Protective Effects Of Ferulic Acid, Angelica Polysaccharide On The Cultured Neural Stem Cells In Hypoxic Injury

Abstract:Objective:To investgate whether the ferulic acid (FA) and angelica Sinensis Polysaccharide(ASP) had any protective effect on neural stem cells (NSCs)in hypoxic injury and its mechanism.Method:(1) NSCs culture, identification and differentiation.①NSCs culture and identification.Take SD rats within 24h after birth,75% alcohol disinfection, remove the brain with aseptic conditions,eye scissors repeatedly cut into pieces,200 mesh stainless strainer filter and centrifuged twice,resuspended in serum-free medium and inoculated in T25 training bottle,every 2-3d semi-medium was changed,6d passage about a separation. Observing the biological characteristics of NSCs under the inverted microscope morphological and immunocytochemistry (SABC) detect nestin.②In vitro differentiation of NSCs.Inducing differentiation by serum medium after 6 days to observe its morphologic changes,SABC to detect neuron-specific enolase(NSE), glial fibrillary acidic protein (GFAP).(2)Hypoxic injury model.The 3rd generation of NSCs were randomly divided into control group and hypoxia 4,8,12,16 and 20h group. Hypoxia group into three gas incubator, the control group were placed in normoxic incubator. CCK-8 compare the activity of NSCs after hypoxia among each group to determine the time.(3)experimental group.The 3rd generation of NSCs were randomly divided into control group, hypoxia group and dosing group(FA group and the ASP group), dosing group was divided into high concentration(500mg/L), concentration(50mg/L) and low concentration (5mg/L)group.Dosing group and hypoxia group removed after 8h, the control group in the normoxic incubator. Collect each group to detected the following indicators:①The morphological changes of NSCs;②CCK-8 detect NSCs activity.③serum medium induced differentiation of NSCs in each group. SABC detect neuronal markers NSE and astrocyte marker GFAP.(4) Image analysis:The immune staining of climbing films by AX07 TRF-A micro-imaging system was observed in 200 times,each taking five random mounting of camera view and count the number of positive cells.(5)Statistical analysis:Statistical analysis software package with SPSS 13.0 processing data. All parameters indicated by mean±standard,the single factor analysis of variance (ANO-WAY), line paired t test.Results:(1)Proliferation of NSCs in vitro.The cells are round and strong refraction on 1 day. Primary cultured 3d, shows nearly spherical cell mass,loose, uneven size and surrounded by slag-like cells,5-6d to connect closely spherical shape increases,refractive index and good pieces with the surrounding cells for fluid decreased.After passage, the proliferation of neural ball speed faster than the original generation,more uniform sphere, clean background.NSCs specific markers Nestin staining was positive.The cells were subcultured to passage 3,cell colony formation and cell proliferation activity both better. (2)The differentiation of NSCs in vitro.Serum medium induct differentiation after 6 days,showing that NSE-positive neurons,blunt-type or polygonal cell body,t one or two processes and GFAP-positive astrocytes,star-shaped cell bodies,cell body large, stubby processes and more branches.(3) Determine the time of hypoxic injury. Put NSCs in the incubator (95% N2,5% CO2)and removed after different times of hypoxia, CCK-8 colorimetric way assay cell activity:The results showed that hypoxia 8 h or more, the optical density values (OD) significantly lower than the control group,the difference was statistically significant (P<0.01),and hypoxic damage was positively correlated with the time. (4) FA and ASP on the NSCs hypoxic injury.①cell morphology:the normal control group,grow well.Hypoxia group,the number of neurospheres are less,ball loose, less refractive, irregularly shaped and serious change was flocculent.Dosing group compared with hypoxia group,except for the low concentration of ASP group,the cell damage in varying degrees of ease,and is inversely proportional to the concentration; Second, the protective effect of FA is better than ASP with the same concentration.②CCK-8 colorimetric way assay activity:hypoxia group compare to the control group is decreased with significant differences.Except for the low concentration of ASP group,other FA groups and ASP groups was to improve the vitality of cells,and is the drug concentration effect relationship.③Differentiation of NSCs:After the intervention of three different doses of FA and ASP,compare the number of NSE, GFAP positive cells.Except the low concentration of ASP group compare with the hypoxic group is not statistically significant(P>0.05)),the other groups compared with the control group are significantly increased (P<0.05), and the concentrations of FA,ASP is greater,the number of nerve cells is more. Conclusions:(1)Under the 95% N2,5% CO2 conditions and 8h, Hypoxic injury model of NSCs can be established which can provide experimental support for the study of hypoxic injury to the NSCs;(2)FA and ASP as the active ingredients of Angelica can play a protection role to the hypoxic injury NSCs, within a certain concentration was positively correlated and its mechanism may be through activate the proliferation of endogenous NSCs which provide important experimental support for Angelica as the protective agent in clinical use of hypoxia diseases.