The Promotive Effects Of Tetrahydroxystilbene Glucoside,an Active Component Of Polygonum Multiflorum Thunb,on The Differentiation Of Neural Stem Cells Into Dopaminergic Neurons And The Potential Molecular Mechanisms

Parkinson's disease(PD)is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine(DA)levels.Current treatments include drug therapy and surgical treatment.However,no efficient methods targeting on arresting dopaminergic neuronal progressive death are usable.Therefore,it is pivotal solution to seek an ideal strategy for replacement or replenishment of lost DA neurons.Neural stem cells(NSCs)have hitherto regarded as powerful perspective candidates for cell replacement in clinical therapy for neural injury and neurodegenerative diseases,owing to its potential to differentiate into a variety of neurons including dopaminergic(DA)neurons.Replacing lost cells by transplanting DA neurons has value to repair the damaged brain.pluripotent cells capable of differentiation into DA neurons,which are the major cell types damaged in PD.Unfortunately,there exists some shortcomings regarding the conventional methods to induce differentiation of NSCs to DA neurons,such as low differentiation efficiency,tedious manipulation,and differentiation instability,and thus limited their therapeutic potential in PD.We have previously demonstrated that tetrahydroxystilbene glucoside(TSG),one of the main active ingredients of Polygonum multiflorum Thunb(PM),is neuroprotective.To date compelling studies demonstrated that TSG can promote the secretion of neurotrophic factors and exert neuroprotection in nervous system.Nevertheless it is unknown whether TSG effectively potentiate the differentiation of NSCs into DA neurons,and the underlying molecular mechanisms remain elusive.The aims of our present experiment are to systematically investigate the differentiation of mesencephalic NSCs into DA neurons with TSG.Thereafter,the morphological characteristics and biochemical phenotype of DA neurons derived NSCs were identified by means of cellular and biochemical assays.Concomitantly,the molecular mechanism of differentiation of NSCs to DA neurons by TSG was further investigated.Thus,the present study will be undertaken to further elucidate the molecular mechanism through Wnt/?-catenin signaling pathway.The present experiment may provide novel theoretical basis for clarification of the molecular mechanism of NSCs differentiation,we will eventually develop an effective strategy for differentiation of NSCs to DA neurons,which could contribute immensely to clinical applications of traditional Chinese medicine combined with cell therapy as a novel alternative to treat the central nervous system injury and the neural degenerative diseases such as PD.?Objective?1.To investigate the promotive effects of TSG,one of the main active ingredients of Polygonum multiflorum Thunb,on the differentiation of NSCs into DA neurons,and harvest biochemical phenotypic and functional DA neurons.2.To elucidate the underlying mechanism of the TSG induction on the differentiation of midbrain NSCs to DA neurons via Wnt/?-catenin signaling pathway.?Methods?1.NSCs culture and identification: NSCs were prepared from the ventral mesencephalic(VM)of mouse embryos at E12,and maintained in serum-free DMEM/F12 medium,supplemented with 1% N2,1% penicillin/streptomycin,20 ng/ml recombinant human epidermal growth factor(EGF),and 20 ng/ml recombinant human fibroblast growth factor(bFGF).Thereafter,these cells were identified using immunofluorescence staining with nestin.2.EdU labelling NSCs: EdU,a new thymidine analogue was used to detect midbrain NSC proliferation by staining labeling assay.3.TSG induction: NSCs were inducted to differentiate by the addition of TGS at different concentration(0,1,5,10,and 20 ?M)after removal of normal culture supernatant.4.Flow cytometry: The effects of TSG on the survival and apoptosis of midbrain NSCs were assessed by using flow cytometry assay.5.Immunofluorescence staining: After differentiation of NSCs in special medium with TSG,immunofluorescence was carried out to observe the morphological change of differentiated cells and the expression of several specific markers positive for DA neuron such as Tuj-1,GFAP,CNPase,TH,DAT,and Nurr1.6.Western Blot: Quantitative analysis of the expression levels of Tuj-1,GFAP,CNPase,TH,DAT,Nurr1,Wnt1,Wnt3 a,Wnt5a,and ?-catenin was performed to further investigate the effects of TSG on the differentiation of NSCs.?Results? Part?: Study on the culture,proliferation and differentiation of NSCs derived from the mibrain.1.Seven days after of culture in condition medium,these cells formed numerous floating clusters termed neurospheres in culture,and the volume of neurosphere gradually expand with the prolongation of culture time.Furthermore the neurosphere were positive by immunostaining with nestin,NSC specific marker,indicating that the cultured cells are NSCs.2.EdU labelling results showed that the number of EdU positive NSCs contiuously increased,and was at time-dose dependent manner within 5 days in vitro.3.Immunofluorescence staining results showed that when NSCs were induced to differentiate using media containing 2% fetal bovine serum for different time,the NSCs lost their own round morphology at 12 hour point,and exhibited were polygonal shapes with neurite extension from soma.Seven days later,these differentiated cells displayed different morphology,such as strong stereo,good refractivity and/or flat cell bodies.Immunofluoresence revealed that differentiated cells expressed Tuj-1(Class III ?-tubulin,Tuj-1),GFAP(glial fibrillary acidic protein,GFAP),and CNPase within 7 d.In comparison,the GFAP positive cells counted for highest proportion of population,followed by the percentage of Tuj-1 positive cells,and the smallest was CNPasa positive cells,suggesting that the cultured NSCs possessed the potential for multi-differentiation.As for the serum-free culture group,no remarkable positive cells mentioned-above were found.In every vision field,the majority of cell adhesion and growth was slow,and exhibited degenerative.4.By means of 2% FBS induction,we can observe GFAP positive cells and Tuj-1 positive cells in the induction system.Notably,the number of these positive cells gradually elevated with prolongation of induction time.At two weeks,the percentages of Tuj-1 positive cells reached peak.In contrast,the number of GFAP positive cells exhibited a rising trend.Part ?: Effects of TSG on the survival,apoptosis and differentiation of midbrain NSCs,and the potential molecular mechanisms involved Wnt/?-catenin signaling.1.It was found from flow cytometry assay that after induction with TSG at 5 different concentrations(0.5,1,5,10,20?M),TSG at1,5,and 10?M significantly promoted survival of NSCs,and reduced cell apoptosis.Compared to other concentration group,TSG at 10?M can exert best protection(P<0.01).2.Immunofluorescence and western blot assay? Immunostaining of neural stem cells with Tuj-1,GFAP and CNPase showed that after 2 weeks of induction,10 ?M TSG supplemented with 2%FBS significantly promoted the differentiation of NSCs to Tuj-1 positive cells,displaying higher number of Tuj-1 positive cells and protein levels when compared to 2% FBS induction.Meanwhile,the expression of GFAP and CNPase decreased.There is a statistical difference(P<0.01).Strikingly,there is same promotive differentiation between 10?M TSG alone and 10 ?M TSG supplemented with 2%FBS.In addition,no cell differencation and subsequent Tuj-1?GFAP and CNPase exression were found in blank group.Namely,it can not cause the differentiation of NSCs without administration of TSG or FBS.? After the examination of three DA neuronal specific markers in differentiated cells induced by 10?M TSG+2%FBS for 2 weeks,we found that the number of TH?DAT and Nurr1 positive cells,and their expression levels were signicantly elevated when compared to 2% FBS induction group(P<0.01).TSG alone has same effects on the differentiation of NSCs with TSG+2% FBS.Western-blot analysis of total cell lysates of the TSG-treated cells showed an increased expression.There is a statistical difference between the two groups and 2%FBS induction group(P<0.01).As for blank group(without any inducers),no any positive for three markers was found in cells.Results indicated TSG as a differentiation inducer for NSCs to neurons.3.The underlying mechanism of differentiation of NSCs induced by TSG NSCs were treated with different concentration(0,1,5,10,and 20?M)of TSG for 2 weeks.Western-blot analysis showed 10?M TSG+2% FBS significantly elevated the expression of Wnt1,Wnt3 a,Wnt5a and ?-catenin.Excitedly,10?M TSG alone can cause same elevation in the molecule expression as 10?M TSG+2% FBS.No significant difference was found between two groups.But there is a statiscal difference between these two treatment and 2% FBS alone(P<0.01).As for no any treatment group,we did not find differentiation of NSCs,and thus no Wnt1,Wnt3 a,Wnt5a and ?-catenin were detected in cells.The differentiation of NSCs induced by TSG was significantly suppressed by application of the Wnt signaling blocker IWR1.At this time,NSC proliferation,migration,and differentiation were slowing down.Moreover,flow cytometary assay revealed that the addition of IWR1 resulted in inhibition of NSC survival,displaying the decrease in the number of live cells and increase in apototic cells.When compared with the TSG group(without the addition of IWR1),a significant difference was found(P<0.01).Furthermore,Immunofluorescence staining revealed that the number of Tuj-1 and TH positive cells was significantly reduced in the addition of IWR1 when compared with the 10?M TSG group(P<0.01),indicating invovlement of Wnt/?-catenin signaling pathway in the differentiation of NSCs induced by TSG.?Conclusion?1.Conventional induction with media containing 2% FBS causes the differentiation of NSCs into neurons,astrocytes and oligodendrocytes.Under the condition,the particular ideal time to induce NSC differentiation into neurons is at two weeks.Conversely,NSCs can not complete neural differentiation and gradually degenerated,revealing fetal bovine serum is a key induction component to direct specific differentiation of NSC.2.TSG at a certain concentration promotes the survival of NSCs from mesencephalon,suppresses cell apoptosis.Amongst these different concentrations,TSG at 10?M was most ideal dose for NSC differentiation.In addition,10?M TSG effectively potentiates the differentiation of NSCs into neural cells.These differentiated cells have possessed mature DA neuron phenotype,morphology features and biochemical characteristics such as expression of TH,DAT,and Nurr1 specific markers for DA neuron.3.TSG promotes the differentiation of NSCs from mesencephalon into DA neurons via triggering the Wnt/?-catenin signaling pathway.