| Objective:Multiple sclerosis is an autoimmune disease of the central nervous system,but its cause is not yet fully clear. The main pathological features is that multiple central nervous system inflammatory demyelination,glial cell proliferation and varying degrees of axonal damage . At present a number of studies have shown that patients with multiple sclerosis exist blood-brain barrier damage at the early stage, and speculate that it may be a critical component of the pathogenesis of multiple sclerosis. Matrix metalloproteinase -9 degradates matrix proteins which is in the vascular endothelial cells,and leading to an increased microvascular endothelial permeability, so that T cells and other inflammatory cells can penetrate through the BBB basement membrane,and finally the BBB is damaged.α-lipoic acid which has been known as the "universal antioxidant" is the most effective natural anti-oxygen agent , it is not only scavenging oxygen free radicals, and reducing the migration ability of the monocyte , but also stabilizing the blood-brain barrier ,and resisting the oxidative stress which is induced by the adhesion of the monocytes.This study is designed to establish a rat model of autoimmune encephalomyelitis,and detect the expression of the MMP-9 levels in the peripheral blood and the central nervous system, while observing the effect ofα-lipoic acid on the level of MMP-9 expression in the peripheral blood and central nervous system of the EAE rat. Discussing the protective effect ofα-lipoic acid on the injury of the central nervous system and its mechanism.Methods:125 female Wistar rats ,6-8 weeks old, weighing from 180g to 220g.80 rats are randomly divided into two groups(the EAE group and the CFA group),and each of the two groups are further divided into eight subgroups(the 6-day group,the 8-day group,the 10-day group,the 12-day group,the 14-day group,the 16-day group,the 18-day group,and the 20-day group) by time,and each sub-group contains five rats. The other 45 rats are randomly divided into three group (the control-EAE group,the low-doseα-lipoic acid-EAE group which is given intraperitoneally 30mg/kg·d ALA from 6th day after immunization and the high-doseα-lipoic acid-EAE group which is given intraperitoneally 100mg/kg·d ALA from 6th day after immunization), and each of the three groups are further divided into three subgroups(8-day, 12-day group and 20-day group) by time, and each sub-group contains five rats. Weighing every day and making clinical score. observing the infiltration of inflammatory cells by HE staining,and evaluating the level of MMP-9 expression in the brain tissue and spinal cord by immunohistochemical staining. Assessing the serum level of MMP-9 by enzyme-linked immunosorbent.Results:1 The clinical appearance: (Fig.1,2)The clinical appearance of the EAE rats has show dynamic changes during the acute stage of the disease.Most of them emerge the clinical appearance at the 11th day after immunization,and reach the peak of the disease at the 14th day after immunization,and then decline.2 Neurological function score: (Table 1)At the peak of the disease,the neurological function score of the high-doseα-lipoic acid-EAE group was significantly lower than the control EAE group and low-doseα-lipoic acid-EAE group (P <0.05).Low-doseα-lipoic acid-EAE group is also lower than the control EAE group (P <0.05),which is statistical significance.3 Histopathological changes: (Fig.3,4)Different degrees of inflammatory lesions appear in the spinal cord and brain tissues in the EAE group(12-day group, 14-day group, 16-day group,and 18-day group). Especially the 14-day group (at the peak of the disease) is the most significant,which appears the sleeve-like dense inflammatory cell infiltration around the blood vessels.4 Immunohistochemical pathology observation: (Fig. 5～10)(Table 2～5)4.1 Comparing the two groups( EAE group and the CFA group )Compared with the CFA group(8-day group, 10-day group, 12-day group, 14-day group, 16-day group), the MMP-9 positive cells in the spinal cord of the EAE group was statistically significant (P <0.05).Compared with the corresponding CFA group(10-day group, 12-day group, 14-day group)the MMP-9 positive cells in the brain tissue of the EAE group is statistically significant (P <0.01).4.2 Comparing between the EAE groupsCompared with the 6-day group, 14-day group, 18-day group, 20-day group, the MMP-9 positive cells in the spinal cord lumbar enlargement of the 10-day group was statistically significant (P <0.05).Compared with the 6-day group, 8-day group, 14-day group, 16-day group, 18-day group, 20-day group, the MMP-9 positive cells in the brain tissue at the optic chiasm of the 10-day group was statistically significant (P <0.05).4.3 Comparing the three groups(the control EAE group,the low-doseα-lipoic acid-EAE group,and the high-doseα-lipoic acid-EAE group)Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).Compared with the low-doseα-lipoic acid-EAE group(12-day group, 20-day group), the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.01).Compared with the low-doseα-lipoic acid-EAE group(12-day group), the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).5 Comparing the serum level of the MMP-9(Table 6,7)Compared with the 12-day group of the CFA group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P <0.01).Compared with the 6-day group, 8-day group, 10-day group, 14-day group, 16-day group, 18-day group, 20-day group of the EAE group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).Compared with the control EAE group, the serum level of the MMP-9 of the low-doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).Compared with the control EAE group, the serum level of the MMP-9 of the high -doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant(P <0.01).Conclusion:1Α-lipoic acid can reduce the incidence of EAE rats ,and also can reduce dysfunction of the nervous system which is resulted by the disease itself.2Α-lipoic acid can reduce the MMP-9 level which is permeability marker of the blood-brain barrier,and have a role in stabling the blood-brain barrier,which is suggesting that it has a promising application prospects in the treatment of human MS.3Α-lipoic acid with a dose-dependent manner in the treatment of the EAE rats, when large doses can be more effective in suppressing the development of the disease, and which can guide clinical practice. 4 The levels of the MMP-9 in the peripheral blood and CNS of the EAE rats shows dynamic changes during the acute phase of the disease,and which is relevant to the clinical manifestations.It points that MMP-9 may play a key role in the occurrence and progress of the disease, and may be considered as the Potential biological marker of the disease.
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