, CD-11b and glial fibrillary acidic protein (GFAP), markers for microglia and astrocytes, were used respectively. The expression of cleaved caspase-3 was study by immunohistochemistry.To underly the possible mechanism, the expression of Nrf2, NF-B, TNF-a, cytochrome-c, Bcl-2 and Bax were investigated by western blotting.ResultsIn this study, oral administration of 60 mg/kg DL-NBP after onset significantly prolonged survival (164.78±16.672 days) compared to vehicle control(140.00±16.892 days) (P<0.05). Interestingly, the dose of 30mg/kg and 120mg/kg treatment also slightly increased this value compared to control, although there was no statistical significance. Oral administration of 60 mg/kg DL-NBP started on 45 days delayed disease onset by 10days (P<0.05). The Rotarod test and hanging wire test revealed rapid deficits in the ALS-SOD1G93Amice that received vehicle following the symptomatic onset of the disease. Similar motor dysfunction was also seen in mice receiving 30 mg/kg and 120mg/kg DL-NBP. In contrast,60mg/kg DL-NBP treatment significantly decreased the progression rate of motor deficits in these animals.60mg/kg DL-NBP treatment elevated the amplitude of the gastrocnemius muscle CMAP in the ALS-SOD1G93Amice compared with vehicle controls; however, the P value was 0.209. There was a significant decrease in MUNE as recorded from the gastrocnemius muscle of vehicle control ALS-SOD1G93Amice.60 mg/kg DL-NBP significantly slowed the rate of MUNE reduction (p<0.01). No difference was observed in 30mg/kg and 120 mg/kg DL-NBP-treated ALS-SOD1G93Amice. At the late symptomatic stage, ALS-SOD1G93Amice showed an extensive loss of large anterior horn cells. In contrast, motor neurons were remarkably preserved in the anterior horns in mice treated with 60mg/kg DL-NBP compared with vehicle control mice at the age of 20 weeks (P<0.05). No difference was observed in 30mg/kg and 120 mg/kg DL-NBP-treated ALS-SOD1G93Amice.Remarkable staining for both CD11b, GFAP and cleaved Caspase-3 was seen in the spinal cord from the late symptomatic stage (20-week old).There was significantly less immunoreactivity detected in the corresponding regions from the wild type littermates. In contrast,60mg/kg DL-NBP treatment significantly reduced CD11b, GFAP and cleaved caspase-3 immunoreactivity compared with vehicle control mice (p<0.05). There were much higher levels of NF-κB, TNF-α,cytochrome-c and Bax in ALS-SOD1G93Amice compared with wild type. However, DL-NBP treatment significantly reduced the abundance of both NF-κB, TNF-a, cytochrome-c and Bax proteins, but slightly increased the level of Nrf2 and Bcl-2 protein in the spinal cord.ConclusionsThis study showed that oral administration of DL-NBP may delay disease onset and prolong survival in ALS-SOD1G93Amice. DL-NBP treatment significantly decreased the progression rate of motor deficits, delayed motor neuron loss; motor unit reduction.DL-NBP had inhibitory effects on glial activation and apoptosis in the spinal cord of ALS-SOD1G93Amice. These results suggest that DL-NBP might be a promising compound in the treatment of ALS. Part 2The expression of superoxide dismutase-1 in peripheral venous blood of healthy peopleBackgroundCu-Zn superoxide dismutase (SOD1) is an important copper enzyme which dismutates the superoxide anion radical to the less toxic hydrogen peroxide and oxygen. It is found intracellularly in most tissues and is one of the major antioxidant in the CNS together with glutathione peroxidase and catalase.SOD could remove toxic oxygen free radicals away from the body to maintain the dynamic balance between the production and clearance of free radicals. Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease that affects motor neurons. A subset of autosomal dominantly inherited familial ALS (FALS) cases is known to possess mutations of the Cu,Zn-superoxide dismutase (SOD1) gene. Transgenic mice that express mutant SOD1, but not wild-type SOD1 or SOD1 knockout mice, develop motor neuron disease, indicating a toxic gain-of-function of the mutant. Study has showed that the expression of SOD1 mRNAs was statistically higher in Parkinson disease (PD) and Alzheimer disease (AD) patients and implicated the involvement of oxidative damage to SOD1 in the pathogenesis of sporadic AD and PD. Previous work suggested that AD, PD, and ALS may share a common or overlapping pathogenic mechanism(s) that could potentially be targeted by similar therapeutic strategies. It is of great value to clarify the expression of SOD 1 in healthy people.ObjectiveThe aim of the present study was to investigate the expression of SOD 1 protein and SOD activity in peripheral venous blood of healthy people in different age.MethodsIn the present study, blood samples were obtained from members of a familial ALS and 120 healthy people from the Medical Center of Peking Union Medical College Hospital. The expression of SOD1 protein was measured by western blotting. The superoxide dismutase (SOD1) activity in serum was determined by Cayman's SOD analysis kit with a spectrophotometric assay.Results The expression of SOD1 protein in peripheral blood mononuclear cells increased with age and reached peak at age between 40-50 years, then decreased after 60 years-old. After birth to 20 year-old, the SOD activity in serum ranged from 0.13 to 1.16U/ml,then decreased after 20 years-old and maintained a relative low level, from 0.07 to 0.08U/ml.ConclusionsThe expression of SOD1 protein in peripheral blood mononuclear cells in healthy people increased with age and reached peak at age between 40-50 years, then decreased after 60 years-old. After birth to 20 year-old, the SOD activity in serum maintained a relative high level, then decreased after 20 years-old.