迷迭香酸對鐵處理的SK-N-SH細胞的神經(jīng)保護作用機制研究
發(fā)布時間：2018-08-06 14:20
【摘要】：帕金森病(Parkinson's disease,PD)是中老年人常見的神經(jīng)系統(tǒng)退行性疾病。其主要臨床特征是靜止性震顫,肌僵直,姿勢反射障礙等。PD的主要病理學改變?yōu)楹谫|(zhì)(substantia nigra, SN)多巴胺(dopamine, DA)神經(jīng)元變性缺失,以及alpha-突觸核蛋白聚集的路易小體的形成。迄今為止,PD的病因及發(fā)病機制仍不清楚。近年來,越來越多的研究發(fā)現(xiàn)PD病人黑質(zhì)中鐵含量增加,鐵的異常增高和鐵誘發(fā)的氧化應激反應可能是PD發(fā)病中的關鍵因素。而alpha突觸核蛋白是PD特征性病理標志Lewy小體的主要成分,其聚集及由此引起的黑質(zhì)多巴胺能神經(jīng)元變性死亡是PD的主要病理改變。前期研究證實鐵可誘導alpha-突觸核蛋白的表達及聚集,alpha-突觸核蛋白的聚集可加重鐵對神經(jīng)元的損傷,提示鐵在alpha-突觸核蛋白聚集中發(fā)揮著重要的作用。 迷迭香酸(rosmarinic acid, RA)是一種天然抗氧化劑,它由一分子咖啡酸和-分子丹參素組成,為水溶性多酚類化合物。RA具有抗炎,抗菌,抗病毒,免疫抑制等多種生物活性。我們前期研究證實迷迭香酸對神經(jīng)毒素6-羥基多巴胺(6-hydroxydopamine,6-OHDA)及1-甲基-4-苯基吡啶陽離子(1-methyl-4-phenylpyridinium ion, MPP+)誘導的細胞損傷具有保護作用,其機制可能與抗氧化應激、抗凋亡、降低黑質(zhì)鐵水平有關。但是其保護作用的分子機制仍不清楚。已經(jīng)證實迷迭香酸在體外可以通過抗氧化作用來抑制alpha-突觸核蛋白纖維的生成,還可以降解已形成的alpha-突觸核蛋白纖維。但是迷迭香酸是否可以通過抑制alpha-突觸核蛋白的聚集在PD中發(fā)揮其保護作用尚不清楚,其對alpha-突觸核蛋白的生成及聚集的調(diào)節(jié)機制也未見報道。因此,本實驗在多巴胺能神經(jīng)細胞SK-N-SH細胞上,應用流式細胞術(flow cytometry, FCM)檢測線粒體膜電位的改變,免疫組織化學檢測alpha-突觸核蛋白的聚集、實時熒光定量PCR及western blots檢測HO-1,alpha-突觸核蛋白,IRP1的表達,以期闡明RA對鐵誘導的alpha-突觸核蛋白聚集的影響及其可能的機制。實驗結(jié)果如下： 1.100μmol/L Fe2+和100μmol/L FAC處理SK-N-SH細胞24h后,細胞線粒體跨膜電位差(mitochondrial transmembrane potential,△Ψm)明顯降低(P0.05)。RA預處理可以阻斷上述改變(P0.05)。 2.1mmol/L Fe2+和FAC分別處理SK-N-SH細胞24h,細胞內(nèi)alpha-突觸核蛋白的聚集較對照組明顯增多。而10-3和10-4mol/L RA預處理可以拮抗鐵誘導的alpha-突觸核蛋白的聚集。 3. FAC(100μmol/L)和RA(10-4mol/L)單獨處理SK-N-SH細胞均可以上調(diào)HO-1mRNA及蛋白水平,與對照組相比,差別有統(tǒng)計學意義(P0.05)；而FAC/RA共孵育,可以使HO-1mRNA及蛋白水平較FAC處理組進一步增加(P0.05)。 4. FAC (100μmol/L)處理SK-N-SH細胞24h, alpha-突觸核蛋白mRNA的表達明顯增加,與對照組相比,差別有統(tǒng)計學意義(P0.05)。RA預處理后能夠顯著的抑制FAC誘導的alpha-突觸核蛋白mRNA的表達增加(P0.05) 5. FAC (100μmol/L)處理SK-N-SH細胞24h, IRP1的蛋白水平下降,與對照組相比,差別有統(tǒng)計學意義(P0.05)。而FAC/RA共孵育,可以使IRP1蛋白水平較FAC處理組明顯上升,差別有統(tǒng)計學意義(P0.05)。 上述實驗結(jié)果表明：高鐵可以引起細胞△Ψm的降低,促進alpha-突觸核蛋白的聚集,并通過IRE/IRP機制,升高鐵誘導的alpha-突觸核蛋白mRNA的表達,引起SK-N-SH細胞的損傷。而RA預處理SK-N-SH細胞,在一定濃度下可以部分逆轉(zhuǎn)細胞內(nèi)高鐵造成的△甲m降低,使HO-1的mRNA及蛋白上升,從而抑制鐵誘導的alpha-突觸核蛋白的聚集,并通過IRE/IRP機制,降低鐵誘導的alpha-突觸核蛋白mRNA的表達,從而減輕高鐵水平對細胞造成的損害。本實驗所研究的RA對DA能神經(jīng)元的保護作用以及對其可能機制的初步探討,為PD的防治提供全新的研究結(jié)果和藥物防治的新策略。
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R742.5
本文編號：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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R742.5
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相關期刊論文 前4條
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，本文編號：2168004
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