NBP Reduce The Neural Damage Of Experimental Autoimmune Encephalomyelitis Mice Through Anti- Apoptotic Effect

Objective:Multiple sclerosis is a chronic inflammatory and demyelinating disease of the central nervous system that the physiopathological features of MS comprise demyelination, loss of oligodendrocytes, neuroaxonal degeneration and progressive neurological dysfunction.The course of multiple sclerosis is repeated and it aggravate the burden of family and society with high rate of disability.Although the exact etiology and pathogenesis is not clear,it is believed that environmental factors,genetic susceptibility,autoimmune and other factors were related.Apoptosis is a programmed cell death through genetic controlling, which involves a series of gene activation, expression and regulation and so on. At present, numerous studies have demonstrated: Mitochondria is the most important Organelles that plays a key regulatory role in the apoptotic signal transduction pathway, and mitochondrial dysfunction can lead to apoptosis, eventually leading to axons and neurons degeneration in the experimental autoimmune encephalomyelitis,which is the animal models of multiple sclerosis.NBP is a synthetic racemic.In 2002, NBP was first approved for stroke patients by China Food and Drug Administration. Experiments show that NBP can play a protective role in the brain through improving blood supply,protecting blood-brain barrier,anti-thrombotic, improving mitochon- drial function and so on. It has achieved good results in the treatment of ischemic cerebrovascular disease. But at home and abroad, there is little research on the treatment of NBP fou multiple sclerosis,.With regard to the influence on mitochondrial structure and function damage and apoptosis of MS, hasn’t been reported.There is currently no clear method of treatment for multiple sclerosis that can improve the long-term neurological defect, this study provide new clinical treatments by the anti-apoptotic role of NBP to experimental autoimmune encephalomyelitis mouse.Experimental autoimmune encephalomyelitis is a kind ofautoimmune disease which is characterized by mononuclear cell infiltration around small vessels of the central nervous system and demyelination--- an ideal animal model for multiple sclerosis research.This experiment ues NBP drug as an intervention, so as to explore the anti-apoptotic role of NBP to experimental autoimmune encephalomyelitis mice by observing the changes of neurological function score and the expresstion of Tunel-positive cells. The method Provide a theoretical basis for new clinical therapeutic approaches.Methods:1 Purchase and breeding of animals: 45 clean and healthy female C57 BL / 6 mice, 6-8 weeks old, weighted 180 ~ 220 g, were purchased from Vital River Laboratory Animal Technology Co, Ltd.(Beijing, China). Housed separately in the Experimental Animal Center of the Second Hospital of Hebei Medical University, a standard diet and water feeding, environment thermostat 20 ~ 25 ℃.2 Experimental animal groups: 36 C57 BL / 6 mice were randomly divided into EAE group(E, NS 0.1ml/d/per mouse), dexamethasone group(D, dexamethasone injection 0.07mg/per mouse), NBP low dose group(L, butylphthalide 80 mg / d /per mouse) and NBP high dose group(H, SBR phthalocyanine 120 mg / d /per mouse). Choose 9 mice as normal group(N, NS 0.1ml / d/per mouse). After onset, according to 0d,7d and 14 d,each group divided into three subgroup.Each subgroup had three mice.3 Established EAE model:The antigen MOG35-55 is diluted with saline to 6mg / ml,then added an equal volume of CFA on the basis of 1: 1. Mycobacterium tuberculosis H37 Ra was added to a final concentration 4mg / ml, thoroughly emulsified. After emulsification, injected hypodermic 0.1ml per mouse on both sides of the spine in four points.At 0h and 48 h post immunization,mice were injected with 0.5ml of PTX.4 Mice neurological score: Since immunization day, two observers weighed mice and evaluate neurological score with internationally accepted Benson(5 points) scoring criterion:Specific scoring criteria are as follows: 0 point:no clinical symptoms. 1 point: Tail tension disappear and show mild clumsy gait. 2 points :double hind limb was weakness,and can be restored after a passive stand up. 3 points: hind limbs paralyzed,and can not be restored after a passive stand, but after giving stimulation,mice can move. 4 double :hind limb paralyzed, forelimb paralysis or muscle weakness associated with urinary incontinence. 5 points on the verge of death, or death.When clinical symptoms between the two scores,lower scores plus 0.5 points indicate the final score.5 After the mice were given 10% chloral hydrate(350mg/kg) by intraperitoneal injection, at the onset of the day(day 0):each of EAE group and normal control group got three.7 days,14 days after onset: each of EAE group, NBP groups, dexamethasone group got three. First with saline and then with 4% paraformaldehyde after cardiac perfusion, take the lumbar enlargement of the spinal cord tissue embedded in paraffin.Determination of the number of apoptotic cells were positive changes by Tunel staining.Results:1 The incidence of post-immunized mice: mice were listlessness, reduced activity, such as fur is not the whole, since about 10 days after immunization onset, showed the rear elevation, weakness, decreased muscle tone, paralysis of hind limbs, partial paralysis and quadriplegia, etc. appear forelimb.2 Animal neurological scoreThe seven days after onset: L7, H7, D7 and E7 group of mice neurological score was 1.67±0.76,1.5±0.5,1.33±0.29,2.67±0.87,there was statistically significant that L7, H7 and D7 mice scores were less than EAE group(P<0.05), L7, H7 and D7 group score was no significant difference(P>0.05).The 14 days after the onset: L14, H14, D14 and E14 group mice neurological scores were 1.17±0.58,1±0.5,1±0,2.17±0.29, there was statistic-ally significant that L14, H14, D14 scores were less than mice EAE group(P<0.05), L14, H14, and D14 group score was no significant difference(P>0.05). 3 The number of Tunel-positive cells:There was barely no Tunel-positive cell in the spinal cord of the normal group’s mice. Each experimental group had Tunel-positive cells,which scattered around the spinal cord and blood vessels. The apoptotic cells may be Neuron oligodendrocytes, inflammatory cells and so on.The Tunel –positive cells of E0 group, E7 group, E14 group were 39.33±4.56,40.37±4.23,34.30± 4.33, there was statistically significant that the number of Tunel-positive cells of E14 group less than E0 group and E7 group(P<0.05).There was no significant difference between E0 group and E7 group(P>0.05).The seven days after onset: the number of Tunel-positive cells of L7, H7, D7 and E7 group were 23.53±4.92,21.53±3.49,22.67±4.06,40.37±4.23, there was statistically significant that L7, H7 and D7 group were less than EAE group(P<0.05),.There was no significant difference between L7, H7 and D7 group(P>0.05).The 14 days after the onset: the number of Tunel-positive cells of L14, H14, D14 and E14 group were 20.30±3.72,20.27±3.47,21.20±3.89,34.30±4.33, there was statistically significant that L14, H14 and D14 group were less than EAE group(P<0.05).There was no significant difference between L14, H14 and D14 group(P>0.05).Conclusion: In this study, through the application of NBP intervention for experimental autoimmune encephalomyelitis mice, EAE mice by observing the neurological score and Tunel staining cells, confirm that NBP can significantly reduce neurological dysfunction of EAE, anti apoptotic, improve neurological deficits of EAE by observing the neurological score and the number of the Tunel-positive cells.Provide new treatment ideas for the clinical treatment of multiple sclerosis.