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目的:通过观察姜黄素对Id2基因表达的影响,探讨姜黄素抗缺氧损伤神经元凋亡的机制。方法:以5×105/cm密度接种于6孔板的原代神经元,培养3 d后随机分为正常对照组、模型组和姜黄素组。各组神经元按不同实验条件进行干预,其中,正常对照组:常氧条件培养30 h;模型组:常氧培养24 h后,再缺氧处理6 h;姜黄素组:给予20μM姜黄素常氧培养24 h后,再缺氧处理6 h。各组干预结束后,采用CCK8方法检测各组神经元的活力,计算分析各组的抑制率;通过Annexin V/PI染色后,行流式细胞术检测各组神经元凋亡率;通过Real time RT-PCR检测神经元Bcl-2、Bax、Caspase-3等凋亡相关基因及Id2mRNA的表达。结果:CCK8结果显示,模型组神经元活力明显降低,与正常对照组相比差异有显著性(P<0.01),经姜黄素预处理后明显提高神经元活性,与模型组相比差异有显著性(P<0.01);流式细胞术结果显示,模型组神经元凋亡率明显升高,与正常对照组相比差异有显著性(P<0.01),经姜黄素预处理后神经元凋亡率降低,与模型组相比差异有显著性(P<0.05);Real time RT-PCR结果显示,模型组神经元的Bcl-2mRNA下调,Bax、Caspase-3、Id2mRNA上调,与正常对照组相比差异有显著性(P<0.01);经姜黄素预处理后,缺氧损伤神经元的Bax、Caspase-3mRNA下调,与模型组相比较差异有显著性(P<0.01),Id2mRNA下调,Bcl-2mRNA上调,与模型组相比较差异有显著性(P<0.05)。结论:中药单体姜黄素抑制缺氧损伤神经元的凋亡,其机制可能与下调Id2mRNA的表达,进而下调Bax、上调Bcl-2mRNA表达,进一步阻断Caspase-3基因的激活有关。
Objective: To observe the effect of curcumin on Id2 gene expression and explore the mechanism of curcumin on neuronal apoptosis induced by hypoxia. Methods: Primary neurons seeded into 6-well plates at a density of 5 × 10 5 / cm 3 were randomly divided into normal control group, model group and curcumin group. The neurons in each group were intervened by different experimental conditions. The normal control group was cultured in normoxia for 30 h, the model group was cultured in normoxia for 24 h and then anaerobically treated for 6 h. The curcumin group: 20 μM curcumin normoxia After culturing for 24 h, re-hypoxia treatment for 6 h. After intervention of each group, the activity of neurons in each group was detected by CCK8 method, and the inhibition rate of each group was calculated and analyzed. The apoptosis rate of neurons in each group was detected by Annexin V / PI staining; Real time The expression of Bcl-2, Bax, Caspase-3 and other apoptosis-related genes and Id2 mRNA in neurons were detected by RT-PCR. Results: The results of CCK8 showed that the activity of neurons in the model group was significantly lower than that in the normal control group (P <0.01). After curcumin pretreatment, the neuronal activity was significantly increased compared with the model group (P <0.01). The result of flow cytometry showed that the apoptosis rate of neurons in model group was significantly higher than that in normal control group (P <0.01). After curcumin pretreatment, (P <0.05). The results of Real time RT-PCR showed that the expression of Bcl-2mRNA, Bax, Caspase-3 and Id2mRNA were up-regulated in the neurons of model group compared with the normal control group (P <0.01). After curcumin preconditioning, the expression of Bax and Caspase-3 mRNA in hypoxia-injured neurons was significantly lower than that in model group (P <0.01), Id2 mRNA was down-regulated, Bcl-2 mRNA was up-regulated, which was significantly different from the model group (P <0.05). CONCLUSION: Curcumin inhibits the apoptosis of neurons induced by hypoxia. The mechanism may be related to down-regulation of Id2mRNA, down-regulation of Bax, up-regulation of Bcl-2mRNA, and further block of caspase-3 gene activation.