The Effect And Mechanism Of Nordihydroguaiaretic Acid On EAE Mice Of The Blood Brain Barrier

Objective: To investigate the effect and mechanism of nordihydroguaiaretic acid(NDGA) on EAE mice of the blood brain barrier(BBB), We use experimental autoimmune encephalomyelitis(EAE), which is an international standard animal model of multiple sclerosis(MS), to explore the potential protection and mechanism of NDGA on EAE BBB. So as to provide a new theoretical basis for the future clinical application of NDGA in central nervous system autoimmune disease, and find new targets for the clinical treatment of MS.Methods: 66 8-10 weeks female inbred C57BL/6 mice(18-20 g weight), were randomly divided into three groups: normal control group mice, mice in group EAE and mice in NDGA group, each group was 18. Each group was further divided into two subgroups of before the onset of the drug group and after the onset of the drug group. The prior to the onset of drug group was divided into 8 days, 14 days of immune two subgroups, each subgroup 2. After the onset of the drug group was divided into administration for 10 days, and 20 days two subgroups.The subgroup of After 10 day treated was 6, and after 20 day treated was12. The antigen MOG35-55 diluted with physiological saline to become 5mg/ml, complete Freund’s adjuvant(final concentration of Mycobacterium tuberculosis 4mg/ml) by an equal volume of adding, mixing evenly, fully emulsified with ether anesthesia, mice, fully fixed, divided into four points on both sides of the spine in mice subcutaneous injection, each point injection of 0.025 ml, total 0.1ml. We intraperitoneal injection of 500 ng PTX on The day of the 0h or the 48 h after immunizition(1000ng/only). The control group was granted immunity.On the first day of immunity we began to give the prior to the onset of drug group interventions. NDGA group was given NDGA 10mg/kg daily intraperitoneal injection; give the EAE group and normal control group of mice 5% DMSO 10ml/kg daily intraperitoneal injection. When each animal developed a clinical score up to 1 points(15 points method) after the onset of the drug group started to give the NDGA group NDGA 10mg/kg daily intraperitoneal injection; the EAE group and normal control group of mice were daily intraperitoneal injection with 5% DMSO(10ml/kg). The animal body weight status, activities, mental state, the glossiness of skin were observed on animal immunity after immunization at 8:00 and afternoon 17:00 daliy.We use 15 points method,which is the international general standard neurological function score of animal. Evans blue staining was used to observe the permeability of blood brain barrier with the application of fluorescence microscopy at different time points after the mice harvest. Immunohistochemistry was used to detect the expression of MMP-9, claudin-5, ZO-1 and occludin in spinal cord tissues of mice in each group after 20d-treated. Expression and localization of the tight junction proteins claudin5, ZO-1 and occludin with confocal method was detected in mouse brain tissues after20d-treated. Real-time fluorescence quantitative PCR was usd for detecting the level of m RNA of MMP-9 and NF-κ B in spinal cord tissues of mice in each group at different time points.Results: 1 Clinical scores in each group: In control group, no mice got sick. Mean clinical scores of mice in NDGA-treated group after 10 d and 20 d were significantly lower than EAE group(P<0.05). 2 EB fluorescence penetrant results: In each time point,there was less fluorescence penetrant in NDGA group than in EAE group. 3 Comparison of the expression TJs in each group3.1 The expression of claudin-5 3.1.1 Compared the expression of claudin-5 in the spinal cord tissues with the method of immunohistochemical in after 20d-treated group mice :compared the EAE control group to control group,claudin-5 in vascular endothelium brown granules is less and the staining is shallow,and did not form a continuous endothelial cells.The expression of claudin-5 in NDGA group was significantly increased than in EAE group. 3.1.2 Compared the expression of claudin-5 in brain tissues with the method of confocal in after 20d-treated gruop mice : In control group, claudin-5 positive cells continuously distributed in cytoplasm of vascular endothelial cells. The expression of claudin-5 protein was decrease in EAE group and NDGA group, and its continuity is destroyed, of which NDGA group were damaged weaker than in EAE. 3.2 The expression of occludin 3.2.1 Compared the expression of occludin in the spinal cord tissues with the method of immunohistochemical in after 20d-treated gruop mice :compared the EAE control group to control group,occludin in vascular endothelium brown granules is less and the staining is shallow,and did not form a continuous endothelial cells.The expression of occludin in NDGA group was significantly increased than in EAE group. 3.2.2 Compared the expression of occludin in brain tissues with the method of confocal in after 20d-treated gruop mice: In control group, occludin positive cells continuously distributed in cytoplasm of vascular endothelial cells. The expression of occluidn protein was decrease in EAE group and NDGA group, and its continuity is destroyed, of which NDGA group were damaged weaker than in EAE. 3.3 The expression of ZO-1 3.3.1 Compared the expression of ZO-1 in the spinal cord tissues with the method of immunohistochemical in after 20d-treated gruop mice :compared the EAE control group to control group,ZO-1 in vascular endothelium brown granules is less and the staining is shallow, and did not form a continuous endothelial cells.The expression of ZO-1 in NDGA group was significantly increased than in EAE group. 3.3.2 Compared the expression of ZO-1 in brain tissues with the method of confocal in after 20d-treated gruop mice : In control group, occludin positive cells continuously distributed in cytoplasm of vascular endothelial cells. The expression of ZO-1 protein was decrease in EAE group and NDGA group, and its continuity is destroyed, of which NDGA group were damaged weaker than in EAE. 4 The expression of MMP-9 in each group 4.1 The results of immunohistochemistry showed: in the group of after 20d-treated mice,there were no MMP-9 positive cells obviously in spinal cord tissues. In EAE group and NDGA group MMP-9 positive cells were observed in the meningeal cells、 small vascular endothelial cells and a small amount in the cytoplasm of glial cells. NDGA group compared with EAE group,there was less MMP-9 positive cells expression. 4.2 Comparison of the level of m RNA of MMP-9 with real-time fluorescence quantitative PCR:compared with the control group, in EAE group and NDGA group at different time point the m RNA level of MMP-9 were upregulated. In NDGA group the MMP-9 m RNA level was significant decreased than in EAE group(P<0.05). 5 Comparison of the level of m RNA of NF-κB with real-time fluorescence quantitative PCR:compared with the control group, in EAE group and NDGA group at different time point the m RNA level of NF-κB were upregulated. In NDGA group the NF-κB m RNA level was significant decreased than in EAE group(P<0.05).Conclusions:1 The destruction of blood brain barrier is in the early stage of EAE mice, and the more serious the condition is, the more obvious the destruction.2 NDGA can reduce the clinical scores of EAE mice, delay the progress of the disease and protect blood brain barrier from disruption. Its mechanism may be closely connected by protecting the integrity of the tight junction proteins and inhibiting of the expression of MMP-9 and NF-κ B.