| Object1. To explore the effects of hydroxysafflor yellow A (HSYA) on neurogenesis in the hippocampal dentate gyrus (HDG) of the organotypic slices after glutamate(Glu) neurotoxicity.2. To investigated the neuroprotective effects of HSYA on Glu induced neuronal death and mitochondrium dysfunction in organotypic hippocampal slice cultures (OHSCs).3. To approach anti-apoptotic effects of HSYA on neurotoxicity of glutamate in primary cultured rat cortical neurons along with its possible mechanism of action through p38MAPK pathway.Materials and Methods1. The organotypic hippocampal slices (300μm) from postnatal6th day SD rats were prepared and randomly divided into four groups(n=6brain slices):(1) saline control group (3.5mM Glu+1ml normal saline intervention, CG);(2) blank control group (normal culture, not modeling+nonintervention. Nor):(3) large doses HSYA group (3.5mM Glu+1ml0.072mg/ml HSYA intervention, HG1);(4) small doses HSYA group (3.5mM Glu+lml0.036mg/ml HSYA intervention, HG2). Each group except Nor was received the same model of Glu intervention and then cultured with different dose of HSYA for72h. Double staining of BrdU and Nestin immunofluorescence according to conventional methods. After that, Neural stem cells(NSCs) in hippocampal dentate gyrus(HDG) were observed under invert microscope or detected by immuno fluorescence staining in different time points(before.24and48h after the application of HSYA). Count NSCs and make the comparison in groups. Repeated measure analysis of NSCs was used in different time points with different intervention, so did in different time in each group; at each time point, comparisons in groups using one-way analysis of variance (One-way ANOVA), two groups’comparisons using LSD method. P<0.05was considered statistically significant.2. The organotypic hippocampal slices (300μm) from postnatal6th day SD rats were prepared and randomly divided into five groups(n=5brain slices):(1) normal control group (normal culture, not modeling+nonintervention, Nor);(2) large doses HSYA group (3.5mM Glu+1ml0.072mg/ml HSYA intervention, HG1);(3) small doses HSYA group (3.5mM Glu+1ml0.036mg/ml HSYA intervention, HG2);(4) saline control group (3.5mM Glu+1ml normal saline intervention, CG);(5) large doses HSYA control group(1ml0.072mg/ml HSYA intervention, OnlyHGl). Each group except Nor and OnlyHG1was received the same model of Glu intervention and then cultured with different dose of HSYA for72h. PI uptake was measured before,24and48h after the application of HSYA. Protein expressions of5-LO, caspase-3, the SOD2and phospho-Akt(P-Akt) were deteced by using western blot analysis. Each of these indicators was compared in groups using one-way analysis of variance (One-way ANOVA). If homogeneity of variance, pairwise multiple comparisons using LSD method within groups, and if not, using Tamhane’s T2method. P<0.05was considered statistically significant. 3. Primary cultures of prefrontal cortical neurons were prepared from embryonic18days old (E18) Sprague-Dawley rats. Cultures were used for challenge with Glu between10and14days in vitro (DIV). Cultures were randomly divided into five groups(n=5walls):(1) normal control group (normal culture, not modeling+nonintervention, Nor);(2) large doses HSYA group (3.5mM Glu+0.072mg/ml HSYA intervention, HG1);(3) small doses HSYA group (3.5mM Glu+0.036mg/ml HSYA intervention, HG2);(4) saline control group (3.5mM Glu+normal saline intervention, CG);(5) large doses HSYA control group(0.072mg/ml HSYA intervention, OnlyHG1). Cell death and apoptosis were detected with MTT assay, Hoechst33258/PI double staining and flow cytometry (Annexin V-FITC/PI double staining). mRNA expressions of caspase-3, ATF2, p38MAPK and MAPKKK were detected by RT-PCR. Each of these indicators was compared in groups using one-way analysis of variance (One-way ANOVA), two groups’ comparisons using LSD method. P<0.05was considered statistically significant.Results1. Low power lens (40x):normal brain slices were brown, uniform and good light transmission, can be clearly distinguished between the cortex, hippocampus (CA1-4and DG District), intraventricular, subcortical nuclei and white matter fiber bundle structure. High power lens (200x):visible cytoplasm and nuclei. NSCs (BrdU+, Nestin+) difference was statistically significant in different time points before or after Glu intervention (F=228.075, P<0.001), HG1, HG2, CG and Nor, F value31.537,181.762,248.345and52.444respectively, all P<0.001. Neural stem cell numbers between the groups there were statistically significant differences (F=947.077, P<0.001); except Glu intervention before, from the time point of view, each time point the number of neural stem cells have the following relationship: Nor>HG1>HG2>CG (P<0.001). Glu intervention different time points and different groups there is interactive effect (F=123.639, P<0.001).2.3.5mM Glu damage after24and48hours caused increasing cell death in hippocampal DG area.Glu damage after24h PI fluorescence values between the groups had significant difference (F=11.305; P=0.002); HG1<CG, HG1<HG2, the differences were statistically significant (both P<0.05).Glu damage after48h PI fluorescence values between the groups had significant difference (F=11.731, P=0.002); HG1<CG, HG2<CG, HG1<HG2, the differences were statistically significant (all P<0.05).5-LO had a significant difference (F=46.702, P=0.000); Nor<HG2, Nor<CG, HG1<CG, HG2<CG, the differences were statistically significant (all P<0.05). Caspase-3had a significant difference (F=19.419, P=0.000); Nor<HG2. Nor<CG, HG1<CG, the differences were statistically significant (all P<0.05). SOD2had a significant difference (F=15.483, P=0.000); Nor>HG1, Nor>HG2, Nor>CG, HG1>CG, HG2>CG the differences were statistically significant (all P<0.05). Phospho-Akt had a significant difference (F=23.299, P=0.000); Nor>HG2, Nor>CG, HG1>CG, HG1> HG2, the differences were statistically significant (all P<0.05).3. The MTT measuring method of cell survival rate differences between the groups was statistically significant (F=17.063, P=0.000); Nor<HG1, Nor<HG2, Nor<CG, HG1<CG, HG2<CG, the differences were statistically significant (all P<0.05). Hoechst33258and PI double staining in HG1, HG2and CG group, cells showed weak blue fluorescent cells reduced, strong blue and weak red double staining increased, and strong red and weak blue increased. Apoptosis rate between the groups had significant difference (F=29.965, P=0.000); Nor<HG1, Nor<HG2, Nor<CG, HG1<CG, HG2<CG the differences were statistically significant (all P<0.05). Flow cytometric analysis between the groups:Early apoptosis rate difference had statistics significance (F=55.089, P=0.000); Nor<HG1, Nor<HG2, Nor<CG HG1<CG, HG2<CG HG1<HG2, the differences were statistically significant (all P<0.05). Late apoptotic rate difference had statistics significance (F=13.519. P=0.000); Nor<HG2, Nor<CG, HG1<CG, HG1<HG2, the differences were statistically significant (all P<0.05). Necrosis rate difference had statistics significance (F=26.957, P=0.000):Nor<HG1, Nor<HG2, Nor<CG, HG1<CG HG2<CG the differences were statistically significant (all P<0.05). RT-PCR results: Caspase-3had a significant difference (F=39.237, P=0.000); Nor<HG2. Nor<CG, HG1<CG, HG2<CG the differences were statistically significant (all P<0.05). ATF2had a significant difference (F=53.137, P=0.000); Nor<HG2, Nor<CG HG1<CG, HG2<CG, the differences were statistically significant (all P<0.05). P38MAPK had a significant difference (F=23.645, P=0.000); Nor<HG2, Nor<CG, HG1<CG, HG2<CG HG1<HG2, the differences were statistically significant (all P<0.05). MAPKKK had a significant difference (F=10.174, P=0.001); Nor<HG2, Nor<CG, HG1<CG, HG1<HG2, the differences were statistically significant (P<0.05).Conclusions1.0.072mg/ml HSYA alleviated Glu intervention on NSCs damage, promoted the regeneration of NSCs.2.0.072mg/ml HSYA reduced Glu induced neuronal death, this protective effect of24h to48h, sustainable. Glu excitotoxicity led to mitochondrial dysfunction,0.072mg/ml HSYA protected mitochondrial function and reduced neuronal cell death through reducing of5-LO and caspase-3, increasing SOD2and phospho-Akt protein expression.3. HSYA mainly by inhibiting the apoptosis induced by Glu to protect nerve cells.0.072mg/ml HSYA inhibited neuronal apoptosis through down-regulating p38MAPK pathway gene expression of MAPKKK, p38MAPK, ATF2, caspase-3multiple loci. |
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