α-硫辛酸联合青霉胺对TX乳鼠神经元P38MAPK通路的调控作用研究
发布时间：2019-07-30 14:26
【摘要】：目的研究α-硫辛酸(alpha-lipoic acid,ALA)联合青霉胺(Penicillamine,PCA)对Wilson病(Wilson disease,WD)模型TX乳鼠神经元P38 MAPK通路的调控机制。方法本实验乳鼠神经元通过原代方法分离培养所得,分为正常对照组、模型组、ALA组、PCA组及联合组。采用流式细胞仪检测药物治疗前后活性氧释放量及JC-I荧光强度的变化;Western blot法检测经药物作用后P38丝裂原活化蛋白激酶、细胞色素C、半胱氨酸天冬氨酸蛋白酶-9、半胱氨酸天冬氨酸蛋白酶-3蛋白的表达。结果经流式细胞仪检测结果可知ALA、PCA及联合用药均能减少神经元内ROS释放量且其MFI分别为59.29±1.22、53.19±1.34及52.46±1.23,增进JC-1的荧光强度(P0.01),且同ALA组和PCA组比,联合组ROS释放量明显降低,JC-1的荧光强度显著增强。Western blot检测发现与正常组相比,模型组乳鼠神经元内P38MAPK,Cyt C,Caspase 9,Caspase 3蛋白含量显著增加(P0.01);与模型组相比,LA组、PCA组及联合组乳鼠神经元内P38 MAPK,Cyt C,Caspase 9,Caspase 3蛋白表达水平明显下降;联合组P38 MAPK,Cyt C,Caspase 9,Caspase 3蛋白含量较LA组及PCA组减少。结论 ALA联合PCA可促进铜排出,降低高铜诱导的线粒体损伤,降低高铜神经毒性,延缓神经元凋亡,改善WD神经系统损伤症状。
[Abstract]:Objective to study the regulatory mechanism of 伪-lipoic acid (alpha-lipoic acid,ALA) combined with penicillamine (Penicillamine,PCA) on P38 MAPK pathway in neonatal TX neurons of Wilson disease (Wilson disease,WD) model. Methods the neurons of neonatal rats were isolated and cultured by primary method and divided into normal control group, model group, ALA group, PCA group and combined group. The changes of reactive oxygen species release and JC-I fluorescence intensity before and after drug treatment were detected by flow cytometry. The expression of P38 mitogen-activated protein kinase, cytochrome C, cystine aspartate protease-9 and cystine aspartate protease-3 protein was detected by; Western blot method. Results the results of flow cytometry showed that both ALA,PCA and combined drugs could reduce the release of ROS in neurons and their MFI were 59.29 卤1.22, 53.19 卤1.34 and 52.46 卤1.23, respectively, which increased the fluorescence intensity of JC-1 (P01). Compared with ALA group and PCA group, the release of ROS and the fluorescence intensity of JC-1 in the combined group were significantly decreased and the fluorescence intensity of JC-1 was significantly increased. Western blot showed that compared with the normal group, the expression of P38MapK in the neurons of the model group was significantly higher than that of the normal group. The content of caspase 3 protein increased significantly in Cyt C, caspase 9 and caspase 3 protein content (P 0.01). Compared with the model group, the expression levels of P38 MAPK,Cyt C, caspase 9 and caspase 3 protein in neonatal rats in LA group, PCA group and combination group were significantly lower than those in LA group and PCA group, and the expression of caspase 3 protein in P38 MAPK,Cyt C group was significantly lower than that in LA group and PCA group. Conclusion ALA combined with PCA can promote copper excretion, reduce the damage of mitochondria induced by high copper, reduce the neurotoxicity of high copper, delay neuronal apoptosis and improve the symptoms of WD nervous system injury.
【作者单位】： 安徽中医药大学神经病学研究所附属医院神经内科;
【基金】：国家自然科学基金项目(编号:81603596) 省自然科学基金项目(编号:1508085MH153)
【分类号】：R596;R747.9
本文编号：2520948
[Abstract]:Objective to study the regulatory mechanism of 伪-lipoic acid (alpha-lipoic acid,ALA) combined with penicillamine (Penicillamine,PCA) on P38 MAPK pathway in neonatal TX neurons of Wilson disease (Wilson disease,WD) model. Methods the neurons of neonatal rats were isolated and cultured by primary method and divided into normal control group, model group, ALA group, PCA group and combined group. The changes of reactive oxygen species release and JC-I fluorescence intensity before and after drug treatment were detected by flow cytometry. The expression of P38 mitogen-activated protein kinase, cytochrome C, cystine aspartate protease-9 and cystine aspartate protease-3 protein was detected by; Western blot method. Results the results of flow cytometry showed that both ALA,PCA and combined drugs could reduce the release of ROS in neurons and their MFI were 59.29 卤1.22, 53.19 卤1.34 and 52.46 卤1.23, respectively, which increased the fluorescence intensity of JC-1 (P01). Compared with ALA group and PCA group, the release of ROS and the fluorescence intensity of JC-1 in the combined group were significantly decreased and the fluorescence intensity of JC-1 was significantly increased. Western blot showed that compared with the normal group, the expression of P38MapK in the neurons of the model group was significantly higher than that of the normal group. The content of caspase 3 protein increased significantly in Cyt C, caspase 9 and caspase 3 protein content (P 0.01). Compared with the model group, the expression levels of P38 MAPK,Cyt C, caspase 9 and caspase 3 protein in neonatal rats in LA group, PCA group and combination group were significantly lower than those in LA group and PCA group, and the expression of caspase 3 protein in P38 MAPK,Cyt C group was significantly lower than that in LA group and PCA group. Conclusion ALA combined with PCA can promote copper excretion, reduce the damage of mitochondria induced by high copper, reduce the neurotoxicity of high copper, delay neuronal apoptosis and improve the symptoms of WD nervous system injury.
【作者单位】： 安徽中医药大学神经病学研究所附属医院神经内科;
【基金】：国家自然科学基金项目(编号:81603596) 省自然科学基金项目(编号:1508085MH153)
【分类号】：R596;R747.9
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