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目的:研究氧化槐定碱对原代培养新生大鼠海马神经元氧糖剥夺再灌注损伤后细胞调亡的影响。方法:以原代培养的新生大鼠海马神经元为研究对象建立氧糖剥夺再灌注损伤模型。Hoechst 33342和TUNEL染色法检测海马神经细胞凋亡,透射电子显微镜术观察凋亡细胞形态学变化,化学比色法测定海马神经细胞Caspase-3、Caspase-9和Caspase-8的活性,免疫蛋白印迹技术和实时荧光定量PCR技术检测凋亡相关因子Cytochrome C、Caspase-3、Bcl-2、Bax和PARP-1的蛋白和mRNA的表达。结果:与正常组比较,损伤组Hoechst 33342和TUNEL染色法显示神经细胞凋亡率明显升高;Caspase-3、Caspase-9和Caspase-8活性显著提高;Cytochrome C、Caspase-3、Bax和PARP-1蛋白和mRNA水平明显上调,Bcl-2蛋白和mRNA水平明显下调,Bcl-2/Bax比率也明显下调。与损伤组比较,氧化槐定碱治疗组(20、5、1.25mg/L)可显著改善氧糖剥夺再灌注损伤所致的神经细胞凋亡的形态学变化,并降低神经细胞凋亡率;明显抑制Caspase-3、Caspase-9和Caspase-8的活性。氧化槐定碱治疗组(20mg/L)可明显抑制Cytochrome C、Caspase-3、Bax和PARP-1蛋白和mRNA表达,增强Bcl-2蛋白和mRNA的表达,使Bcl-2/Bax比率上升。结论:氧化槐定碱通过抗凋亡作用对氧糖剥夺再灌注损伤的大鼠海马神经细胞发挥保护作用。
OBJECTIVE: To study the effects of sophoridine on apoptosis of primary cultured neonatal rat hippocampal neurons after oxygen-glucose deprivation-reperfusion injury. Methods: The primary cultured neonatal rat hippocampal neurons were used to establish a model of oxygen deprivation and reperfusion injury. The apoptosis of hippocampal neurons was detected by Hoechst 33342 and TUNEL staining. Morphological changes of apoptotic cells were observed by transmission electron microscopy. The activities of Caspase-3, Caspase-9 and Caspase-8 in hippocampal neurons were determined by chemical colorimetry. The expression of Cytochrome C, Caspase-3, Bcl-2, Bax and PARP-1 protein and mRNA were detected by real-time fluorescence quantitative PCR and real-time PCR. Results: Hoechst 33342 staining and TUNEL staining showed that the apoptotic rate of neurons was significantly increased, the activities of Caspase-3, Caspase-9 and Caspase-8 were significantly increased compared with the normal group. The expressions of Cytochrome C, Caspase-3, Bax and PARP -1 protein and mRNA levels were significantly up-regulated, Bcl-2 protein and mRNA levels were significantly down-regulated, Bcl-2 / Bax ratio was significantly down-regulated. Compared with the injury group, the oxidized sophoridine group (20, 5, 1.25 mg / L) could significantly improve the morphological changes of neurocyte apoptosis caused by oxygen deprivation and reperfusion injury and reduce the apoptosis rate of nerve cells; Significantly inhibited the activity of Caspase-3, Caspase-9 and Caspase-8. Oxalicarbine treatment group (20mg / L) significantly inhibited the expression of Cytochrome C, Caspase-3, Bax and PARP-1 protein and mRNA, increased the expression of Bcl-2 protein and mRNA, and increased the ratio of Bcl-2 / Bax. CONCLUSION: Oxalicarbine exerts a protective effect on neurons in hippocampus of rats with oxygen-glucose deprivation and reperfusion injury through anti-apoptotic effects.