Experimental Study On The Effects And Mechanisms Of Astragalus Polysaccharide In Regulating Remyelination And Directional Differentiation Of Neural Stem Cells In Demyelinated Mice

Objective:To observe the effects of astragalus polysaccharide(APS)on remyelination in demyelinated mice,and to investigate the regulation on the directional differentiation of neural stem cells and its possible mechanisms.Methods:Animal experiments:1.C57BL/6 mice were divided into 3 groups by random number table: normal group,cuprizone(CPZ)group and APS group.To induce experimentally demyelination,CPZ group and APS group mice were fed with standard chow containing 0.2%(w/w)CPZ powder for 5 weeks.After model identification,CPZ was removed and replaced with standard chow for 2 weeks allowing for spontaneous remyelination.APS group mice received APS 500 mg/kg/day in oral starting from the first day of week 5 until the end of week 7 while CPZ group mice received normal saline 0.2 m L at the same time.Normal group mice were fed with standard rodent for 7 weeks.2.Turn-back time in pole test and beam-walking time in beam walking test were recorded once a week,so as to evaluate the effects of APS on neurologic dysfunctions in demyelinated mice.Luxol fast blue(LFB)staining was employed to detect the myelin density in corpus collosum(CC)in 3 group mice,in order to observe the effect of APS on remyelination.3.Real-time PCR,Western blot and immunohistochemistry were applied to detect the m RNA and protein expression level of Nestin,GFAP,MBP and Neu N,the specific biomarkers of neural stem cells,astrocytes,oligodendrocytes and neurons in the CC of 3 group mice,so as to investigate the regulatory effects of APS in vivo.4.Real-time PCR and Western blot were used to measure the m RNA and protein expression level of ligand Shh,receptor Ptch1,receptor Smo,transcription factor Gli1 in the CC of 3 group mice,the key molecules of Sonic hedgehog signaling pathway,in order to investigate the molecular mechanism in the regulation induced by APS in vivo.Cell experiments:1.The culture system and differentiation model of C17.2 mouse neural stem cell line were established.After model identification,0.1 ?g/m L,1 ?g/m L,10 ?g/m L,100?g/m L and 1 mg/m L APS were added to differentiation medium,so as to intervene the differentiation process for 4 days.PBS group was set as control at the same time.2.Real-time PCR and Western blot were used to detect the m RNA and protein expression level of Nestin,GFAP,MBP and Neu N in 6 group cells,so as to investigate the regulatory effects of APS on C17.2 neural stem cell differentiation in vitro.3.Real-time PCR and Western blot were used to detect the m RNA and protein expression level of Shh,Ptchl,Smo and Gli1 in 6 group cells,in order to investigate the molecular mechanism in the regulation induced by APS in vitro.Results:1.In animal experiments,compared with CPZ group mice,turn-back time and beam-walking time of APS group mice were shorter(P<0.01).Myelin density in CC was higher(P<0.01).These results showed that APS could relieve the neurologic dysfunctions and promote remyelination in demyelinated mice.2.In animal experiments,compared with CPZ group mice,the expression level of Nestin m RNA and protein in CC and subventricular zone(SVZ)of APS group mice were downregulated(P<0.01)which indicated that APS could inhibit the stemness maintenance of neural stem cells in vivo,and promote them to go into the differentiation state.The expression level of GFAP m RNA and protein in CC of APS group mice were downregulated(P<0.05).The expression level of MBP and Neu N m RNA and protein in CC of APS group mice were upregulated(P<0.05).These results suggested that APS could effectively regulate the directional differentiation of neural stem cells in vivo.In detail,APS could promote neural stem cells differentiate into oligodendrocytes and neurons directionally,and inhibit them differentiate into astrocytes.3.In animal experiments,compared with CPZ group mice,the expression level of Smo and Gli1 m RNA and protein in CC of APS group mice were upregulated(P<0.01)while the expression of Ptch1 protein was downregulated(P<0.01).These results suggested that APS could facilitate the activation of Sonic hedgehog signaling pathway which might be one of the possible molecular mechanisms in the regulatory process in vivo.4.In cell experiments,compared with control group,with the increase of APS concentration,the expression level of Nestin m RNA and protein downregulated gradually in the differentiation model cells(P<0.01).The result indicated that APS could inhibit the stemness maintenance of C17.2 neural stem cells in vitro,and promote them to go into the differentiation state.The expression level of GFAP m RNA and protein downregulated gradually in the differentiation model cells while the expression of MBP and Neu N m RNA and protein upregulated gradually(P<0.01).These results suggested that APS could effectively regulate the directional differentiation of C17.2 neural stem cells in vitro.In detail,APS could inhibit C17.2neural stem cells differentiate into astrocytes directionally,and promote them differentiate into oligodendrocytes and neurons directionally.5.In cell experiments,compared with control group,with the increase of APS concentration,the expression level of Shh,Smo and Gli1 m RNA and protein upregulated gradually in the differentiation model cells.The expression of Ptch1 m RNA and protein downregulated gradually(P<0.01).These results suggested that APS could facilitate the activation of Sonic hedgehog signaling pathway which might be one of the possible molecular mechanisms in the regulatory process in vitro.Conclusions:1.APS can relieve the neurologic dysfunctions and promote remyelination in demyelinated mice.2.The mechanism of promoting remyelination induced by APS is related to regulating neural stem cells differentiating into oligodendrocytes directionally.3.Facilitating the activation of Sonic hedgehog signaling pathway may be one of the molecular mechanisms in the regulatory process mediated by APS.