The Protection Of Methylene Blue On Experimental Autoimmune Encephalomyelitis Mice

Objective: Experimental autoimmune encephalomyelitis(EAE) is an international standard animal model of multiple sclerosis(MS). We use it to observe the protective effect of methylene blue(MB) on EAE mice and explore the effect of MB on the m RNA levels of ROR?t, STAT3, Foxp3, IL-1?, IL-6, IL-17 A, IL-10, TGF-? and protein concentration of IL-1?, IL-6, IL-10, IL-17 A, TGF-? in the spleen of EAE mice. This experiment is aimed at exploring the protective mechanism of MB. The purpose of this study is to provide experimental basis for the treatment of MS.Methods:1 24 female C57BL/6 mice(8 to 10 weeks old, 18 g to 20 g body weight), were randomly divided into 3 different groups: 8 mice in group Control, 8 mice in group EAE and 8 mice in group MB. The animal model was established with MOG35-55, Mycobacterium tuberculosis, Complete Freund's Adjuvant and Pertussis Toxin, but the control group was granted immunity. The day on which the mice were immunized was recorded as Day 0. The mice in MB group received the solution of MB(20mg/kg) by peritoneal injection every day from Day 1. Mice in other groups received equal volume of physiological saline by peritoneal injection every day from Day 1. The weight and neurological score of mice were observed two times every day. We use 24 mice to observe EAE clinical course.2 30 female C57BL/6 mice(8 to 10 weeks old, 18 g to 20 g body weight), were randomly divided into 3 different groups: 10 mice in group Control, 10 mice in group EAE and 10 mice in group MB. The animal model was established with MOG35-55, Mycobacterium tuberculosis, Complete Freund's Adjuvant and Pertussis Toxin, but the control group was granted immunity. The day on which the mice were immunized was recorded as Day 0. The mice in MB group received the solution of MB(20mg/kg) by peritoneal injection every day from Day 1. Mice in other groups received equal volume of physiological saline by peritoneal injection every day from Day 1. The weight and clinical of mice symptoms were observed two times every day. 25 days post the immunition, the mice of each group were sacrificed to obtain the spleens. The m RNA levels of ROR?t, STAT3, Foxp3, IL-1?, IL-6, IL-10, IL-17 A, TGF-? in spleen were detected with qrt PCR, and the protein concentration of IL-1?, IL-6, IL-10, IL-17 A and TGF-? in the supernant of spleenocytes were tested by ELISA.Results:1 All mice in MB and EAE group developed EAE, while no mice in control group got sick. Compared to EAE group, the mean neurological score of mice in MB group on Day 25 were significantly lower(P<0.05).2 25 days post the immunition, the m RNA levels of inflammatory cytokines in spleen in three groups: comparing to the control group, the m RNA levels of ROR?t, STAT3, IL-1?, IL-6, IL-17 A were significantly increased in EAE group(P<0.05); however, the m RNA levels of Foxp3, IL-10, TGF-? were significantly decreased in EAE group(P<0.05); comparing to the EAE group, the m RNA levels of ROR?t, STAT3, IL-1?, IL-6, IL-17 A were significantly decreased in MB group(P<0.05), however, the m RNA levels of Foxp3, IL-10, TGF-? were significantly increased in MB group(P<0.05).3 25 days post the immunition, the protein concentration of inflammatory cytokines in the supernatant of spleenocytes in three groups: comparing to the control group, the protein concentration of IL-1?, IL-6, IL-17 A were significantly increased in EAE group(P<0.05), however, the protein concentration of IL-10 and TGF-? were significantly decreased in EAE group(P<0.05); comparing to EAE group, the protein concentration of IL-1?, IL-6, IL-17 A were greatly decreased in MB group(P<0.05), however the protein concentration of IL-10 and TGF-? was significantly increased in MB group(P<0.05).Conclusions:1 Methylene blue could delay the onset time and reduce the neurological score of EAE. These result indicate methylene blue has a protective effect against the development of EAE. 2 Methylene blue could decrease the expression of proinflammatory cytokines ROR?t, STAT3, IL-1?, IL-6, IL-17 A and upregulate the expression of anti-inflammatory cytokines Foxp3, IL-10 and TGF-? in the EAE spleen tissue. 3 Methylene blue may plays a neuroprotective protective role by regulating the balance of immune inflammation.