| ObjectiveTo research the protective effects and mechanism of ginsenosides on proliferation and differentiation of endogenous neural stem cells(NSCs)based on NSCs niche through regulation of NSCs,astrocytes(AC)and brain microvascular endothelial cells(EC)after cerebral ischemia/reperfusion in vitro.We further investigated the role of ginsenosides in proliferation and differentiation of NSCs after cerebral ischemia/reperfusion in vitro based on NSCs niche by regulating HIF-1α-VEGF signaling pathway in a manner of autocrine and paracrine.Methods1 Hippocampal NSCs were isolated from the brain of 15-17 days SD pregnant rat embryos in vitro.We used immunofluorescence to identify NSCs,Nestin labeled as the specific marker of NSCs,BrdU labeled as proliferation marker of NSCs,Tuj-1 labeled as neural progenitor cell specific marker and Vimentin labeled as astrocytes progenitor cell specific markers.Astrocytes and brain microvascular endothelial cells were isolated from the newborn 1-3 days SD rats’ cerebral cortex in vitro.We used immunofluorescence to identify Astrocytes and brain microvascular endothelial cells.GFAP labeled as the specific marker of astrocytes and Factor Ⅷ labeled as the specific marker of brain microvascular endothelial cells.2 MTS assay was used for determination of the optimal dose and the best time of ginsenosides for promoting the proliferation of NSCs,astrocytes and brain microvascular endothelial cells.3 NSCs were transfected by lentiviral vector carrying GFP gene and the positive rate of GPF was observed to determine the transfection rate of NSCs.The optimal transfection index MOI value and the optimal transfection time of lentivirus transfected NSCs were screened.According to the previous modeling method,we used oxygen glucose deprivation/reperfusion(OGD/R)models to simulate cerebral ischemia/reperfusion injury.The maximum loss time of NSCs caused by OGD was 4h.Western Blot was used for the detecting the expression of HIF-1α in NSCs nucleus after lentiviral transfected NSCs and calculated the silence rate of HIF-la.We used immunofluorescence to detect expression of HIF-1α,identification,proliferation and differentiation of NSCs after lentivirus transfection.4 The experiment was divided into 6 groups:NSCs normal group,NSCs model group,NSCs ginsenoside group(1.00μg/mL),NSCs(LV-NSCs)normal group,LV-NSCs model group,LV-NSCs ginsenoside group(1.00μg/mL).OGD/R model was used to simulate cerebral ischemia/reperfusion injury,NSCs model group and NSCs ginsenoside group were hypoxia cultured for 4h and then reoxygenation cultured 2h,4h and 6h,respectively.MTS was used to detect the survival rate of NSCs in each group.Immunofluorescence double staining was used to analyze the proliferation and differentiation of NSCs.Western Blot was used to analyze the expression of HIF-1α in NSCs nucleus.Determination of VEGF content in culture medium was detected by ELISA.5 Lentiviral transfected NSCs(LV-NSCs)and astrocytes were co-cultured using Transwell co-culture system.The experiment was divided into 6 groups:NSCs/AC normal group,NSCs/AC model group,NSCs/AC ginsenoside group(12.50μg/mL),LV-NSCs/AC normal group,LV-NSCs/AC model group,LV-NSCs/AC ginsenoside group(12.50μg/mL).MTS was used to detect the survival rate of NSCs in each group.Immunofluorescence double staining was used to analyze the proliferation and differentiation of NSCs.We used Western Blot to analyze the expression of HIF-1α in astrocytes nucleus and determination of VEGF content in co-culture medium was detected by ELISA.6 Lentiviral transfected NSCs(LV-NSCs)and brain microvascular endothelial cells were co-cultured using Transwell co-culture system.The experiment was divided into 6 groups:NSCs/EC normal group,NSCs/EC model group,NSCs/EC ginsenoside group(12.50μg/mL),LV-NSCs/EC normal group,LV-NSCs/EC model group,LV-NSCs/EC ginsenoside group(12.50μg/mL).MTS was used to detect the survival rate of NSCs in each group.Immunofluorescence double staining was used to analyze the proliferation and differentiation ofNSCs.Western Blot was used to analyze the expression of HIF-1α in brain microvascular endothelial cells nucleus and determination of VEGF content in co-culture medium was detected by ELISA.Results1 Primary culture and identification of NSCs,astrocytes and brain microvascular endothelial cellsNSCs,astrocytes and brain microvascular endothelial cells were isolated and cultured in vitro successfully,and the purity was over 95%.2 The optimal dose and the best time of ginsenosides to promot the proliferation of NSCs,astrocytes and brain microvascular endothelial cellsThe optimal dosage of ginsenoside to promote the proliferation of NSCs was 1.00μg/mL and the optimal time of ginsenoside administration was 24h.The optimal dose of ginsenoside to promote the proliferation of astrocytes was 12.50μg/mL and the optimal time of ginsenoside administration was 72h.The optimal dosage of ginsenoside to promote the proliferation of brain microvascular endothelial cells was 12.50μg/mL and the optimal time of ginsenoside administration was 24h.3 The optimal MOI value and the optimal transfection time of lentivirus transfected NSCsNSCs were successfully transfected with lentiviral vector,and the optimal MOI value was 10,and the optimal transfection time was 144h.Compared with the control group,the transfection rate of NSCs was 90.47%(P<0.01).4 Detection of HIF-la expression in NSCs after transfection of lentiviral vector by Western BlotUnder hypoxia condition,HIF-la in the NSCs transfected group was not expressed,while HIF-1α in NSCs normal group and negative control group were positive expressed.Compared with NSCs normal group and negative control group,the expression of HIF-1αwas significantly lower in NSCs transfected group(P<0.01).After calculation,the HIF-la silence rate was 88.5%.5 Immunofluorescence detected the expression of HIF-1α after OGDUnder hypoxia,NSCs normal group showed that HIF-la could be expressed in the NSCs nucleus.In the negative control group,the lentiviral vector carrying GFP green fluorescence could be successfully transfected into NSCs,and NSCs was able to express HIF-1 after transfection.NSCs transfection group showed that GFP green fluorescence could be expressed in the nucleus and the expression of HIF-la was not expressed.The results showed that lentivirus could successfully transfect NSCs and silence the expression of HIF-1α.6 Immunofluorescence staining was used to identify the proliferation and differentiation of NSCs after lentivirus transfected NSCsAfter transfection,NSCs was able to express NSCs specific marker Nestin.BrdU,Tuj-1 and Vimentin were positive expressed after transfection.The results showed that NSCs transfected by lentivirus could proliferate and differentiate into progenitor cells of neurons and astrocytes.7 Immunofluorescence double staining was used to detect the proliferation and differentiation of NSCs after OGD(1)For NSCs each groups:NSCs reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the NSCs normal group,area density,optical density,the number of positive cells of the NSCs model group were decreased(P<0.01);compared with the NSCs model group,area density,optical density,the number of positive cells of NSCs ginsenoside group were increased(P<0.01).(2)For LV-NSCs each groups:LV-NSCs reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the LV-NSCs normal group,area density,optical density,the number of positive cells of the LV-NSCs model group were decreased(P<0.01);compared with the LV-NSCs model group,area density,optical density,the number of positive cells of LV-NSCs ginsenoside group were increased(P<0.01).8 Detection of the expression of HIF-1α in NSCs nucleus in each group by Western Blot after OGD(1)For NSCs each groups:In normal condition,there was a lower expression of HIF-la in NSCs;compared with NSCs normal group,the expression of HIF-la of NSCs model group was increased(P<0.01);compared with NSCs model group,the expression of HIF-la of NSCs ginsenoside group was increased(P<0.01).(2)For LV-NSCs each groups:In normal condition,there was a lower expression or not expression of HIF-1α in LV-NSCs.Compared with LV-NSCs normal group,the expression of HIF-1α of LV-NSCs model group was increased slightly or scarcely increased,the difference was not statistically significant.Compared with LV-NSCs model group,the expression of HIF-la of LV-NSCs ginsenoside group was increased slightly or scarcely increased,the difference was not statistically significant.(3)Compared with NSCs ginsenoside group,the expression of HIF-1α of LV-NSCs ginsenoside group was significantly reduced(P<0.01).9 Determination of VEGF content in cell culture medium was detected by ELISA(1)For NSCs each groups:In normal condition,the content of VEGF was low.Compared with NSCs normal group,the content of VEGF in cell culture medium of NSCs model group was increased(P<0.01).Compared with NSCs model group,the content of VEGF in cell culture medium of NSCs ginsenoside group was increased(P<0.01).(2)For LV-NSCs each groups:In normal condition,the content of VEGF was low.Compared with LV-NSCs normal group,the content of VEGF of LV-NSCs model group was increased slightly or scarcely increased,the difference was not statistically significant.Compared with LV-NSCs model group,the content of VEGF of LV-NSCs ginsenoside group was increased slightly or scarcely increased,the difference was not statistically significant.(3)Compared with NSCs ginsenoside group,the content of VEGF of LV-NSCs ginsenoside group was significantly reduced(P<0.01).10 Detection of NSCs survival rate by MTS method after NSCs and LV-NSCs co-cultured with astrocytes(1)For NSCs co-cultured with astrocytes(NSCs/AC)each groups:NSCs/AC reoxygenation of 2h,4h and 6h respectively after OGD,compared with NSCs/AC normal group,the survival rate of NSCs/AC model group decreased(P<0.05);compared with NSCs/AC model group,the survival rate of NSCs/AC ginsenoside group increased(P<0.05).(2)For LV-NSCs co-cultured with astrocytes(LV-NSCs/AC)each groups:LV-NSCs/AC reoxygenation of 2h,4h and 6h respectively after OGD,compared with LV-NSCs/AC normal group,the survival rate of LV-NSCs/AC model group decreased(P<0.05);compared with LV-NSCs/AC model group,the survival rate of LV-NSCs/AC ginsenoside group increased(P<0.05).11 Immunofluorescence double staining was used to detect the proliferation and differentiation of NSCs after NSCs and LV-NSCs co-cultured with astrocytes(1)For NSCs/AC each groups:NSCs/AC reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the NSCs/AC normal group,area density,optical density,the number of positive cells of the NSCs/AC model group were decreased(P<0.01);compared with the NSCs/AC model group,area density,optical density,the number of positive cells of NSCs/AC ginsenoside group were increased(P<0.01).(2)For LV-NSCs/AC each groups:LV-NSCs/AC reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the LV-NSCs/AC normal group,area density,optical density,the number of positive cells of the LV-NSCs/AC model group were decreased(P<0.01);compared with the LV-NSCs/AC model group,area density,optical density,the number of positive cells of LV-NSCs/AC ginsenoside group were increased(P<0.01).12 Detection of the expression of HIF-1α in astrocytes after NSCs and LV-NSCs co-cultured with astrocytes by Western Blot(1)For NSCs/AC each groups:In normal condition,there was a lower expression of HIF-la in astrocytes.Compared with NSCs/AC normal group,the expression of HIF-1αincreased in NSCs/AC model group(P<0.01).Compared with NSCs/AC model group,the expression of HIF-1α in NSCs/AC ginsenoside group increased significantly(P<0.01).(2)For LV-NSCs/AC each groups:In normal condition,there was a lower expression of HIF-1α in astrocytes.Compared with LV-NSCs/AC normal group,the expression of HIF-1αincreased in LV-NSCs/AC model group(P<0.01).Compared with LV-NSCs/AC model group,the expression of HIF-1α in LV-NSCs/AC ginsenoside group increased significantly(P<0.01).13 Determination of VEGF content in cells co-cultured medium was detected by ELISA(1)For NSCs/AC each groups:In normal condition,the content of VEGF was low.Compared with NSCs/AC normal group,the content of VEGF of NSCs/AC model group was increased(P<0.05).Compared with NSCs/AC model group,the content of VEGF of NSCs/AC ginsenoside group was increased(P<0.05,P<0.01).(2)For LV-NSCs/AC each groups:In normal condition,the content of VEGF was low.Compared with LV-NSCs/AC normal group,the content of VEGF of LV-NSCs/AC model group was increased(P<0.05).Compared with LV-NSCs/AC model group,the content of VEGF of LV-NSCs/AC ginsenoside group was increased(P<0.05,P<0.01).14 Detection of NSCs survival rate by MTS method after NSCs and LV-NSCs co-cultured with brain microvascular endothelial cells(1)For NSCs co-cultured with brain microvascular endothelial cells(NSCs/EC)each groups:NSCs/EC reoxygenation of 2h,4h and 6h respectively after OGD,compared with NSCs/EC normal group,the survival rate of NSCs/EC model group decreased(P<0.05);compared with NSCs/EC model group,the survival rate of NSCs/EC ginsenoside group increased(P<0.05).(2)For LV-NSCs co-cultured with brain microvascular endothelial cells(LV-NSCs/EC)each groups:LV-NSCs/EC reoxygenation of 2h,4h and 6h respectively after OGD,compared with LV-NSCs/EC normal group,the survival rate of LV-NSCs/EC model group decreased(P<0.05);compared with LV-NSCs/EC model group,the survival rate of LV-NSCs/EC ginsenoside group increased(P<0.05).15 Immunofluorescence double staining was used to detect the proliferation and differentiation of NSCs after NSCs and LV-NSCs co-cultured with brain microvascular endothelial cells(1)For NSCs/EC each groups:NSCs/EC reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the NSCs/EC normal group,area density,optical density,the number of positive cells of the NSCs/EC model group were decreased(P<0.01);compared with the NSCs/EC model group,area density,optical density,the number of positive cells of NSCs/EC ginsenoside group were increased(P<0.01).(2)For LV-NSCs/EC each groups:LV-NSCs/AC reoxygenation of 2h,4h and 6h respectively after OGD,the positive expression of Nestin,BrdU,Tuj-1 and Vimentin in normal group,model group and ginsenoside group.Compared with the LV-NSCs/EC normal group,area density,optical density,the number of positive cells of the LV-NSCs/EC model group were decreased(P<0.01);compared with the LV-NSCs/EC model group,area density,optical density,the number of positive cells of LV-NSCs/EC ginsenoside group were increased(P<0.01).16 Detection of the expression of HIF-1α in brain microvascular endothelial cells after NSCs and LV-NSCs co-cultured with brain microvascular endothelial cells by Western Blot(1)For NSCs/EC each groups:In normal condition,there was a lower expression of HIF-1α in brain microvascular endothelial cells.Compared with NSCs/EC normal group,the expression of HIF-1α increased in NSCs/EC model group(P<0.05,P<0.01).Compared with NSCs/EC model group,the expression of HIF-1α in NSCs/EC ginsenoside group increased significantly(P<0.05,P<0.01).(2)For LV-NSCs/EC each groups:In normal condition,there was a lower expression of HIF-1α in brain microvascular endothelial cells.Compared with LV-NSCs/EC normal group,the expression of HIF-1α increased in LV-NSCs/EC model group(P<0.05,P<0.01).Compared with LV-NSCs/EC model group,the expression of HIF-la in LV-NSCs/EC ginsenoside group increased significantly(P<0.05,P<0.01).17 Determination of VEGF content in cells co-cultured medium was detected by ELISA(1)For NSCs/EC each groups:In normal condition,the content of VEGF was low.Compared with NSCs/EC normal group,the content of VEGF of NSCs/EC model group was increased(P<0.05,P<0.01).Compared with NSCs/EC model group,the content of VEGF of NSCs/EC ginsenoside group was increased(P<0.05,P<0.01).(2)For LV-NSCs/EC each groups:In normal condition,the content of VEGF was low.Compared with LV-NSCs/EC normal group,the content of VEGF of LV-NSCs/EC model group was increased(P<0.05,P<0.01).Compared with LV-NSCs/EC model group,the content of VEGF of LV-NSCs/EC ginsenoside group was increased(P<0.05,P<0.01).ConclusionGinsenosides promoted the proliferation of NSCs and induced the differentiation of NSCs into progenitor cells of neurons and astrocytes after ischemia/reperfusion injury through regulating NSCs,astrocytes and brain microvascular endothelial cells in NSCs niche in vitro.The neuroprotective mechanism of ginsenosides related to the regulation of HIF-1α-VEGF signaling pathway in a manner of autocrine and paracrine. |
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