Anti-oxidative Stress Effect Of Nordihydroguaiaretic Acid And Tanshinone Ⅱ A On Experimental Autoimmune Encephalomyelitis Mouse Central Nervous System

Objective: Multiple sclerosis (MS) is one of autoimmune diseases,which main pathological characteristics is demyelination in central nervoussystem. As we all know the ideal animal model to study human multiplesclerosis is experimental autoimmune encephalomyelitis (EAE). Experimenthas confirmed that sulforaphane can activate the Nrf2pathway, exert aneuroprotective effect by its anti-oxidative effect. But sulforaphane can notapply to the human body, we reviewed the literature to findnordihydroguaiaretic acid (NDGA) and tanshinone Ⅱ A which can activatethe Nrf2pathway and be applied in human body. We simulated the normalcourse of diagnosis and treatment of patients,"onset–doctors’ office visiting-treatment". We gave the mice intervention drugs after neurological deficitsymptoms appearance(in previous studies, they gave modeling drugs andintervention drugs at the same time).We explored the anti-oxidative effect ofnordihydroguaiaretic acid (NDGA) and tanshinone Ⅱ A in EAE mousecentral nervous system. Detected the possibility of NDGA and tanshinone ⅡA used in the clinical treatment of MS and its mechanism.Methods:1Groups: female C57BL/6mice bred to8-10weeks, weighing18-20g,were randomly divided into DMSO group, NDGA group and TanshinoneⅡAgroup, there were9mice in every group.2Preparation of EAE Model: Diluted antigen MOG35-55withphysiological saline into5mg/ml and added in a1:1volume of Freund’scomplete adjuvant (the final concentration of tuberculosis was4mg/ml),mixedcompletely, and emulsified. Injected subcutaneously in four points on both sides of the mice spine with0.1ml. Injected peritoneal500ng PTX at0hourand at48hours after ether anesthesia and fixed.3After immunization, we measured the nervous system function everyday. When the mouse nervous system function was impaired (score≥1),mice of DMSO group were injected5%DMSO1ml/mg/dayintraperitoneally, mice of NDGA group were injected NDGA10mg/kg/dayintraperitoneally, mice of TanshinoneⅡA group were injected TanshinoneⅡA20mg/kg/day intraperitoneally.4Evaluate the neurological function score:.Two investigators hadassessed the clinical signs of mice twice a day until20days after onset.5Measured the concentration of MDA in the brain tissues: The micewere sacrificed. Removed brain tissues quickly on ice, stripped the dura.Washed away the blood with4℃saline, dried with filter paper, andweighted the tissue. According to the ratio of1:9added4℃saline.Centrifuged for15min at4℃and2000r/min. Stored the supernatant at-80℃.6Measuring the content of Nrf2, HO-1in brain tissues: After anesthesia,irrigated the mice with normal saline. Then fixed the brain tissue with4%paraformaldehyde for24hours, paraffin-embedded. Cut the brain tissue intosections (5um), and observed immunohistochemical staining positive cellswith an optical microscope and counting.Results:1Clinical manifestations:Neurological function score: The respectively mean Neurologicalfunction score of DMSO group、NDGA group and TanshinoneⅡA group was:2.50±0.40,1.25±0.29,1.33±0.19. Neurological function score of NDGAgroup and TanshinoneⅡA group were significantly lower than DMSO group,the differences were statistically significant (P <0.05).2The MDA content in brain tissue: The respectively mean MDA contentof DMSO group、NDGA group and TanshinoneⅡA group was:41.05±8.23,23.28±3.02,27.03±3.51. The MDA content of NDGA group and TanshinoneⅡ A group were significantly lower than DMSO group, the differences werestatistically significant (P <0.05).3The Nrf2expression in brain tissue: The respectively Nrf2immunoposition cells of DMSO group、NDGA group and TanshinoneⅡAgroup was:19.13±2.81,84.41±3.48,76.15±3.18. The Nrf2immunopositioncells of NDGA group and TanshinoneⅡA group were significantly higherthan DMSO group, the differences were statistically significant (P <0.05).4The HO-1expression in brain tissue: The respectively HO-1immunoposition cells of DMSO group、NDGA group and TanshinoneⅡAgroup was:21.46±2.86,86.04±11.22,83.00±8.76. The HO-1immunopositioncells of NDGA group and TanshinoneⅡA group were significantly higherthan DMSO group, the differences were statistically significant (P <0.05).Conclusions:1NDGA and TanshinoneⅡA can reduce neurological damage EAE mice.2NDGA and Tanshinone Ⅱ A may be via antioxidant to performneuroprotective effects.3NDGA and TanshinoneⅡA antioxidant effects may be due to activationof Nrf2/ARE pathway which increased expression of HO-1.