Effects Of Ginsenosides On Proliferation And Differentiation Of Oxygen-stained And Reperfused (OGD/R) Neural Stem Cells

ObjectiveGinseng saponin is the main effective component of the dry root of traditional Chinese medicine ginseng.Pharmacological experiments and clinical application of modern Chinese medicine have proved that ginsenosides can promote the growth of nerve tissue and the establishment of neural network.Ginsenosides can up-regulate the HIF-1? protein and VEGF protein levels in brain tissue after ischemia-reperfusion,and induce the proliferation and differentiation of neural stem cells through paracrine and autocrine forms,thereby promoting the repair of brain damage structure and function.In this study,based on the previous work of the research group,we investigated and discussed the effects of ginsenosides on the proliferation and differentiation of neural stem cells(NSCs)in fetal rat hippocampus undergone oxygen-glucose deprivation and reperfusion(OGD/R).Methods1 Isolation,culture and identification of neural stem cells from fetal rat hippocampusNeural stem cells of rat embryonic hippocampal were isolated and cultured in SD rats aged 15-17 days in vitro.The proliferation of neural stem cells were identified though Nestin and BrdU by Immunofluorescence Staining Tuj-1 and Vimentin were separately used to identify the neuronal ancestors cell and astrocyte progenitor cells.2 Establishment of OGD/R model of neural stem cells in fetal rat hippocampusA single cell suspension was prepared from fetal rat hippocampus,P2 neural stem cells that had been identified by Immunofluorescence Staining with a purity of 95%.The cells were counted under a microscope and the cell density was adjusted to 1×105 cells/mL,and evenly inoculated.The normal group and the model group were set.The normal group was cultured with the neural stem cell complete culture medium and in a normal cell culture incubator(37?,5%CO2).The model group was cultured with a Earle culture medium containing without glucose.Prior to the start of OGD,a three-gas incubator was circulated in advance to remove residual oxygen;the cultured neural stem cells were placed in glucose-free Earle and cultured at 37?,1%O2,5%CO2,94%N2 for 4h;and neural stem cells were taken out.The culture medium was replaced with a culture solution of neural stem cells;the cells were placed in a 37?,5%CO2 incubator for 4 hours to generate reperfusion injury.3 Ddetermination the optimal effective concentration and time point of ginsenosides on proliferation of neural stem cells undergone oxygen-glucose deprivation and reperfusion(OGD/R)by MTSA single cell suspension of P2 neural stem cells was collected and prepared,10 concentration gradients(200,100,50,25,12.5,5,2.5,1,0.5,and 0 ?g/mL)of ginsenosides were set,and added to the cell culture fluid,respectively.The cells were cultured and a blank control group was set up.The OD value at 490 nm wavelength was measured by MTS.The effect of different concentrations on the proliferation of neural stem cells was calculated,and the optimal effective concentration of ginsenosides for promoting the proliferation of neural stem cells was selected.Similarly,ginsenosides were cultured in single-cell suspensions of neural stem cells at the optimal concentration,and 10 different time points(0,1,3,6,12,24,48,72,96,120h)were set.In the blank control group,the best effective time point of ginsenosides in promoting the proliferation of neural stem cells was also determined by MTS.4 The effects of ginsenosides on the proliferation and differentiation of neural stem cells undergone oxygen-glucose deprivation and reperfusion(OGD/R)by Immunofluorescence StainingA single cell suspension of P2 neural stem cells was collected and prepared,and the normal group,the model group,and the ginsenosides group were set.The normal group was subjected to conventional cell culture,the model group and the ginsenosides group were cultured for 4 hours in a three-gas incubator,then reperfusion for 4 hours.The cells were taken out and the ginsenosides were added to the above three groups at the optimal concentration to continue the culture with the best effect time.Immunofluorescence staining was used to detect the expressions of specific markers:Nestin,BrdU,Tuj-1,and Vimentin related to the proliferation and differentiation of neural stem cells in each group.Results1 Isolation,culture and identification of neural stem cells from fetal rat hippocampus The neural stem cells of fetal rat hippocampus were successfully isolated and cultured in vitro and their purity was over 95%.2 The optimal effective concentration and time point of ginsenosides for promoting the proliferation of neural stem cells undergone oxygen-glucose deprivation and reperfusion(OGD/R)?The optimal effective concentration of ginsenosides for promoting the proliferation of neural stem cells undergone OGD/R was 1 ?g/mL.?The optimal effective time point of ginsenosides for promoting the proliferation of neural stem cells undergone OGD/R was 48h.3 The effects of ginsenosides on the proliferation and differentiation of neural stem cells undergone OGD/R by Immunofluorescence StainingCompared with the normal group,the number of positive cells which labeled Nestin and BrdU/Tuj-1/Vimentin decreased in the model group,with/or the number of protrusions and the cell volume decreased.Compared with the model group,the number of positive cells in the ginsenosides group was lower than that in the model group,and(or)the number of protrusions and volume increased significantly,but less than the normal group.Therefore,ginsenosides had the effect to promote the proliferation and differentiation of neural stem cells undergone oxygen-glucose deprivation and reperfusion(OGD/R).Conclusions1?g/mL and 48h were the optimal effective concentration and time point for ginsenosides to promote the proliferation of neural stem cells from fetal rat hippocampus undergone OGD/R.Ginsenosides could increase the number of positive cells and protrusions of neural stem cells from fetal rat hippocampus undergone OGD/R,and differentiate into neurons and astrocyte progenitors,which suggested that ginsenoside have the effect to repair brain tissue damage undergone cerebral ischemia through promoting the proliferation and differentiation of neural stem cells.