Study On The Effects Of Ginsenosides Rg1 And Rb1 On The Proliferation And Protection Of Neural Stem Cells

Ginseng is a precious traditional Chinese medicine which has been confirmed to have multi-effects,such as invigoration,resisting fatigues,expanding blood vessels,growing intelligence and anti-cancers.Ginseng in China has been applied for over 2000 years, drawn world-wide attention and well-known as "the King of Medicine".Ginsenosides is one of the main effective ingredients of Ginseng,which belong to triterpenoid saponin and are divided into three species:one is diol-type of Ginsenosides (including Rb1,Rb2,Rc,Rd,Rh2),the second is triol-type of Ginsenosides(including Rg1,Re,Rf,Rg2,Rh1),and the third is oleanolic acid type(including Ro,Rh3,Ri). The majority of Ginsenosides are diol-type of Ginsenosides and tri-type of Ginsenosides, which are regarded as the sources of main active constitutents of ginseng plants.STATs is a group of transcription factors activated by the ligands of polypeptide of cytokines and growth factors.The ligand-dependent continuous activation of STATs is closely related to the differentiation,growth and proliferation of cells.As a member of STATs,STAT3 participates in the processes of differentiation,proliferation and apoptosis. Janus kinase signal transducers and activators of transcription(JAK/STAT) widely participates in the process of differentiation,proliferation and degeneration of neural cells.The conception of neural stem cells has thoroughly changed the notion that central nervous system cannot be regenerated.NSCs is a special population of cells which can self-renewal and regenerate that are widely existing in central nervous systems in mammals.Anterior subventricular zone is regarded as the most integrating part of NSCs in central nervous systems.Research shows that a narrow passage-Rostral migratory stream (RMS) is formed following the NSCs shifting from SVZa to OB.In the process of shift, neural stem cells are in proliferation state with little differentiation of the properties.The stem cells becomes differentiated maturely after shifting to OB.The unique way of shifting of neural stem cells of SVZa has provided an perfect model for studying the biological characters of neural stem cells.The application of NSCs as the donators is limited due to some reasons such as ethics consideration,limited proliferation of NSCs and so on.At present,most studies are being carried on applying various kinds of growth factors to promoting the proliferation of NSCs. However,it is restricted for these factors to use as the clinical drug on the whole body because of its high expense in a large dose,no penetration to blood brain barrier and poisonous side effects,etc.And therefore,it is of great value to find small molecular natural medicinal herbs of low cost,less poison and easy to permeate blood brain barrier. At present,ginsenoside Rg1 and Rb1 has been selected from over 200 herbs and is proved to have effectiveness of protection,proliferation and anti-degeneration of neural cells.Our experiment aims to observe and study the mechanism of ginsenoside Rg1 and Rb1 on the proliferation and protection of SVZa-NSCs.A clean level of SD pregnant rat in embryonic 17 days is used in this experiment. Adopting techniques of SVZa NSCs obtaining,primary culture and subculturing methods, the sub-cultured NSCs are divided into three groups(the control group,Rg1 group and Rb1 group).The proliferation of NSCs was tested by Cytometry and MTT assay,and the expression of STAT3 in NSCs was detected by immunocytochemistry.The effect of ginsenoside Rg1 and Rb1 on the proliferation of SVZa-NSCs was observed in above techniques.Meanwhile,neurotoxic model of NSCs has been set up by use of Glu and sub-cultured NSCs was divided into four groups(normal group,Glu model group,Rg1 group and Rb1 group) as the target,to study the protective effect of ginsenoside Rg1 and Rb1 on the SVZa-NSCs damaged by Glu,main research methods were adopted in our experiment including the surviving rate tested by MTT assay,Trypan blue rejection essay and the apoptosis rate measured by TUNEL stainning as well as the STAT3's expression of NSCs detected by immunocytochemistry.The results are as follows:A.The effect of Ginsenoside Rg1 and Rb1 on the proliferation of neural stem cells1.Observation of cell growth under inverted microscope:Suspended single cells are growing and proliferating and reaches the highest density at the 4th day when parts of the cells begin to move to the walls and differentiates.The proliferation of the cells is thus resisted.Comparing with the control,ginsenoside Rg1 and Rb1 can promote proliferation and differentiation of suspending NSCs.The proliferation of the cells reaches the peak with ginsenoside Rg1 and Rb1 density at 40uM.Most of the cells start to move to the walls and differentiate after several hours in the culture medium with 5%BSA.And no obvious difference is observed for the differentiation of groups with ginsenoside Rg1 and Rb1 contrasting with the control.2.Cell numbering method for test of the number of cloned:The number of cloned NSCs in all groups of ginsenoside Rg1 and Rb1 was increased significantly compared with the control(P＜0.05) except that Rg1 was used in a dose of 160uM and Rb1 was used in a dose of 5uM and 160uM.Among different groups of ginsenoside Rg1 and Rb1,it has the strongest promotive effect for the number of cloned NSCs to proliferate in 40uM group in which proliferated cells reachs the highest number at the fourth day with obvious difference contrasting to the control(P＜0.01).Compared between Rg1 and Rb1 groups,obvious difference of the increase of cloned(P＜0.05).3.To test the rate of NSCs proliferation by MTT assay:On the 4th day,applying Rg1 and Rb1,comparing SuM group with 10-160uM groups respectively,the rate of NSCs proliferation increase obviously(p＜0.01).Comparing Rg1 groups with Rb1 groups,the difference is remarkable(p＜0.01).4.the expression of STAT3 in NSCs:Comparing Rg1 and Rb1 groups with the control groups,STAT3 expression of SVZa-NSCs and the percentage of the positive cells increase obviously(p＜0.01). Comparing Rg1 groups with Rb1 groups,there is no difference statistically.B.Protection for neural stem cell from Glu injuring by ginsenoside Rg1 and Rb11.Observation of the cells under inverted microscope:It was found that the cells in the Glu group have poor refractivity,swelling body. Some cells smash,and so there are a large number of cellular pieces,and there are only a few cells alive.The cells in the Rg1 group have good refractivity,most of them body keeps intact,and few smashes and pieces.The Rb1 group has the same result.2.Measuring the survival rate of the cell through trypan blue repulsion assay:Trypan blue makes the dead cells blue,but makes ones alive no color.In the control group,most cells smash and die,and only a few ones survive.Trypan blue makes most cells blue in the control group with cell survival rate of 11.3%.While most of the cells of RG1 group and RB1 group are not colored with cell survival rate of 58.6%and 62.3% respectively,which are obviously different with the Glu groups(P＜0.01).The results of RG1 and RB1 groups have no statistical differences.3.Testing the OD values through MTT assay:Activity of cells can be reflected by OD values.OD value of the control group is 0.325±0.062 and that of the Glu group is 0.200±0.031.Statistics difference exist(P＜0.01).After adding Rg1 and RB1,the OD values rise to 0.285±0.041 and 0.276±0.019, respectively.Compared with the Glu groups,there are significant differences.4.Measuring the rate of apoptosis cells by TUNEL assay:Most of the cells in RG1 and RB1 groups survive with rate of apoptosis cells of 21.6%and 20.2%respectively,which show obviously difference with the Glu groups(65.3%)(P＜0.01).The results of RG1 and RB1 groups have no statistical differences.5.The expression of STAT3 of NSCs:Only a few cells,about 4.52%of NSCs,express STAT3 in the control group.Most cutured cells proliferate and express STAT3 and its percnetage of positive cells reaches 30.81%in the Rg1 group.Rg1 group has obvious difference with Glu group(P＜0.01). The expression rate of STAT3 positive cells of Rb1 group is 29.83%,which has no difference with Rg1 group in statistics.Conclusions:1.Ginsenoside Rg1 and Rb1 can promote the proliferation of SVZa-NSCs:the optimal concentration is 40uM,and the best time is the 4th day.The proliferation of NSCs is possibly caused by the promotion of STAT3 expression and percentage of the positive cells.2.Ginsenoside Rg1 and Rb1 have protective effect to SVZa-NSCs:Ginsenoside Rg1 and Rb1 are able to help restore the number,morphology of SVZa-NSCs injured by Glu. Ginsenoside Rg1 and Rb1 can protect SVZa-NSCs by anti-neurotoxicity of Glu,which may be related to the increase of STAT3 expressions.3.Observation shows that Ginsenoside Rg1 and Rb1 have no influence on the differentiation of SVZa-NSCs:The densities and lengths of the differentiated cells in all groups of Genoside Rg1 and Rb1 treatment have no difference with those of the comparison group.The positive cells' percentage of GFAP andβ-Tubulin in Rb 1 and Rg1 treated group have no statistic difference with the comparison group.This statement need further studies to be confirmed.In this study through selection of Ginsenoside Rgl and Rbl of cheap,low poisonous and easy to cross the blood-brain barrier,the proliferation and protection of Rg1 and Rb1 on the SVZa-NSCs were examined systemically.It is found that Ginsenoside Rg1 and Rb1 can promote proliferation of NSCs,and protect the injured NSCs by the anti-neurotoxicity of Glu.It has not been observed that the effect of Ginsenoside Rg1 and Rb1 on the differentiation of NSCs in this experiment.This research provides a theoretical reference for the application of Ginseng to therapy CNS injuries and diseases as well as functional rehabilitations.