Neuroprotective And Immunoregulative Effect Of Buyang Huanwu Decoction On Experimental Autoimmune Encephalomyelitis

Objective: In this study, we use buyang huanwu decoction(BHD) to treat experimental autoimmune encephalomyelitis(EAE) mices induced by myelin oligodendrocyte glycoprotein peptides 35-55(MOG35-55), observe the clinical symptoms, pathological changes and the influence of BHD on the central nervous system( CNS) and peripheral immune system. The results of this study will provide reliably theoretical and expermental basis for treatment of multiple sclerosis(MS).Methods: Twenty-four female adult C57BL/6 mice were immunized with MOG35-55 to induce chronic EAE. The mice were randomly divided into EAE saline control group(12) and BHD group(12). Normal saline and BHD were respectively administered by oral route once a day from day 3 post-immunization( p.i.) to day 27 p.i.. Animals were weighed and clinical score was evaluated every day. Mice were sacrificed on day 28 p.i.,Splenocytes were separated and the expression of CD4+IL17+, CD11b+IL-12+,CD11b+CD16/32+, CD4+CD25+, CD4+IL-10+, CD4+TGF-?+, CD4+IFN-?+ were analyzed by flow cytometry. The levels of IL-6, IL-1?, TNF-? were analyzed by ELISA kits.Frozen sections of spinal cords were obtained for HE staining, myelin staining and immunohistochemistry staining to measure CD4, CD68, GFAP, BDNF, GDNF, NG2, Neun,MBP, MAP-2 and TLR4, Myd88, p-NF-?B/p65, p-38, JNK were measured by Western blotting. The experimental data were analyzed and disposed by Graphpad Prism 5.0Software.Results: 1. C57BL/6 mice with EAE induced by MOG35-55 were established successfully. Compared with EAE control mice, oral administration of BHD can not only delay the onset of EAE, but also relieve the clinical symptoms of EAE. And degrees of the body weight loss mice were fewer in BHD group(p <0.05,p <0.01,p <0.001).2. The treatment of BHD can promote remyelination of demyelinated axons and expression of Neun and MAP-2(p <0.05).3. BHD enhances expression of Neu N protein MAP2 in EAE and promotes neuroprotection(p <0.05).4. BHD enhanced the expression of neurotrophic factors. Compared with EAE control mice, BHD enhanced the expression of BDNF, GDNF(p <0.05).5. BHD inhibits the inflammatory infiltration in the spinal cord. HE staining andimmunohistochemistry staining showed that there were infiltration of inflammatory cell in various degrees within white matter of spinal cords in EAE(p <0.05).6. BHD inhibits aggregation of astrocytes in the spinal cord(p <0.05).7. BHD inhibits inflammatory signaling pathway in the spinal cord and reduced the expression of TLR4, Myd88, p-NF-?B/p65, p-38 and JNK(p <0.05).8. BHD modulates peripheral immune responses. Compared with EAE control mice,BHD can downregulate the expression of CD4+IL17+, CD11b+IL-12+, CD11b+CD16/32+ in the peripheral immune system, upregulate the expression of CD4+CD25+, CD4+IL-10+,CD4+TGF-?+, CD4+IFN-?+, and inhibit the release of inflammatory cytokines IL-6, IL-1?,TNF-?(p <0.05).Conclusion: C57BL/6 mice with EAE induced by MOG35-55 were established successfully. BHD has a nice effect in treating EAE. BHD can not only delay the onset of EAE, but also relieve the clinical symptoms of EAE. Oral administration of BHD can obviously relieve the demyelination and increase the expression of MBP, NG2 to repair the damaged myelin and promote remyelination. BHD can enhance expression of Neun,MAP-2 and can protect nerve cells and promote the expression of neurotrophic factors,which play a neuroprotective role. BHD can reduce the inflammatory infiltration of the CNS, such as CD4, CD68 and astrocyte GFAP. BHD may also mediate the TLR4 signaling pathway, and reduce the expression of TLR4, Myd88, p-NF-?B/p65, p-38, and JNK.Meanwhile, BHD downregulated the expression of CD4+IL17+, CD11b+IL-12+,CD11b+CD16/32+ in the peripheral immune system, upregulate the expression of CD4+CD25+, CD4+IL-10+, CD4+TGF-?+, CD4+IFN- ? +, and inhibit the release of inflammatory cytokines IL-6, IL-1? and TNF-?. The effective mechanism of BHD may be related to the anti-inflammatory effect, immune regulation and enhanced the expression of neurotrophic factors, thus inhibiting the immune inflammatory reaction of CNS and peripheral immune system via the inhibition of TLR4/ NF-?B signaling pathway.Eventually, the occurrence and development of EAE can be suppressed.