Study On The Treatment Of Hydroxyfasudil And Its Influence On Expression Of ROCK-Ⅱ, INF-γ, IL-10 And IL-17 In Experimental Autoimmune Encephalomyelitis Mice

Objective:To explore the effective treatment for multiple sclerosis (MS) and to investigate the mechanism of occurrence and development of experimental autoimmune encephalomyelitis (EAE), meanwhile, in order to approach the possible therapeutic mechanism of Fasudil hydrochloride, we designed the present study to treat EAE mice by Fasudil hydrochloride, observing the expression of ROCK-Ⅱand detecting changes of proliferation response of splenic lymphocytes induced by myelin oligodendrocyte glycoprotein peptides 35-55 (MOG35-55)and the secretion of cytokines in C57BL/6 mice with EAE.Methods:Sixty female C57BL/6 mice were divided by average body weight into three groups:EAE group (20), Fasudil hydrochloride treated group (20) and Adjuvant group (20). Mice in EAE and treated groups were immunized subcutaneously with MOG35-55. At the same time, adjuvant group was injected with normal saline instead of MOG35-55. Fasudil hydrochloride was intraperitoneally injected in the dosage of 50mg/kg/d in treated group from day 6 post-immunization (p.i), and followed by 14days. Mise in EAE and adjuvant group were injected with normal saline as control. The general conditions, body weight changes and clinical manifestations of each groups were observed and compared. On day 18p.i, splenic lymphocytes were isolated under asepsis from eight mise in each group respectively, and splenic lymphocyte suspensions were prepared. The proliferation assay was determined by MTT. The supernatants were harvested for the detection of INF-γ, IL-17 and IL-10 by ELISA. Mice were sacrificed on days 18-20 p.i. The spinal cords were dissected for HE and Luxol Fast Blue staining. The brains were dissected for Western blot to detect the expression of ROCK-Ⅱ. Data were analyzed with SPSS 13.0 software.Results:1. C57BL/6 mice with EAE induced by MOG35-55 were established successfully. HE and Luxol Fast Blue staining showed marked inflammatory cell infiltration and demyelination in spinal cords. The differences of incidence, change of body weight and mean maximum clinical scores between EAE group and treated group were statistically significant (P<0.05). After intervention of Fasudil hydrochloride, the inflammatory cell infiltration and demyelination were decreased obviously.2. Proliferation response (P<0.01)and cytokines secretion of splenic lymphocytes such as INF-γand IL-17(P<0.05) were marked increased in EAE group. Fasudil hydrochloride may obviously inhibit the secretion of those two cytokines and promote the secretion of IL-10 (P<0.05)3. ROCK-Ⅱexpressed in brain of mice in each group. The expression of ROCK-Ⅱin EAE group increased obviously compared with that of adjuvant group (P<0.05). After intervention of Fasudil hydrochloride, the expression of ROCK-Ⅱwas decreased obviously compared with that of EAE group (P< 0.05).Conclusion:1. Fasudil hydrochloride could significantly decrease the incidence rate, relieve the clinical severity of EAE and lessen inflammatory cell infiltration and demyelination.2. Fasudil hydrochloride may suppress proliferation response of splenic lymphocytes and secretion of INF-y and IL-17 and promote the secretion of IL-10, which may be one of mechanism that Fasudil hydrochloride can treat EAE.3. The another possible mechanism that fasudil hydrochloride treats EAE is the inhibition of ROCK-Ⅱ.