Effects Of Ginsenoside On Proliferation And Differentiation Of Neural Stem Cells After Stroke

Neural stem cells existing in adult mammalian brain can proliferate, self-renew and multi-differentiate into neurons, astrocytes and oligodendrocytes. Since the sebiological characteristics, neural stem cells can be used to treat some brain injury diseases such as stroke. Under normal circumstances, neural stem cells are in a resting state. When the brain is injured, NSCs can proliferate and migrate to the injured section, and then differentiate into corresponding nerve cells which involved in the formation of neural circuits, and promote structural and functional brain injury repair.In this study, we try to discuss the influence of ginsenosideson the proliferation and differentiation of post-stroke neural stem cells by regulating HIF-la, and investigate the function mechanisms of Chinese active ingredientsin promoting the proliferationand differentiation of neural stem cells at the molecularlevel. We hope that this study can provide a theoretical basis for Chinese medicine applications in the interventions of stem cells transplantation, promoting new neurons survival and treatment of neurological diseases.1 Isolation, culture and identification of fetal rat hippocampal neural stem cellsHippocampal neural stem cells were isolated from the brain of 17 days SD pregnant rat embryos to be serum-free cultured in vitro, and induced to differentiate with 10% fetal bovine serum. Then they were identified by immunofluorescence staining method.Nestin and Brdu positive neural stemcells with proliferation ability were obtained, and after the induced differentiation by serum, Tuj-1 and Vimentin positive neurons and astrocytes were obtained. It is suggested that neural stem cells which have the ability of growing steady in vitro and multi-differentiation can be obtained by adding specific growth factors without serum.2 The function of several Chinese medicinemonomers and active ingredientson the proliferation and differentiation of neural stem cellsFetal rat hippocampaltissue isolated was serum-freecultured in vitro, and several traditional Chinese medicinemonomers (ginsenosides, Astragaloside, ginsenosidesRgl, Rb2, Re, Rf, RadixsaponinD) in different concentrations were added into the single cell suspension of neural stem cells. After 3 days, MTT method was used to determine the function of various drugs in different concentrationson the proliferation and differentiation of neural stem cells.Ginsenosides in measured optimum concentration was added into the single cell suspension of neural stem cells, and after different culturetimes, MTT methods was used to determine the influence of ginsenosideson the proliferation and differentiationof neural stem cells at different time points. It was determined that the optimum concentration of ginsenosides was 1μg/ml, and compared with the control group, the difference was statistically significant (P<0.05).The optimum concentration of RadixsaponinD.Astragaloside, ginsenoside Re, Rg1, Rb2andRf in promoting neural stemcell proliferation and differentiation was 0.3μg/ml,0.25μg/ml,0.005ug/ml,0.32μg/ml,0.01μg/ml and 0.32μg/ml, respectively. Compared with the controlgroup, the difference was statistically significant (P<0.05).Drugs promoted the proliferation and differentiationof neural stem cells after 3h, and compared with the control group, the difference was statistically significant (P<0.05). It is believed that ginsenosides, Astragaloside, Radix saponinD, ginsenosideRe, Rgl, Rb2andRf all can promote the proliferation and differentiation of neural stem cells, and ginsenosides works after 3h.3 The function of ginsenosides on the proliferation and differentiation of ischemia -reperfusion fetal rat hippocampal neural stem cells by regulating HIF-1αOxygen glucose deprivation/reperfusion (OGD/R)cellmodel was used to simulatecerebral injury caused by ischemia-reperfusion. We divided neural stemcells cultured in vitro into nine groups:A total of three time points, and normal group, model group (ischemia-reperfusion) and drug-treated group (ginsenosides 1μg/ml) at every time point. Normal group was culturedin normal, and the latter two groups cells were hypoxia cultured firstly for 4h, then normally cultured for 2h,4h,6h, respectively.After every experiment, Western blot was used to detect the HIF-la protein content in cell nucleus, and enzyme-linked immunosorbent assay (ELI SA) was used to detect the vascular endothelial growth factor(VEGF)levels in cell culture supernatant; We also used immunofluorescence double labelled staining method to detect the expression of neural stem cell specificmarker such as Nestin,neural stem cells proliferation marker(Brdu).neuron specificmarker(Tuj-1)and astrocytemarker(Vimentin).Then we obtained statistical data of number, optical density, area density and gradation values of positive cellsin each group.As a result,the expression of HIF-la and VEGF was less in normoxic state and increased after hypoxia, and compared with the normal group, the difference was statistically significant (P<0.05); The expression of Nestin,Brdu,Tuj-1 and Vimentin arose in all of normal group, model group, drug-treated group; Compared with normal group, there was more HIF-la protein and VEGF protein content in model group, and the number, optical density, surface density and gamma values of positive cells also increased significantly (P<0.05) in model group; Compared with the model group, there was more HIF-la protein and VEGF protein content in drug-treated group, and the number, optical density, surface density and gamma values of positive cells also increased significantly (P<0.05) in drug-treated group.There were three reoxygenation time points:2h,4h,6h.As the extension of reoxygenation time, the content of HIF-la protein and VEGF protein increased gradually inmodel group and drug-treated group, so were the number,optical density, surface density and gray value of positive cells. It is believed that after ischemia reperfusion, the transcription and translation of HIF-la gene in neural stem cell nucleus enhanced, resulting to the enhancement of the transcription and translation of VEGF, its downstream target genes. As a result, the HIF-la protein and VEGF protein expression increased, thus contributing to the proliferation and differentiation of neural stem cells;Ginsenosidescan further facilitate the increase of HIF-1α protein and VEGF protein levels after ischemia reperfusion, and play a part in the brain protection by promoting the proliferation and differentiation of neural stem cells.In conclusion, Nestin positive neural stem cells can be obtained by serum-free cell culture method; Ginsenosides can not only promote the proliferation and differentiation of normal-cultivated neural stem cells, but also have a protective effect on the neural stem cells of ischemia reperfusion model. By investigating the mechanism, this study provides a theoretical basis for the treatment of ischemic stroke. It can also provide new ideas and new drug therapeutic targets for revealingdeep function mechanism of Chinese medicine in the treatment of nervous system diseases and developingnew drugs.