| ObjectiveAstragalus is a commonly used traditional Chinese medicine for cerebral ischemic stroke.Astragaloside ? is the main active component of astragalus,which has a good protective effect on the nervous system.Astragaloside ? can promote the regeneration and repair of the central nervous system,improve the nerve regeneration ability,and finally repair the damaged part of the nerve.At present,the application of traditional Chinese medicine to treat ischemic brain injury has become a research hotspot.This experimental study is based on the early experimental conclusions of the research group.We explored the effect of astragaloside ? on up-regulating the expression of HIF-1?-VEGF pathway.Under oxygen sugar deprivation/reperfusion(OGD/R),we verified the effect of astragaloside ? on proliferation and differentiation of neural stem cells.Methods1.Isolation and culture neural stem cells of fetal rat hippocampalRats were intraperitoneally injected with 10%chloral hydrate for anesthesia,the fetuses were removed under sterile conditions,the cerebral cortex was separated,the pia mater was removed,the hippocampal tissue was separated,rinsed with DMEM/F12 medium,the tissue was shredded,and repeated pipetting,Digest with 0.05%trypsin solution,set the centrifuge at 1000 rpm/min for 5 min,aspirate the excess liquid,add DMEM/F12 culture solution,and pipet evenly to make a single cell suspension.Observed under a microscope,the cell density was adjusted to 106/ml,inoculated in a 75ml cell culture flask,and placed in a constant temperature incubator(37?,5%CO2)for cultivation.After 5-6 days,the cells were spherical and neural stem cells were passaged.Immunofluorescence identification of neural stem cells:After dissolving the BrdU solution with the culture solution,the cultured neural stem cells are added.The final concentration was determined to be 10 ?g/ml,cultured for 4 days,the density was adjusted to 5 X 105/ml,and inoculated into a 24-well plate coated with PLL-covered coverslips.Wash with PBS;fix with 4%PPL;block with 0.1%bovine serum albumin(BSA);add primary antibody(rabbit anti-Nestin 1:200,murine anti-BrdU 1:200,murine anti-Tuj-11:200,murine anti Vimentin 1:200),refrigerator overnight at 4?;add secondary antibody(goat anti-rabbit IgG-Cy3 1:50,goat anti-mouse IgG-FITC 1:50);add DAPI staining solution with a final concentration of 100ng/ml,mount,Observed under a laser confocal microscope.2.Establishing an OGD/R model of neural stem cells in vitroNormal group:Take the complete culture medium of neural stem cells and put them in a cell incubator(37? 5%CO2);Model group:Open the three-gas incubator to circulate the gas first,remove the residual oxygen to produce 37?,5%CO2,95%N2 environment;take DMEM culture medium without glucose,after 4 hours of culture,add complete medium and continue to maintain in the incubator for 4 hours until reperfusion injury occurs.3.MTS method to detect the best concentration and time of Astragaloside ? on OGD/R model neural stem cellsSet 10 concentration gradients of astragaloside:50,20,10,5,2,1,0.5,0.25,0.05,0.01(?g/ml),set blank control group,add cell culture solution to culture,and measure OD Value,the data of different concentrations are obtained through statistics,then the optimal concentration of astragaloside ? is selected.According to the above principle,the isolated neural stem cells were cultured at the optimal concentration,and set different time points(blank control group,1h,3h,6h,12h,24h,48h,72h,96h),and screened the best Time of action.4.Immunofluorescence cell staining method was used to detect the effect of astragaloside ? on the proliferation and differentiation of oxygen-sugar deprivation reperfusion(OGD/R)neural stem cells.The normal group,model group and astragaloside IV administration group were set up.The cells in the normal group were routinely cultured.The model group and the astragaloside? administration group were cultured in a three-gas incubator for 4 hours under hypoxia and then reperfused for 4 hours.The cells were removed and the best concentration of astragaloside ? was added to the three groups respectively Cultured at the time of action,using immunofluorescence cell staining to detect the expression of the specific markers of neural stem cell proliferation and differentiation in each group:Nestin,BrdU,Tuj-1 and Vimentin.NSCs were labeled with Nestin,neural stem cell proliferation was labeled with BrdU,neurons were labeled with Tuj-1,and progenitor cells of astrocytes were labeled with Vimentin.Immunofluorescence staining was performed.Fix with 4%paraformaldehyde for 30 min,0.01%TritonX-100 for membrane rupture for 10 min,and 0.1%bovine serum albumin(BSA)for 30 min.200),BrdU antibody(1:200),Tuj-1 antibody(1:200),Vimentin antibody(1:300),overnight at 4?.Add goat anti-rabbit-IgG-Cy3(1:50),goat anti-mouse IgG-FITC(1:50)mixed secondary antibody,incubate at room temperature in the dark for 30 min,DAPI stained nucleus,glycerol mount,observe by laser confocal microscope Staining results.5.Western blot detection of HIF-1 ? protein expression in the nucleus of neural stem cellsCollect all the cells in the plate of normal group,model group and astragaloside IV group,centrifuge at 1000r/min for 5min,discard the supernatant,add 40?L of protein lysate and lyse on ice for 30min.The lysate was centrifuged at 12000r/min at 4? for 5 minutes,and the supernatant was taken and packed in centrifuge tubes.Nuclear protein was extracted according to the nuclear protein-cytoplasmic protein extraction kit.Protein quantification was performed using the Bradford method.Denature by boiling with water for 1Omin.After electrophoresis on SDS-PAGE gel,it was transferred to PVDF membrane,non-specific background was blocked with 5%skim milk powder,and the room temperature was 1h.The mouse anti-HIF-1? antibody(1:500)and mouse anti-?-actin antibody(1:1000)were diluted with PBST solution,and the membrane was placed in the primary antibody dilution solution at 4? overnight.PBST solution was washed 3 times at room temperature for 10min each time.The membrane was placed in horseradish enzyme-labeled goat anti-mouse secondary antibody(1:5 000)and incubated at room temperature for 1h.Use ECL luminescent liquid to develop color in the dark room,dry the film,and scan the result with a scanner.The image analysis software IPP 6.0 performs grayscale analysis on the image.The expression level of HIF-1? protein was expressed as the ratio of the average optical density value of HIF-1? protein and internal reference ?-actin in each group at each time point.6.Enzyme-linked immunosorbent assay(ELISA)to detect the expression of VEGF protein in the culture medium of neural stem cellsRefer to the VEGF ELISA test kit to collect cells and operate sequentially.Use a microplate reader to detect the OD value at 450nm wavelength.The average OD value of the VEGF standard and the sample to be tested is subtracted from the OD value of the blank control group.The measured concentration and the measured OD value are used as a standard curve to calculate the concentration of the sample to be measured.Results1.Isolation,culture and identification of fetal rat hippocampal neural stem cellsThe fetal rat hippocampal neural stem cells were successfully isolated and cultured in vitro.The purity was identified to be more than 95%.2.The best concentration and time of astragalosideThe optimal concentration and time of astragaloside ? to promote the proliferation and differentiation of OGD/R neural stem cells were 0.25 ?g/mL and 24h respectively.3.Immunofluorescence staining methodCompared with the normal group,the number of positive cells labeled by Nestin and BrdU,Vimentin,Tuj-1 and the number of protrusions in the model group were reduced,and the volume of the cells was reduced;compared with the model group,the positive in the astragaloside group The number of cells,number of protrusions and volume increased significantly,and were less than the normal group.Astragaloside ? can promote the proliferation and differentiation of oxygen-sugar deprivation reperfusion(OGD/R)neural stem cells.4.The effect of astragaloside on the expression of HIF-1?Western blot detection of the expression of HIF-1? protein in the NSCs nucleus of each group:cultured under normal aerobic and sugar conditions,the expression of HIF-1?protein in the nucleus was less;compared with the normal group,the model after hypoxia and hypoglycemia treatment Compared with the model group,the HIF-1? protein content in the astragaloside group increased and the difference was statistically significant(P<0.05).5.The effect of astragaloside on the expression of VEGF protein under normal aerobic and sugar conditions,the VEGF content in the cell culture fluid is less;compared with the normal group,after hypoxia and glucose deficiency treatment,in the model group cell culture fluid The VEGF content increased,the difference was statistically significant(P<0.05);compared with the model group,the VEGF content in the cell culture fluid of the astragaloside group increased,the difference was statistically significant(P<0.05).ConclusionAstragaloside ? promotes oxygen glucose deprivation reperfusion(OGD/R)model.The optimal concentration of neural stem cell proliferation and differentiation in fetal rat hippocampus is 0.25 ? g/mL,and the optimal time point is 24h.Astragaloside ? can increase the number of positive cells in the hippocampal neural stem cells of the fetal rat in oxygen-glucose deprivation reperfusion(OGD/R)model,and the number of protrusions increases significantly,which promotes its differentiation into astrocytes and progenitor neurons.Astragaloside ? upregulates the HIF-1?-VEGF signaling pathway to enhance the proliferation and differentiation of neural stem cells in oxygen-glucose deprivation reperfusion. |
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