迷迭香酸对铁处理的SK-N-SH细胞的神经保护作用机制研究
发布时间：2018-08-06 14:20
【摘要】：帕金森病(Parkinson's disease,PD)是中老年人常见的神经系统退行性疾病。其主要临床特征是静止性震颤,肌僵直,姿势反射障碍等。PD的主要病理学改变为黑质(substantia nigra, SN)多巴胺(dopamine, DA)神经元变性缺失,以及alpha-突触核蛋白聚集的路易小体的形成。迄今为止,PD的病因及发病机制仍不清楚。近年来,越来越多的研究发现PD病人黑质中铁含量增加,铁的异常增高和铁诱发的氧化应激反应可能是PD发病中的关键因素。而alpha突触核蛋白是PD特征性病理标志Lewy小体的主要成分,其聚集及由此引起的黑质多巴胺能神经元变性死亡是PD的主要病理改变。前期研究证实铁可诱导alpha-突触核蛋白的表达及聚集,alpha-突触核蛋白的聚集可加重铁对神经元的损伤,提示铁在alpha-突触核蛋白聚集中发挥着重要的作用。 迷迭香酸(rosmarinic acid, RA)是一种天然抗氧化剂,它由一分子咖啡酸和-分子丹参素组成,为水溶性多酚类化合物。RA具有抗炎,抗菌,抗病毒,免疫抑制等多种生物活性。我们前期研究证实迷迭香酸对神经毒素6-羟基多巴胺(6-hydroxydopamine,6-OHDA)及1-甲基-4-苯基吡啶阳离子(1-methyl-4-phenylpyridinium ion, MPP+)诱导的细胞损伤具有保护作用,其机制可能与抗氧化应激、抗凋亡、降低黑质铁水平有关。但是其保护作用的分子机制仍不清楚。已经证实迷迭香酸在体外可以通过抗氧化作用来抑制alpha-突触核蛋白纤维的生成,还可以降解已形成的alpha-突触核蛋白纤维。但是迷迭香酸是否可以通过抑制alpha-突触核蛋白的聚集在PD中发挥其保护作用尚不清楚,其对alpha-突触核蛋白的生成及聚集的调节机制也未见报道。因此,本实验在多巴胺能神经细胞SK-N-SH细胞上,应用流式细胞术(flow cytometry, FCM)检测线粒体膜电位的改变,免疫组织化学检测alpha-突触核蛋白的聚集、实时荧光定量PCR及western blots检测HO-1,alpha-突触核蛋白,IRP1的表达,以期阐明RA对铁诱导的alpha-突触核蛋白聚集的影响及其可能的机制。实验结果如下： 1.100μmol/L Fe2+和100μmol/L FAC处理SK-N-SH细胞24h后,细胞线粒体跨膜电位差(mitochondrial transmembrane potential,△Ψm)明显降低(P0.05)。RA预处理可以阻断上述改变(P0.05)。 2.1mmol/L Fe2+和FAC分别处理SK-N-SH细胞24h,细胞内alpha-突触核蛋白的聚集较对照组明显增多。而10-3和10-4mol/L RA预处理可以拮抗铁诱导的alpha-突触核蛋白的聚集。 3. FAC(100μmol/L)和RA(10-4mol/L)单独处理SK-N-SH细胞均可以上调HO-1mRNA及蛋白水平,与对照组相比,差别有统计学意义(P0.05)；而FAC/RA共孵育,可以使HO-1mRNA及蛋白水平较FAC处理组进一步增加(P0.05)。 4. FAC (100μmol/L)处理SK-N-SH细胞24h, alpha-突触核蛋白mRNA的表达明显增加,与对照组相比,差别有统计学意义(P0.05)。RA预处理后能够显著的抑制FAC诱导的alpha-突触核蛋白mRNA的表达增加(P0.05) 5. FAC (100μmol/L)处理SK-N-SH细胞24h, IRP1的蛋白水平下降,与对照组相比,差别有统计学意义(P0.05)。而FAC/RA共孵育,可以使IRP1蛋白水平较FAC处理组明显上升,差别有统计学意义(P0.05)。 上述实验结果表明：高铁可以引起细胞△Ψm的降低,促进alpha-突触核蛋白的聚集,并通过IRE/IRP机制,升高铁诱导的alpha-突触核蛋白mRNA的表达,引起SK-N-SH细胞的损伤。而RA预处理SK-N-SH细胞,在一定浓度下可以部分逆转细胞内高铁造成的△甲m降低,使HO-1的mRNA及蛋白上升,从而抑制铁诱导的alpha-突触核蛋白的聚集,并通过IRE/IRP机制,降低铁诱导的alpha-突触核蛋白mRNA的表达,从而减轻高铁水平对细胞造成的损害。本实验所研究的RA对DA能神经元的保护作用以及对其可能机制的初步探讨,为PD的防治提供全新的研究结果和药物防治的新策略。
[Abstract]:Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease of middle and old people. The main clinical features are static tremor, muscular stiffness, and postural reflex disorder, and the main pathological changes of.PD are the degeneration and deletion of substantia nigra (SN) dopamine (dopamine, DA), and alpha- synaptic nuclear protein aggregation. The formation of the Louis corpuscle. So far, the etiology and pathogenesis of PD remains unclear. In recent years, more and more studies have found that the iron content in the substantia nigra of PD patients is increased, the abnormal increase of iron and the oxidative stress induced by iron may be the key factors in the pathogenesis of PD. Alpha synapse is the main pathological marker of PD, the main body of Lewy corpuscle. Components, their aggregation and the resulting degeneration and death of dopaminergic neurons in the substantia nigra are major pathological changes in PD. Earlier studies have confirmed that iron can induce the expression and aggregation of alpha- synuclein. The aggregation of alpha- synuclein can aggravate the damage of iron to neurons, suggesting that iron plays an important role in the aggregation of alpha- synapses. Use.
Rosmarinic acid (RA) is a natural antioxidant consisting of a molecular caffeic acid and molecular Danshensu. It is a water-soluble polyphenolic compound,.RA, which has many biological activities, such as anti-inflammatory, antibacterial, antiviral, immunosuppressive and other biological activities. Our previous study confirmed that rosmarinic acid to neurotoxin 6- hydroxy dopamine (6-hydroxydopamine, 6-OH). DA) and 1- methyl -4- phenyl pyridine cations (1-methyl-4-phenylpyridinium ion, MPP+) induced cell damage have protective effects. The mechanism may be related to antioxidant stress, anti apoptosis, and lowering the level of substantia nigra. But the molecular mechanism of its protective effect is still unclear. It has been proved that rosemary acid can be used in vitro through antioxidant action. To inhibit the formation of alpha- synuclein fibers, it can also degrade the formed alpha- synuclein fibers. But it is not clear whether rosemary can play its protective role in PD by inhibiting the aggregation of alpha- synaptic nuclear proteins. The regulation mechanism for the formation and aggregation of alpha- synuclein has not been reported. In this experiment, the changes of mitochondrial membrane potential were detected by flow cytometry (flow cytometry, FCM) on SK-N-SH cells of dopaminergic neurons. Immunohistochemistry was used to detect the aggregation of alpha- nucleoprotein. Real-time quantitative PCR and Western blots were used to detect HO-1, alpha- process, nucleoprotein and IRP1 expression. The effect of pha- synuclein aggregation and its possible mechanism are as follows:
After 1.100 mol/L Fe2+ and 100 mol/L FAC treated SK-N-SH cells 24h, the mitochondrial transmembrane potential difference (mitochondrial transmembrane potential, delta m) decreased significantly (P0.05).RA preconditioning could block the above changes.
SK-N-SH cell 24h was treated with 2.1mmol/L Fe2+ and FAC respectively. The aggregation of alpha- synuclein in cells increased significantly than that in the control group. 10-3 and 10-4mol/L RA pretreatment could antagonize the aggregation of iron induced alpha- synuclein.
3. FAC (100 mol/L) and RA (10-4mol/L) treated SK-N-SH cells could increase the level of HO-1mRNA and protein. Compared with the control group, the difference was statistically significant (P0.05), while FAC/RA co incubation could increase the level of HO-1mRNA and protein in FAC treatment group (P0.05).
4. FAC (100 mol/L) treatment of SK-N-SH cells 24h, alpha- synuclein mRNA expression significantly increased, compared with the control group, the difference was statistically significant (P0.05).RA pretreatment can significantly inhibit the FAC induced alpha- synaptic nuclear protein mRNA expression increased (P0.05)
5. FAC (100 mol/L) treatment of SK-N-SH cells 24h, the protein level of IRP1 decreased, compared with the control group, the difference was statistically significant (P0.05). While FAC/RA co incubation, the level of IRP1 protein can be significantly higher than the FAC treatment group, the difference is statistically significant (P0.05).
The above results show that high iron can cause the decrease of delta m, promote the aggregation of alpha- synaptic nuclear protein, and increase the expression of alpha- induced nucleoprotein mRNA through the IRE/IRP mechanism, and cause the damage of SK-N-SH cells. While RA pretreatment SK-N-SH cells can partly reverse the intracellular high iron at a fixed concentration. Delta m reduces the mRNA and protein of HO-1, thereby inhibiting the aggregation of alpha- induced nucleoprotein, and reducing the expression of iron induced alpha- synuclein mRNA through the IRE/IRP mechanism, thus reducing the damage caused by the high iron level on the cells. The protective effect of RA on the DA neurons in this experiment and its possibility are possible. The preliminary study of mechanism will provide new research results and new strategies for drug control for the prevention and treatment of PD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
本文编号：2168004
[Abstract]:Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease of middle and old people. The main clinical features are static tremor, muscular stiffness, and postural reflex disorder, and the main pathological changes of.PD are the degeneration and deletion of substantia nigra (SN) dopamine (dopamine, DA), and alpha- synaptic nuclear protein aggregation. The formation of the Louis corpuscle. So far, the etiology and pathogenesis of PD remains unclear. In recent years, more and more studies have found that the iron content in the substantia nigra of PD patients is increased, the abnormal increase of iron and the oxidative stress induced by iron may be the key factors in the pathogenesis of PD. Alpha synapse is the main pathological marker of PD, the main body of Lewy corpuscle. Components, their aggregation and the resulting degeneration and death of dopaminergic neurons in the substantia nigra are major pathological changes in PD. Earlier studies have confirmed that iron can induce the expression and aggregation of alpha- synuclein. The aggregation of alpha- synuclein can aggravate the damage of iron to neurons, suggesting that iron plays an important role in the aggregation of alpha- synapses. Use.
Rosmarinic acid (RA) is a natural antioxidant consisting of a molecular caffeic acid and molecular Danshensu. It is a water-soluble polyphenolic compound,.RA, which has many biological activities, such as anti-inflammatory, antibacterial, antiviral, immunosuppressive and other biological activities. Our previous study confirmed that rosmarinic acid to neurotoxin 6- hydroxy dopamine (6-hydroxydopamine, 6-OH). DA) and 1- methyl -4- phenyl pyridine cations (1-methyl-4-phenylpyridinium ion, MPP+) induced cell damage have protective effects. The mechanism may be related to antioxidant stress, anti apoptosis, and lowering the level of substantia nigra. But the molecular mechanism of its protective effect is still unclear. It has been proved that rosemary acid can be used in vitro through antioxidant action. To inhibit the formation of alpha- synuclein fibers, it can also degrade the formed alpha- synuclein fibers. But it is not clear whether rosemary can play its protective role in PD by inhibiting the aggregation of alpha- synaptic nuclear proteins. The regulation mechanism for the formation and aggregation of alpha- synuclein has not been reported. In this experiment, the changes of mitochondrial membrane potential were detected by flow cytometry (flow cytometry, FCM) on SK-N-SH cells of dopaminergic neurons. Immunohistochemistry was used to detect the aggregation of alpha- nucleoprotein. Real-time quantitative PCR and Western blots were used to detect HO-1, alpha- process, nucleoprotein and IRP1 expression. The effect of pha- synuclein aggregation and its possible mechanism are as follows:
After 1.100 mol/L Fe2+ and 100 mol/L FAC treated SK-N-SH cells 24h, the mitochondrial transmembrane potential difference (mitochondrial transmembrane potential, delta m) decreased significantly (P0.05).RA preconditioning could block the above changes.
SK-N-SH cell 24h was treated with 2.1mmol/L Fe2+ and FAC respectively. The aggregation of alpha- synuclein in cells increased significantly than that in the control group. 10-3 and 10-4mol/L RA pretreatment could antagonize the aggregation of iron induced alpha- synuclein.
3. FAC (100 mol/L) and RA (10-4mol/L) treated SK-N-SH cells could increase the level of HO-1mRNA and protein. Compared with the control group, the difference was statistically significant (P0.05), while FAC/RA co incubation could increase the level of HO-1mRNA and protein in FAC treatment group (P0.05).
4. FAC (100 mol/L) treatment of SK-N-SH cells 24h, alpha- synuclein mRNA expression significantly increased, compared with the control group, the difference was statistically significant (P0.05).RA pretreatment can significantly inhibit the FAC induced alpha- synaptic nuclear protein mRNA expression increased (P0.05)
5. FAC (100 mol/L) treatment of SK-N-SH cells 24h, the protein level of IRP1 decreased, compared with the control group, the difference was statistically significant (P0.05). While FAC/RA co incubation, the level of IRP1 protein can be significantly higher than the FAC treatment group, the difference is statistically significant (P0.05).
The above results show that high iron can cause the decrease of delta m, promote the aggregation of alpha- synaptic nuclear protein, and increase the expression of alpha- induced nucleoprotein mRNA through the IRE/IRP mechanism, and cause the damage of SK-N-SH cells. While RA pretreatment SK-N-SH cells can partly reverse the intracellular high iron at a fixed concentration. Delta m reduces the mRNA and protein of HO-1, thereby inhibiting the aggregation of alpha- induced nucleoprotein, and reducing the expression of iron induced alpha- synuclein mRNA through the IRE/IRP mechanism, thus reducing the damage caused by the high iron level on the cells. The protective effect of RA on the DA neurons in this experiment and its possibility are possible. The preliminary study of mechanism will provide new research results and new strategies for drug control for the prevention and treatment of PD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
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相关期刊论文 前4条
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