Study On The Mechanism Of Yougui Pills With Reinforcing Yang From Yin In Promoting Nerve Regeneration Based On The Nerve Growth Guidance Cues In Mice With Experimental Autoimmune Encephalomyelitis

Part One: Regulation of Yougui Pills on neural guidance cues and Rho GTPases in mice with experimental autoimmune encephalomyelitisObjective: The regulation of Yougui Pills(YGPs) on netrin1/DCC, slit2/Robo1 and Rho A/Rac1/Cdc42 expressions in brain and spinal cord of mice were observed to elucidate the underlying molecular mechanism of YGPs on promoting nerve regeneration in experimental autoimmune encephalomyelitis(EAE) mice.Methods: 70 female C57BL/6 mice were divided into seven groups, including the normal control(NC), model(MO), prednisone acetate(PA)-, Catalpol(CA)- and 1.2, 2.4 or 4.8 g/kg YGPs-treated EAE groups. The EAE model was established by injecting the mice subcutaneously with MOG35–55 antigen. Mice were treated with oral administration once a day for successively 40 days. The body weights and the neurological functions scores were recorded in each group. Mice were sacrificed for brain and spinal cord harvest on day 40 post-immunization(PI). The pathology and morphology of the brain and spinal cord was examined. The expressions of MAP-2 were detected by immunofluorescent staining(IF). The levels of netrin1, DCC, slit2, Robo1, Rho A, Rac1, and Cdc42 were assayed by immunohistochemistry(IHC),real-time quantitative polymerase chain reaction(qRTPCR) and Western blot(WB).Results: YGPs reduced neurological function scores in EAE mice. The inflammatory infiltration was decreased and the axon and myelin damage in both brain and spinal cord was alleviated by YGPs treatments. IF analysis showed that YGPs increased the expressions of MAP-2. IHC, q RT-PCR and WB results showed that netrin1, DCC, slit2, Robo1, Rac1 and Cdc42 were increased, while Rho A was reduced following YGPs treatments.Conclusion: YGPs exhibited a neuroprotective effect in EAE mice induced by MOG35-55. YGPs also promoted nerve regeneration, and netrin1/DCC, slit2/Robo1 and Rho A/Racl/Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.Part Two: Regulation of YGPs and their decomposed formulas nourishing yin(NY), warming yang(WY) on neural guidance cues and Rho GTPases in EAE miceObjective: To further evaluate the influence of compatibility of YGPs “reinforcing yang from yin” on nerve regeneration in EAE mice.Methods: Female C57BL/6 mice were divided into seven groups, including the NC, MO, PA-, CA-, YGPs-, nourishing yin(NY)- and warming yang(WY)-treated EAE groups. The methods of establishment of EAE miceand administration were the same as the part one, and so did the detective indexes and experimental methods.Results: YGPs, NY or WY formulas treatments reduced neurological function scores, eased the autoimmune inflammatory and alleviated the axon and myelin damage in EAE mice. Three formulas increased the expressions of MAP-2, netrin1, DCC, slit2 and Robo1. The expressions of Rac1 and Cdc42 were increased, while Rho A was reduced following YGPs, NY or WY treatments. In the study of decomposed formulas, NY was effective in reducing the neurological functions scores in EAE mice at acute phase, lessening pathological damage and good at promotion of neural cues slit2, Robo1 and Rho A in remission phase, while WY was good at promotion of neural cues netrin1, DCC, Rho A, Rac1 and Cdc42 in remission phase. The effect of YGPs had a significant moderating role on all aspects as indicated.Conclusion: YGPs, NY and WY had neuroprotective effects and promotion of nerve repair by regulation of netrin1/DCC, slit2/Robo1 and Rho A/Racl/Cdc42 in EAE mice. The effects of nourishing yin and warming yang focused on neuroprotective effects and promoting nerve repair differently, and YGPs had a significant moderating role on neuroprotective effects and promoting nerve regeneration.Part Three: Identification and Quantification of the main chemical components in YGPs and its decomposed formulasObjective:The aim of study was to provide the evidence for the quality control of YGPs, NY and WY formula and the material basis.Methods: The major chemical components of YGPs, NY and WY were identified by UPLC-LTQ-Orbitrap-MS. The quantification analysis of loganin and hyperoside in YGPs was performed on LC-ESI-MS/MS.Results: The identified compounds of YGPs were songorine, chiorogenic acid, fuziline, loganin, neoline, sweroside, hyperoside and quercitrin, meanwhile these of NY were chiorogenic acid, loganin, sweroside, hyperoside and quercitrin, and these of WY were songorine, chiorogenic acid, fuziline, neoline, hyperoside and quercitrin. The content of loganin and hyperoside in YGPs was 51.8 mg/g and 43 mg/g, respectively.Conclusion: The identification and quantification of the main chemical components in YGPs and its decomposed formulas provided the evidence for the quality control and the material basis.In summary, YGPs exhibited a neuroprotective effect on promoting nerve regeneration in EAE mice. Netrin1/DCC, slit2/Robo1 and Rho A/Racl/Cdc42 were involved in these effects. Nourishing yin and warming yang laid particular emphasis on the neuroprotective effects and promoting nerve repair, and YGPs had a significant moderating role on all aspacts, which involved some scientific implications on the compatibility of YGPs “reinforcing yang from yin”.