α-硫辛酸聯(lián)合青霉胺對(duì)TX乳鼠神經(jīng)元P38MAPK通路的調(diào)控作用研究
發(fā)布時(shí)間：2019-07-30 14:26
【摘要】：目的研究α-硫辛酸(alpha-lipoic acid,ALA)聯(lián)合青霉胺(Penicillamine,PCA)對(duì)Wilson病(Wilson disease,WD)模型TX乳鼠神經(jīng)元P38 MAPK通路的調(diào)控機(jī)制。方法本實(shí)驗(yàn)乳鼠神經(jīng)元通過原代方法分離培養(yǎng)所得,分為正常對(duì)照組、模型組、ALA組、PCA組及聯(lián)合組。采用流式細(xì)胞儀檢測(cè)藥物治療前后活性氧釋放量及JC-I熒光強(qiáng)度的變化;Western blot法檢測(cè)經(jīng)藥物作用后P38絲裂原活化蛋白激酶、細(xì)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蛋白的表達(dá)。結(jié)果經(jīng)流式細(xì)胞儀檢測(cè)結(jié)果可知ALA、PCA及聯(lián)合用藥均能減少神經(jīng)元內(nèi)ROS釋放量且其MFI分別為59.29±1.22、53.19±1.34及52.46±1.23,增進(jìn)JC-1的熒光強(qiáng)度(P0.01),且同ALA組和PCA組比,聯(lián)合組ROS釋放量明顯降低,JC-1的熒光強(qiáng)度顯著增強(qiáng)。Western blot檢測(cè)發(fā)現(xiàn)與正常組相比,模型組乳鼠神經(jīng)元內(nèi)P38MAPK,Cyt C,Caspase 9,Caspase 3蛋白含量顯著增加(P0.01);與模型組相比,LA組、PCA組及聯(lián)合組乳鼠神經(jīng)元內(nèi)P38 MAPK,Cyt C,Caspase 9,Caspase 3蛋白表達(dá)水平明顯下降;聯(lián)合組P38 MAPK,Cyt C,Caspase 9,Caspase 3蛋白含量較LA組及PCA組減少。結(jié)論 ALA聯(lián)合PCA可促進(jìn)銅排出,降低高銅誘導(dǎo)的線粒體損傷,降低高銅神經(jīng)毒性,延緩神經(jīng)元凋亡,改善WD神經(jīng)系統(tǒng)損傷癥狀。
[Abstract]:Objective to study the regulatory mechanism of 偽-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 鹵1.22, 53.19 鹵1.34 and 52.46 鹵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.
【作者單位】： 安徽中醫(yī)藥大學(xué)神經(jīng)病學(xué)研究所附屬醫(yī)院神經(jīng)內(nèi)科;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào):81603596) 省自然科學(xué)基金項(xiàng)目(編號(hào):1508085MH153)
【分類號(hào)】：R596;R747.9
本文編號(hào)：2520948
[Abstract]:Objective to study the regulatory mechanism of 偽-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 鹵1.22, 53.19 鹵1.34 and 52.46 鹵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.
【作者單位】： 安徽中醫(yī)藥大學(xué)神經(jīng)病學(xué)研究所附屬醫(yī)院神經(jīng)內(nèi)科;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào):81603596) 省自然科學(xué)基金項(xiàng)目(編號(hào):1508085MH153)
【分類號(hào)】：R596;R747.9
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