Effect Of Ginsenoside Rg1 On Proliferation And Differentiation Of Neural Stem Cells After Brain Injury And Its Epigenetic Regulation Mechanism

Neural stem cells can proliferate and replicate,and can differentiate into neurons,astrocytes and oligodendrocytes,which constitute the nervous system.Neural precursor cells(neural progenitor cells,NPCs)are the primary differentiation of neural stem cells during differentiation.In the early stage of the embryo,the ventricular NSCs is in a state of symmetrical division,thereby increasing the number of targets.Then,the neural stem cells,through asymmetric division,divided into neural stem cells and neurons to continue the process of neurogenesis.During embryonic development,part of the neural stem cells have the ability to divide into a neural stem cell and a NPCs.In the later stage of neurogenesis,NSCs can not only differentiate into neurons but also astrocytes and oligodendrocytes.The above process is affected by many factors adjust to happen,the regulation of epigenetics is the important factor.Epigenetics refers to the genetic variation of genes or proteins that do not involve changes in DNA sequence,and can be stably transmitted during cell development and cell proliferation,a type of gene regulation that can be passed from one cell to its daughter cells.The changes of epigenetic traits are inherited and reversible.Epigenetics can regulate the proliferation and differentiation of neural stem cells under the control of environmental factors and transcription factors Epigenetics including many ways can affect the proliferation and differentiation of neural stem cells.Therefore,researching how to regulate the proliferation and differentiation of neural stem cells is of great significance for the treatment of neurodegenerative diseases.1.Double-labelling immunofluorescence to detect Ginsenosides Rgl on the effect of differentiation of neural stem cell proliferation after brain injury.Using oxygen deprivation/reperfusion sugar(OGD/R)model to simulate Brain injury induced by cerebral ischemia reperfusion injury.The neural stem cells isolated and cultured in vitro of 2h,4h and 6h were separated into 3 groups:normal group,model group and medication group(ginseng saponin Rgl 1 mu g/ml).The normal group were normally cultured.The model group and medication group were firstly treated by Hypoxia for 4 h,then normally cultured for 2 h,4 h,6 h.After each time point,double-labelling immunofluorescence is applied to detect the expression of each specific marker nest protein(Nestin),neural stem cells,neural stem cells proliferation markers(Brdu),neuron specific markers(Tuj-1)and the expression of astrocytes markers(Vimentin).The number of positive cells,optical density and surface density were statistied among all groups.The results show that under normal circumstances,Nestin,Tuj-1 and Vimentin all can express in neural stem cells.Compared with normal group,the number of positive cells,light density and surface density in model group increased significantly under immunofluorescence observation,the difference was statistically significant(P<0.05);Ginseng saponin Rgl compared with model group,the number of positive cells,light density and surface density of the neural stem cells increased significantly,the difference was statistically significant(P<0.05).Moreover,as the prolongation of the reoxygenation time,the number of positive cells,light density and surface density of neural stem cells increase.Morphologically ginsenoside Rgl can promote the proliferation and differentiation of neural stem cells to protect neural stem cells under the condition of ischemia and hypoxia.2.Ginseng saponin Rgl through micromas-21/210 to influence the differentiation of neural stem cell proliferation after brain injuryReal-time fluorescent quantitative PCR method was used to detect the relationship between expression level of micrornas-21/210 and proliferation and differentiation of neural stem cell.Compared with normal group,miR-21 in OGD model group present higher expression at 4 h and 6 h after oxygen;miR-210 get higher expression at three time points,statistically significant difference(P<0.05),suggesting that the hypoxia may promote the expression of miR-21 and miR-210,thus promote the proliferation of neural stem cells.After intervention with ginseng saponin Rgl,compared with model group,miR-21 in drug group present a higher expression at 4 h and 6 h after oxygen;miR-210 present a higher expression at three time points,with statistically significant difference(P<0.05),suggesting that ginseng saponin Rgl can promote the expression of miR-21 and miR-210 of neural stem cells in anoxic condition,promote the restoration of neural stem cells in hypoxia state,which shows ginseng saponin Rgl may be involved in the repairation of neural stem cells in brain damage by miR-21 and miR-210.3.Effects of ginseng saponin Rgl on proliferation and differentiation of neural stem cells after brain injury by DNMT1/DNMT3aReal-time fluorescent quantitative PCR showed that the expression of DNMT1 and DNMT3a in the OGD model group were increased compared with the normal group at three time points,and the difference was statistically significant(p<0.05).However,after treated with ginseng saponin Rgl intervention,the expression of DNMT1 in drug group was not significantly different at the three time points compared with the model group;while the expression of DNMT3a show a significant increase,and the difference was statistically significant(p<0.05).Western blot experiments suggest that the expression of DNMT3a of neural stem cells in OGD model group are increased at three time points even in hypoxia ischemia condition compared with normal group,statistically significant difference(P<0.05).This shows DNMT3 a may promote self-repairation and proliferation of neural stem cells in hypoxia ischemia condition.Under the intervention of ginseng saponin Rgl,compared with model group,the expression of DNMT3 a at three time points was also increased,illustrating ginseng saponin Rgl could promote the self-renewal and proliferation of neural stem cells by DNMT3a.To sum up,the ginseng saponin Rgl can promote the proliferation and differentiation of neural stem cells under the condition of ischemia hypoxia,promote the expression of miR-21,miR-210 and DNMT3a of neural stem cells in the anoxic condition,and promote the restoration of neural stem cells in hypoxia state.This shows ginseng saponin Rgl was participated in the repairation of neural stem cells after brain damage with the assistance of miR-21/miR-210 and miR-DNMT3a.