Mechanisms Of Ginkgolide B And K On Myelin Protection And Regeneration In CPZ-induced Demyelinating Mice

Objective:The main purpose of this study was to investigate the effects of oral Ginkgolide B and Ginkgolide K on myelin protection and regeneration of demyelinating mouse models induced by Cuprizone(CPZ)and the possible mechanisms.This study provides scientific theoretical and experimental basis for the application of GB and GK in the treatment of multiple sclerosis(MS)and other demyelinating diseases,and also provides a new idea for the protective and regenerative effects of GB and GK on the myelin sheath of central demyelinating diseases.Methods:Thirty-two 8-week-old C57BL/6 male mice were fed adaptive for one week,and then randomly divided into normal group(CON),model group(CPZ),GB group(GB),and GK group(GK),with 8 mice in a group.A demyelinating mouse model was prepared by feeding 0.2%(w/w)CPZ for 6 weeks.Mice in normal group were fed normal diet.From the 3rd week of modeling,mice in the GB administration group were given GB gavage,and mice in the GK administration group were given GK gavage for 4consecutive weeks.Normal group and CPZ model group were given 0.5% CMC-NA solvent gavage.At the end of the experiment,the mice were subjected to behavioral tests,and anxiety,depression,and cognitive behavior were assessed by the elevated plus maze,forced swimming,and T-maze tests,respectively.The animals were treated at the end of the experiment,on day 42,with half of each group randomly receiving brain tissue for frozen sections and the other half for protein extraction.In vitro,BV2 cells,GL261 astrocytes and primary astrocytes were treated by LPS,and GL261 astrocytes and primary astrocytes were stimulated by PAF.Cell culture supernatant was collected and protein was extracted.Luxol Fast Blue(LFB)staining,Black Gold II staining and MBP immunofluorescence staining were used to observe the demyelination of myelin sheath.Western blot was used to detect the expression of related proteins.The release of cell culture supernatant and related inflammatory factors in brain were determined by ELISA.The changes of glial cells were observed by immunofluorescence staining.All experimental data were statistically analyzed using Graph Pad Prism 5.0 statistical software.Results:1.GB and GK can effectively improve the behavioral abnormalities and demyelination of the corpus callosum in CPZ-induced demyelinating mice.2.In vitro and in vivo,GB and GK inhibited the activation of microglia and the release of TNF-α,IL-6,IL-1β and NO.3.In vivo and in vitro experiments,GB and GK enhanced the enrichment of astrocytes and the expression of CD206 and S100A10,inhibited the expression of PAFR by astrocytes,and may enhance the secretion of neurotrophic factors BDNF and CNTF by antagonizing PAF-PAFR pathway.And reduce the secretion of inflammatory factors TNF-α,IL-6,IL-1β.4.GB and GK can promote the production of NG2 and PDGFRα +OPCs,increase the expression of Ki67,inhibit the expression of Notch 1 and Wnt signaling molecules,and enhance the expression of MBP.Conclusions:1.Oral administration of GB and GK improved the behavioral disorders such as anxiety,depression and cognition in CPZ-induced demyelinating mice,and significantly improved the demyelination of corpus callosum.2.GB and GK inhibit the activation of microglia and its associated inflammatory response and oxidative stress.3.GB and GK enhance the enrichment of astrocytes,promote their transformation to A2 phenotype,and may enhance the secretion of neurotrophic factors BDNF and CNTF and inhibit inflammatory response by antagonizing PAF-PAFR pathway.4.GB and GK can promote the formation and proliferation of OPCs,and may promote the differentiation of OPCs into mature oligodendrocytes and myelin sheath regeneration by inhibiting the activation of Wnt and Notch 1 pathways.