Efficacy Evaluation And Mechanism Of Carbazole Alkaloid Derivative CZ-7 On Myelin Injury Induced By CPZ

Objective:The mechanism of demyelinating diseases is controversial,while demyelination and remyeliantion disorder is the acknowledged etiology and therapeutic target.Until now,there is no efficient therapy for these diseases.In our previous study,carbazole alkaloid derivative CZ-7 has neuroprotective effect on the damage of gray and white matter injury caused by vascular dementia.However,the protective effect of CZ-7 and its specific mechanism during the process of demyelination and remyelination is still unclear.To clarify the neuroprotective effect of CZ-7 on myelin injury in the central nervous system,we constructed the myelin injury mice model.In our study,we conducted to investigate the therapeutic effect of CZ-7 on CPZ-induced demyelinating model,and its associated pro-remyelination mechanism.Methods:1.Male C57BL/6 mice were divided into three groups randomly.The groups are control group(Control),model group(CPZ),CZ-7 treatment groups(CPZ+CZ-7).The mice were subjected to CPZ(300 mg/kg)through intragastric gavage and were orally administered CZ-7(20 mg/kg)meanwhile.After 4 weeks,the pole test,grip strength test and beam walking test were conducted to detect the ability of motor integration and limb coordination in mice.All mice were sacrificed for further study in the end.2.LFB staining was mainly employed to inquiry the morphological structure changes and pathological injury of myelin.MBP,a marker to detect the myelin structure,was observed by immunohistochemistry staining.The mRNA expression level of genes related to oligodendrocyte maturation and myelination(MBP,MOG,MOBP,PLP1)were examined by QPCR analysis.3.Both dMBP staining and oil red O staining were employed to check the effect of CZ-7 on removal of detached myelin debris in the corpus callosum.4.Immunofluorescent staining of Iba1 was employed to inquiry the number and morphology of microglia in CC.The mRNA expressions of phagocytosis-related genes(C3,C4)were inspected by QPCR analysis.The immunofluorescent double staining of MBP and Iba1 was used to further prove the phagocytosis of microglia.5.We investigated the microglia phenotype in CC via immunofluorescence staining of CD16/32 and CD206,which represents for M1 phenotype and M2 phenotype marker respectively.We also detected the mRNA expression of M1 markers iNOS and M2 markers Arg1 in CC by QPCR analysis to confirm our results.6.To validate the effect of CZ-7 on remyelination after the CPZ treatment,we decteded its effect on OPCs differentiation and maturation at two sequential stages in the oligodendroglia maturation process by the early marker(NG2,O4)of OPCs.7.Primary SD rat OPCs were cultured and purified and treated by different concentrations of CZ-7 after three days.The differentiation of OPCs was explored by immunofluorescence,Western blot and qPCR.Results:CZ-7 ameliorates CPZ-induced behavior defects by shortening the time climbing down from the top of pole,increasing the grip strength and shortening traversing time on beam walking in the behavior tests.CZ-7 significantly ameliorates myelin injury in the CPZ model mice.CZ-7 enhances the clearance of degraded myelin debris accumulated in CC.The removal and elimination of degraded myelin debris by CZ-7 treatment attributes to more phagocytic microglia assembling in CC.The microglia displaying ameboid and phagocytic shape in CC mostly tend to M2-polarized phenotype.CZ-7 may facilitate more OPCs assembling into CC to differentiate into muture oligodendrocytes and then facilitate remyelination of the demyelinating mice.Conclusion : CZ-7 promotes the change of microglia into M2-polarized phenotype and enhances the phagocytic ability of microglia,thereby promoting the removal of harmful myelin debris and promoting the remyelination at lesion sites in the pathological situation.Also,CZ-7 promotes the differentiation and maturation of OPCs.The comprehensive effect of CZ-7 significantly promotes remyelination in CPZ-induced demyelinating model.