枸杞多糖对小鼠海马神经元细胞系缺糖缺氧损伤保护作用及机制研究

Protective effects of Lycium barbarum polysaccharides on oxygen-glucose deprived mouse hippocampal neurons

目的探讨枸杞多糖(LBP)对小鼠海马神经元细胞系HT22细胞缺糖缺氧损伤保护作用的机制。方法采用含1 mmol/L连二亚硫酸钠的无糖DMEM培养基建立HT22细胞缺糖缺氧损伤模型,设对照组、模型组和LBP高、中、低剂量(100、50、25 mg/L)组,以CCK8法检测细胞活性,流式细胞术检测检测细胞凋亡情况;Western blotting检测Bcl-2、Bax蛋白表达情况;利用实时荧光定量PCR(RT-qPCR)检测TNF-α、NF-κB、I-κBα、Caspase-9基因转录活性。结果与对照组比较,模型组HT22细胞存活率显著降低、凋亡率显著增加(P<0.05);TNF-α、NF-κB、Caspase-9基因转录活性显著上调,I-κBα基因转录活性显著下调(P<0.05);Bax蛋白表达水平显著升高,Bcl-2的蛋白表达水平显著降低(P<0.05)。与模型组比较,LBP各剂量组细胞存活率显著上升、凋亡率显著下降(P<0.05);TNF-α、NF-κB、Caspase-9基因转录活性显著下调,I-κBα基因转录活性显著上调(P<0.05);Bax蛋白表达水平显著降低,Bcl-2的蛋白表达水平显著增高(P<0.05)。结论 LBP可能是通过下调TNF-α、NF-κB、Caspase-9基因转录活性以及Bax蛋白表达,增强I-κBα基因转录活性及Bcl-2的蛋白表达,抑制缺糖缺氧损伤诱导神经细胞凋亡的发生。

Objectives To observe the protective effects of Lycium barbarum polysaccharides(LBP) on oxygen-glucose deprived mouse hippocampal neurons. Methods Cultured mouse hippocampal neurons exposed to 1 mmol/LNa2S2O4 to establish oxygenglucose deprivation model, assigned to control group, model group and LBP group with different doses(100, 50 and 25 mg/L), the cell activity rate was measured by CCK8 assay, and the apoptosis rate was analyzed by Flow Cytometry;the protein expression of Bcl-2 and Bax was measured by Western blotting;RT-qPCR assay was used to evaluate the mRNA expression of TNF-α, NF-κB, I-κBα, Caspase-9. Results Compared with control group, HT22 cell survival rate and apoptosis rate in model group were significantly reduced(P < 0.05);TNF-α, NF-κB, caspase-9 gene transcription activity was significantly increased, I-κBα gene transcription activity was significantly decreased(P < 0.05);Bax protein expression level was significantly increased, Bcl-2 protein expression level was significantly decreased(P < 0.05). Compared with control group, LBP can significantly increase cell viability(P < 0.05);reduce the cell apoptosis rate(P < 0.05);the expression of TNF-α, NF-κB, Bax and Caspase-9 were effectively down regulated;induce an increase of I-κBα mRNA leve and Bcl-2 protein level(P < 0.05). Conclusion LBP have significantly protective effects on oxygen-glucose deprivation, such protective effects may be mediated by its action on the downstream TNF-α, NF-κB, Bax, Caspase-9, and up-regulation of Bcl-2 and I-κBα.