黄芩苷对氧糖剥夺再复氧损伤模型大鼠海马神经干细胞的保护作用

Baicalin protection of rat hippocampal neural stem cells from oxygen-glucose deprivation/reoxygenation-induced injury in vitro

目的探讨黄芩苷对氧糖剥夺再复氧(OGD/R)损伤模型大鼠海马神经干细胞(NSCs)的保护作用。方法体外悬浮培养新生大鼠海马NSCs,将培养的海马NSCs分为对照组、OGD/R组、黄芩苷组。对照组采取完全培养基正常条件下悬浮培养,OGD/R组用无糖Earle's液缺氧后正常培养,黄芩苷组在OGD/R基础上加用黄芩苷(30μmol/L)培养。四甲基偶氮唑盐(MTT)法测定NSCs细胞存活率,比色法检测乳酸脱氢酶(LDH)漏出率,4',6-二脒基-2-苯基吲哚(DAPI)染色检测NSCs细胞凋亡,巢蛋白(Nestin)/5-溴脱氧尿嘧啶核苷(BrdU)染色检测NSCs细胞增殖。结果悬浮培养细胞呈Nestin阳性表达;与对照组比较,OGD/R组细胞OD值[(0.56±0.08)比(0.92±0.15)]、存活率[(60.87±6.67)%比(100.01±7.71)%]、Nestin/BrdU阳性表达面密度[(144.27±16.52)比(401.15±44.89)]及细胞数[(8.02±1.31)个比(40.20±5.03)个]显著降低,乳酸脱氢酶(LDH)漏出率[(28.33±4.13)%比(4.05±0.55)%]显著增加(P<0.01),DAPI核染提示OGD可诱导细胞凋亡。与OGD/R组比较,黄芩苷组细胞OD值[(0.75±0.09)比(0.56±0.08)]、存活率[(81.52±9.44)%比(60.87±6.67)%]、Nestin/BrdU阳性表达面密度值[(205.57±24.26)比(144.27±16.52)]及细胞数[(19.25±2.51)个比(8.02±1.31)个]明显增多,LDH漏出率[(13.89±1.94)%比(28.33±4.13)%]明显减少(P<0.01),DAPI核染显示黄芩苷可减轻OGD/R所引起的细胞凋亡。结论黄芩苷能减轻OGD/R对大鼠海马NSCs损伤,并促进其增殖。

Objective To explore the protective effect of Baicalin on rat hippocampal neural stem cells(NSCs)against the oxygen-glucose deprivation/reoxygenation(OGD/R)-induced injury.Methods The hippocampal NSCs from neonatal rats were isolated and cultured in suspension and then divided into the control(cultured in complete medium),OGD/R(cultured in a glucose-free Earle's minimal essential medium under a hypoxia condition first and then normal condition),and OGD/R plus Baicalin treatment groups.The NSCs viability was analyzed using the MTT assay,NSCs leakage rate of lactate dehydrogenase(LDH)was detected colorimetrically,NSCs apoptosis was assayed using DAPI staining,and NSCs proliferation was determined using Nestin/5-bromodeoxyuracil nucleoside(BrdU)immunofluorescence staining.Results NSCs in the suspension culture expressed Nestin.Compared with the control group,cells in the OGD/R group showed a significant decrease in cell optical density(OD)value(0.56±0.08 vs.0.92±0.15),cell viability(60.87±6.67 vs.100.01±7.71),Nestin/BrdU positive density(144.27±16.52 vs.401.15±44.89),and cell number(8.02±1.31 vs.40.20±5.03),while a significantly increase in the LDH leakage rate(28.33±4.13 vs.4.05±0.55;P<0.01).The DAPI staining suggested that OGD induced NSCs apoptosis.Furthermore,compared with the OGD/R group,the OD value(0.75±0.09 vs.0.56±0.08),cell viability(81.52%±9.44 vs.60.87±6.67),Nestin/BrdU positive density(205.57±24.26 vs.144.27±16.52),and cell number(19.25±2.51 vs.8.02±1.31)in the Baicalin group were significantly increased,whereas the LDH leakage rate(13.89±1.94 vs.28.33±4.13)was significantly decreased(P<0.01).The DAPI staining showed that baicalin was able to reduce OGD/R-induced NSCs apoptosis.Conclusion Baicalin could protect rat hippocampal NSCs against OGD/R0induced injury and promote NSCs proliferation.