Pathogenesis Of HO/CO System In The Relapsing-remitting Experimental Autoimmune Encephalomyelitis

ObjectiveMultiple sclerosis(MS) is an inflammatory demyelination disease of the central nervous system(CNS) that causes relapsing and progressive neurological impairment. It's pathogenesis is not very clearly. Recognized point of view is that MS caused by factor about environment, inheritance and infection. It is our main aim that we investigate the pathogenesis of MS and seek effective therapeutic scheme for MS by setting up an animal model.Increasing evidence shows that oxidative stress play an important role in the pathogenesis of experimental autoimmune encephalomyelitis(EAE),an animal model of the human disease,MS.Oxidative stress is a fundamental mechanism underlying a host of different diseases, and is implicated in the pathogenesis of many neurological diseases.oxidative injury is important in EAE because the CNS, notably the oligodendrocyte, is inherently vulnerable to oxidative damage.Organisms employ a multitude of endogenous antioxidant mechanisms to defend against the deleterious effects of oxidative stress. Heme oxygenase-1(HO-1)is a potent one among these mechanisms. HO-1 is a heat shock protein induced by oxidative stress.HO-1 metabolizes heme to the antioxidant bilirubin and carbon monoxide,and represents a powerful endogenous defensive mechanism against free radicals in many diseases.HO-1 is induced by various stimuli, including heat shock and oxidative stress, and represents one powerful endogenous protective mechanism against free radicals in a variety of pathological conditions, including inflammation, ischemic stroke and many neurodegenerative diseases.However, only a few studies have reported the expression of HO-1 in EAE, and the role of this important enzyme in EAE remains unresolved.In this research, we detected the induction of HO-1 in EAE, and evaluated its effect on this disease.We administrateα-LA(alpha-lipoic acid ) and SNPP(Tin-protoporphyrin)to the rats of EAE in order to investigate the therapeutic potential in treatment of neuroinflammatory diseases like MS. Simultaneously,we will search the genes related to EAE by using the gene chip technique.MethodsA total of 100 adult healthy female Wistar rats with body weighing between 180 and 200g were divided randomly into five groups: normal control group(6 rats), Other rats were immunized subcutaneously in the four foot pads by fresh guinea pig spinal cord homogenate(GPSCH) and complete Freund's adjuvant(CFA). After they first paroxysm,divided randomly out 6 rats as disease group,others divided randomly into EAE group, dexamethasone(DXM) group, alpha-lipoic acid (α-LA) group,Tin-protoporphyrin(SNPP) group, and this four groups were divided into 7 day group and 14 day group, separately.Clinical signs of EAE were assessed the mean of twice daily by two observations. Scores were assigned on the basis of the following symptoms: 1, tail weakness; 2, tail weakness plus limb asthenia; 3, mild limb paralysis; 4, severe limb paralysis; 5,moribund/dead. EAE group, DXM group, LA group and SNPP group were injected intraperitoneal respectively NS 0.5mL/d,DXM 2 mg·kg-1·d-1,LA 100 mg·kg-1·d-1,SNPP 100μmol·kg-1·d-1. This treatment was started on the first day of EAE outbreak and continued daily for 7 day. During the experiment, the mean maximal score of animals at different time point and the relapse of disease were observed as results.Rats were sacrificed after anesthesia with intraperitoneal injection. Tissues of the spinal cord were fixed with 4﹪formalin, then the tissues were embedded in paraffin and sectioned at 4μm thickness. Some of the sections were stained with HE to assess lymphocyte infiltration and inflammation. Some of the sections were stained with immunohistochemistry as described below. The slides were incubated with anti-HO-1 antibody(1:1000), then the tissues were further incubated with the second antibody, avidin-biotin-perxidase complex. Among these approaches, we should wash these specimens with PBS, at last, the results were analyzed with microscopy.The expression of cGMP in the spinal cord of rats was measured by cGMP 125I-RIA Kit. The MDA and SOD level activity in the tissues of rats was measured by MDA kit and SOD kit.Extract the general RNA from the spinal cord of rats , transcripted to cDNA by RT-PCR and labelled probe by UTP. The probe were mixed and hybridized with the signal transduction pathway finder gene chips . Detected by the chemiluminescent kit , acquire image and use the GEArray Expression Analysis Suite to complete the data analysis.Results1 The relapse of disease in different group Comparison of the relapse of disease in four groups, we found the frequency of EAE attacks in EAE group were higher that the treatment groups (P <0.05).2 Clinical profile of EAE in different group Neurological deficits scores of DXM group,LA group and SNPP group were lower than those of EAE group (P <0.01).3 Neuropathological findings The results demonstrated that some monocytes infiltration was observed in the tissues of spinal cords before the clinical signs emerging. The degree of infiltration was associated with the severity of EAE. The extent of inflammation in EAE group was higher than DXM group,LA group and SNPP group. (P <0.05).4 Expression of HO-1The number of HO-1 positive cells in spinal cord of rats in EAE group is more than other groups (P <0.05). The level of expression of HO-1 in spinal cord of rats in SNPP group is the lowest (P<0.01).5 The cGMP level in spinal cordThe cGMP level in spinal cord of rats in EAE group is higher than those of other groups (P <0.05).6 The MDA and SOD level in the tissuesMDA level in the tissues of rats in EAE group is higher than other groups (P <0.05). SOD level in the tissues of rats in EAE group is lower than other groups (P <0.05).7 The result of gene chip analysisThe result of gene chip analysis showed the consistent different genes expression throughout the EAE mice model. There are ten genes express in different levels along with the different stage of EAE. Among this ten genes, eight are related to DXM treatment, three are related to LA treatment, five are related to SNPP treatment.Conclusions1 LA and SNPP reduces the morbidity, and protects the rats from the severity of the disease. Especially, the healing effect of SNPP is similar to DXM.2 SNPP treatment can reduce the expression of HO-1 in spinal cord of EAE rats.3 HO-1,cGMP in spinal cord and MDA,SOD in serum of EAE rats are associated with the activity of EAE.4 The consistent different genes expression throughout the EAE mice model,and some are related to treatments.