WAR5,A Novel Rho Kinase Inhibitor, Exhibits Therapeutic Mechanism In Experimental Autoimmune Encephalomyelitis By Intraperitoneal Injection

Objective:In this experimental, we attempt to explore and discuss the curative effect of a new Rho kinase inhibitor(ROCK) WAR5, a Fasudil derivative, and possible mechanism in myelin oligodendrocyte glycoprotein 35–55(MOG35–55)-induced experimental autoimmune encephalomyelitis(EAE). Research and development of WAR5 for treatment of nervous system inflammatory degenerative diseases such as MS will provide reliably theoretical and experimental basis.Methods:Female C57BL/6 mice were randomly ruled into saline control group and WAR5 treatment group. EAE model was induced by MOG35–55 immunization in two group.Normal saline and WAR5 were separately given by intraperitoneal route twice a day from the third day to the 27 th day of post-immunization(p.i.). Animals were weighed and clinical score was evaluated every other day. On day 28 p.i., mice were executed, spinal cords were acquired for hematoxylin-eosinand myelin staining. Splenocytes were detached and the phenotype of M1 and M2 spleen macrophages was determinated by flow cytometry(FCM).Protein were extracted from brains and spinal cords for the measurement of inducible nitric oxide synthase(i NOS) by Western blot. The experimental data was analyzed and disposed by Graphpad Prism 5.0 software.Results:1. Compared with EAE control mice, intraperitoneal injection of WAR5 can not only postpone the start of EAE obviously, but the clinical symptoms can also relieve obviously.Onset time were prolonged and degrees of body weight loss were fewer in WAR5 group(P<0.05, and P<0.01, respectively).2. WAR5 inhibited invasion of inflammatory cells and demyelination in spinal cord.HE staining showed that there were infiltration of inflammatory cells in varying degrees within white matter of spinal cords in EAE group. Myelin staining showed that there were different degrees of demyelination of nerve fibers within white matter of spinal cords.WAR5 can obviously inhibit the invasion of inflammatory cells and demyelination of nerve fibers.3. WAR5 can relieve the expression of CD16/32 of M1 positive macrophages in the peripheral immune system(P<0.01). Meanwhile, the expression of CD206 of M2 macrophages have a increasing tendency, but there is no statistical significance between two groups(P=0.29).4. WAR5 can suppress the level of i NOS in brains and spinal cords, thereby reducing demyelinating lesions and clinical symptom caused by inflammation in spinal cords.Conclusions:Our results showed that novel Rho kinase inhibitor WAR5 derivatives have a good result in treating EAE. Intraperitoneal injection of WAR5 can obviously relieve CNS inflammatory cell invasion and demyelination, lower the expression of CD16/32 positive macrophages, and still can make the expression of CD206 positive macrophages have an increasing tendency. WAR5 can inhibit the expression of i NOS in brains and spinal cords.The effective mechanism of WAR5 may be related to the inhibiting of Rho kinase activity of the CNS, prompting conversion of M1 macrophages to M2 macrophages, and suppressing demyelination and inflammatory cell infiltration. Eventually, the occurrence and development of EAE can be suppressed.