姜黄素对鱼藤酮诱导的帕金森病SH-SY5Y细胞模型的抗氧化应激作用及其机制
发布时间：2018-08-03 07:46
【摘要】：背景与目的帕金森病(parkinson disease,PD)是一种神经系统变性疾病,其主要病理特征是黑质多巴胺(Dopamine,DA)神经元细胞死亡和黑质纹状体通路退化。由于黑质-纹状体通路的耗尽,黑质对丘脑产生明显的抑制作用使对肌肉运动和肌张力的控制减弱,最终出现静止性震颤、肌强直和行动迟缓等临床症状。目前研究表明,多种因素如氧化应激、线粒体功能损伤、炎症反应、蛋白酶体功能障碍等参与PD的发生,但具体机制尚未完全清楚。姜黄素是姜黄的有效成分,后者已被广泛用作食物调料和传统草药。姜黄素是从姜黄根茎中提取的一种酚性色素,具有多种药理作用如抗炎、抗氧化、清除自由基等研究。研究表明姜黄素能够通过诱导Bcl-2的表达来抑制MPP+诱导的细胞内活性氧累物质的生成,从而维持线粒体膜电位的稳定性,减少细胞色素C的释放,最终起到对抗氧化应激的作用。同时姜黄素能增加谷胱甘肽的水平,抑制脂质过氧化反应等起到保护多巴胺神经元的作用。赖氨酸残基乙酰化对蛋白翻译后修饰和调节起到重要作用,它在生物体内普遍存在。有文献报道,线粒体内超过20%的蛋白存在乙酰化现象。Sir T3是一种依赖于烟酰胺腺嘌呤二核苷酸(NAD)的Ⅲ类去乙酰化酶,主要存在于心脏、脑、肾脏及肝脏等细粒体含量丰富的组织器官中,是Sirtuin家族成员之一。近年来,人们发现Sirt3能够对那些乙酰化的线粒体蛋白进行脱乙酰化从而起到维持线粒体正常生理功能的作用,能够有效降低氧化应激负荷引起的疾病的发生。而其降低氧化应激损伤的机制可能通过乙酰化作用激活激活叉头框转录因子O亚族3a(FOXO3a,forkhead box O 3a)从而上调ROS清除系统如MnSOD和过氧化氢酶catalase(CAT)的表达有关。前期实验已经证实,姜黄素能够通过调控Sirt3的表达对鱼藤酮诱导慢性PD SD大鼠模型起到保护作用。那么姜黄素能否通过激活Sirt3激活FOXO3a进而上调MnSOD和CAT表达来抑制鱼藤酮诱导的SH-SY5Y细胞的损伤或者凋亡,是本实验研究所要探讨问题之一。为此,本研究选择姜黄素处理鱼藤酮诱导的SH-SY5Y的细胞模型,检测细胞活力,ROS含量,SIRT3、FOXO3a、Mn-SOD和CAT蛋白的表达情况,探讨姜黄素对PD细胞模型的保护作用及姜黄素、SIRT3、FOXO3a与ROS清除系统的关系及其所介导的相关机制,为临床治疗提供理论依据和新的思路。材料与方法首先建立SH-SY5Y细胞模型,再将SH-SY5Y细胞,按随机区组法分为对照组(不加药物处理)、鱼藤酮(终浓度0.1uM)模型组、姜黄素预处理组(终浓度分别为0.5μM、1.0μM、5.0μM、10.0μM)共6组。采用MTT检测细胞活力,流式细胞仪检测ROS含量,Western-blot法检测细胞内SIRT3、FOXO3a和Mn-SOD及CAT蛋白的表达。结果1.MTT检测细胞活性:姜黄素为0.5umol/L-1.0umol/L时开始出现对鱼藤酮致细胞损伤的保护作用,细胞活力较鱼藤酮损伤组增加差异均具有显著意义(P0.01),且1.0umol/L时作用最强;5.0umol/L时其保护作用开始下降,单独作用于SH-SY5Y细胞使其活力降低,但和鱼藤酮共同作用于SH-SY5Y细胞则使其活力较鱼藤酮损伤组增加,仍然发挥了保护细胞的作用,而没显示出协同损伤作用,但差异无统计学意义(P0.05)。2.流式细胞仪检测细胞内ROS含量的影响:0.1μmol/L鱼藤酮处理细胞24h后,细胞内ROS水平较对照组升高(P0.01);0.5μmol/L、1.0μmol/L姜黄素处理组较空白对照组和鱼藤酮组细胞内ROS降低(P0.01);5.0μmol/L、10μmol/L姜黄素处理组与鱼藤酮组比较差异无统计学意义(p0.05)3.Western-blot检测SIRT3、FOXO3a、Mn-SOD及CAT蛋白表达:鱼藤酮处理组较对照组表达降低,0.5μmol/L、1.0μmol/L姜黄素处理组较对照组、鱼藤酮组表达升高,差异有统计学意义(P0.01);5.0μmol/L、10.0μmol/L组较鱼藤酮组差异无统计学意义(P0.05)。结论1.鱼藤酮具有明显的神经毒性,其毒性作用呈浓度依赖性:适宜浓度能用来制备SH-SY5Y细胞的PD模型,较高浓度时会直接造成细胞的死亡。2.鱼藤酮诱导的SH-SY5Y细胞的PD模型中细胞的活力降低,细胞内ROS含量增多,Sirt3、FOXO3a、Mn-SOD和CAT的表达降低。3.姜黄素可能通过诱导SIRT3表达,使其去乙酰化激活FOXO3a,继而上调MN-SOD及CAT的表达使细胞内ROS含量减少,从而参与了PD的神经保护。
[Abstract]:Background and objective Parkinson disease (PD) is a neurodegenerative disease whose main pathological feature is the death of the neurons of the substantia nigra (Dopamine, DA) and the degeneration of the nigrostriatal pathway. The depletion of the nigrostriatal pathway and the obvious inhibitory effect of the substantia nigra on the thalamus to the muscle movement and muscle tension The results show that many factors such as oxidative stress, mitochondrial dysfunction, inflammatory response, and proteasome dysfunction are involved in the occurrence of PD, but the specific mechanism is not completely clear. Curcumin is the effective component of curcumin and the latter has been widely used. Used as a food seasoning and traditional herbal medicine. Curcumin is a phenolic pigment extracted from the rhizomes of turmeric. It has a variety of pharmacological effects such as anti-inflammatory, antioxidant, and free radical scavenging. The study shows that curcumin can inhibit the formation of active MPP+ induced intracellular oxygen by inducing the expression of Bcl-2 to maintain the mitochondrial membrane. The stability of the potential, reducing the release of cytochrome C, eventually plays a role in antagonism to oxidative stress. Meanwhile curcumin can increase the level of glutathione, inhibit lipid peroxidation and protect the dopamine neurons. Lysine residue acetylation plays an important role in the modification and regulation of protein translation. It is in vivo It has been reported that more than 20% of the mitochondria in mitochondria have acetylation,.Sir T3 is a kind of class III deacetylase dependent on nicotinamide adenine dinucleotide (NAD), which is one of the members of the Sirtuin family, which is one of the members of the Sirtuin family, which are mainly in the heart, brain, kidney and liver. Sirt3 can deacetylation of those acetylated mitochondrial proteins to maintain the normal physiological function of mitochondria, and can effectively reduce the occurrence of diseases caused by oxidative stress load. The mechanism for reducing oxidative stress damage may activate the O subgroup 3A (FOXO3a) by acetylated activation. Forkhead box O 3a) up regulate the expression of ROS scavenging system such as MnSOD and catalase catalase (CAT). Earlier experiments have proved that curcumin can protect the rat model of rotenone induced chronic PD SD rat by regulating the expression of Sirt3. The expression of T to inhibit the damage or apoptosis of rotenone induced SH-SY5Y cells is one of the problems to be discussed in this study. Therefore, this study selected curcumin to treat the cell model of rotenone induced SH-SY5Y, and detected the expression of cell vitality, ROS content, SIRT3, FOXO3a, Mn-SOD and CAT protein, and explored the model of curcumin to the PD cell model. The protective effect of curcumin, the relationship between curcumin, SIRT3, FOXO3a and ROS scavenging system and its related mechanism, provide theoretical basis and new ideas for clinical treatment. Materials and methods first set up SH-SY5Y cell model, and then divide SH-SY5Y cells into control group (without drug treatment), and rotenone (final concentration 0.1uM) model. Group, curcumin pretreatment group (the final concentration was 0.5 mu M, 1 mu M, 5 mu M, 10 M) in 6 groups. The cell viability was detected by MTT, the content of ROS was detected by flow cytometry, and the expression of SIRT3, FOXO3a and Mn-SOD and CAT protein in cell was detected by Western-blot method. The protective effect of ketone induced cell damage was significantly higher than that of rotenone injury group (P0.01), and the effect of 1.0umol/L was the strongest. When 5.0umol/L, the protective effect began to decrease, and the activity of SH-SY5Y cells was reduced, but with rotenone, the activity of rotenone in SH-SY5Y cells was more than the damage of rotenone. Group increased, still play the role of protective cells, but did not show synergistic damage, but the difference was not statistically significant (P0.05).2. flow cytometry to detect the effect of intracellular ROS content: after 0.1 u mol/L rotenone treated cells 24h, the level of intracellular ROS was higher than the control group (P0.01); 0.5 mu mol/L, 1 mu mol/L curcumin treatment group is more blank. The cell ROS in the control group and rotenone group decreased (P0.01), and the difference was not statistically significant between the 5 mu mol/L, 10 mol/L curcumin treatment group and the rotenone group (P0.05) 3.Western-blot detection SIRT3, FOXO3a, Mn-SOD and CAT protein expression: the rotenone treatment group was lower than the control group, 0.5 Mu mol/L, 1 mu mol/L curcumin treatment group compared with the control group, the fish Teng group. The expression of ketone group was higher, the difference was statistically significant (P0.01). There was no significant difference between 5 and 10 mol/L groups compared with rotenone group (P0.05). Conclusion 1. rotenone has obvious neurotoxicity, its toxic effect is concentration dependent: the appropriate concentration can be used to prepare PD model of SH-SY5Y cells, and the cell death will be directly caused by high concentration. .2. in the PD model of rotenone induced SH-SY5Y cells, the activity of cells decreased, the content of ROS in the cells increased. The expression of Sirt3, FOXO3a, Mn-SOD and CAT reduced.3. curcumin by inducing SIRT3 expression by inducing its deacetylation activation FOXO3a. Protect.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5
本文编号：2161081
[Abstract]:Background and objective Parkinson disease (PD) is a neurodegenerative disease whose main pathological feature is the death of the neurons of the substantia nigra (Dopamine, DA) and the degeneration of the nigrostriatal pathway. The depletion of the nigrostriatal pathway and the obvious inhibitory effect of the substantia nigra on the thalamus to the muscle movement and muscle tension The results show that many factors such as oxidative stress, mitochondrial dysfunction, inflammatory response, and proteasome dysfunction are involved in the occurrence of PD, but the specific mechanism is not completely clear. Curcumin is the effective component of curcumin and the latter has been widely used. Used as a food seasoning and traditional herbal medicine. Curcumin is a phenolic pigment extracted from the rhizomes of turmeric. It has a variety of pharmacological effects such as anti-inflammatory, antioxidant, and free radical scavenging. The study shows that curcumin can inhibit the formation of active MPP+ induced intracellular oxygen by inducing the expression of Bcl-2 to maintain the mitochondrial membrane. The stability of the potential, reducing the release of cytochrome C, eventually plays a role in antagonism to oxidative stress. Meanwhile curcumin can increase the level of glutathione, inhibit lipid peroxidation and protect the dopamine neurons. Lysine residue acetylation plays an important role in the modification and regulation of protein translation. It is in vivo It has been reported that more than 20% of the mitochondria in mitochondria have acetylation,.Sir T3 is a kind of class III deacetylase dependent on nicotinamide adenine dinucleotide (NAD), which is one of the members of the Sirtuin family, which is one of the members of the Sirtuin family, which are mainly in the heart, brain, kidney and liver. Sirt3 can deacetylation of those acetylated mitochondrial proteins to maintain the normal physiological function of mitochondria, and can effectively reduce the occurrence of diseases caused by oxidative stress load. The mechanism for reducing oxidative stress damage may activate the O subgroup 3A (FOXO3a) by acetylated activation. Forkhead box O 3a) up regulate the expression of ROS scavenging system such as MnSOD and catalase catalase (CAT). Earlier experiments have proved that curcumin can protect the rat model of rotenone induced chronic PD SD rat by regulating the expression of Sirt3. The expression of T to inhibit the damage or apoptosis of rotenone induced SH-SY5Y cells is one of the problems to be discussed in this study. Therefore, this study selected curcumin to treat the cell model of rotenone induced SH-SY5Y, and detected the expression of cell vitality, ROS content, SIRT3, FOXO3a, Mn-SOD and CAT protein, and explored the model of curcumin to the PD cell model. The protective effect of curcumin, the relationship between curcumin, SIRT3, FOXO3a and ROS scavenging system and its related mechanism, provide theoretical basis and new ideas for clinical treatment. Materials and methods first set up SH-SY5Y cell model, and then divide SH-SY5Y cells into control group (without drug treatment), and rotenone (final concentration 0.1uM) model. Group, curcumin pretreatment group (the final concentration was 0.5 mu M, 1 mu M, 5 mu M, 10 M) in 6 groups. The cell viability was detected by MTT, the content of ROS was detected by flow cytometry, and the expression of SIRT3, FOXO3a and Mn-SOD and CAT protein in cell was detected by Western-blot method. The protective effect of ketone induced cell damage was significantly higher than that of rotenone injury group (P0.01), and the effect of 1.0umol/L was the strongest. When 5.0umol/L, the protective effect began to decrease, and the activity of SH-SY5Y cells was reduced, but with rotenone, the activity of rotenone in SH-SY5Y cells was more than the damage of rotenone. Group increased, still play the role of protective cells, but did not show synergistic damage, but the difference was not statistically significant (P0.05).2. flow cytometry to detect the effect of intracellular ROS content: after 0.1 u mol/L rotenone treated cells 24h, the level of intracellular ROS was higher than the control group (P0.01); 0.5 mu mol/L, 1 mu mol/L curcumin treatment group is more blank. The cell ROS in the control group and rotenone group decreased (P0.01), and the difference was not statistically significant between the 5 mu mol/L, 10 mol/L curcumin treatment group and the rotenone group (P0.05) 3.Western-blot detection SIRT3, FOXO3a, Mn-SOD and CAT protein expression: the rotenone treatment group was lower than the control group, 0.5 Mu mol/L, 1 mu mol/L curcumin treatment group compared with the control group, the fish Teng group. The expression of ketone group was higher, the difference was statistically significant (P0.01). There was no significant difference between 5 and 10 mol/L groups compared with rotenone group (P0.05). Conclusion 1. rotenone has obvious neurotoxicity, its toxic effect is concentration dependent: the appropriate concentration can be used to prepare PD model of SH-SY5Y cells, and the cell death will be directly caused by high concentration. .2. in the PD model of rotenone induced SH-SY5Y cells, the activity of cells decreased, the content of ROS in the cells increased. The expression of Sirt3, FOXO3a, Mn-SOD and CAT reduced.3. curcumin by inducing SIRT3 expression by inducing its deacetylation activation FOXO3a. Protect.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5
【参考文献】
相关期刊论文 前10条
1 陈春暖;陈祥荣;蔡若蔚;曹学兵;王涛;;鱼藤酮建立帕金森病细胞模型的方法研究[J];中国神经免疫学和神经病学杂志;2015年06期
2 竺飞燕;张雄;王百辰;胡智伟;;鱼藤酮诱导PC12细胞凋亡及线粒体膜电位变化[J];中国药理学通报;2014年02期
3 赵江浩;赵斌;;细胞外α-突触核蛋白的特点及其与帕金森病的关系[J];医学理论与实践;2012年18期
4 王国恩;翟羽佳;何蓉蓉;栗原博;;SirT3调节氧化应激作用[J];中国药理学通报;2012年10期
5 赵素玲;王洪新;周振华;喻晓春;鲁美丽;宋莹;张晶;;黄芪多糖对异丙肾上腺素诱导的乳大鼠心肌细胞肥大的保护作用[J];中国药理学通报;2011年12期
6 王少君;罗琴;王加才;万金城;张江伟;李增潮;张洵岳;韩霞;彭国光;;α-synuclein表达增高与免疫炎性反应在帕金森病发病机制中的作用[J];中国老年学杂志;2011年01期
7 崔群力;孙圣刚;;姜黄素通过抗氧化作用拮抗鱼藤酮致PC12细胞损伤的研究[J];华中科技大学学报(医学版);2010年01期
8 余美荣;蒋福升;丁志山;;姜黄素的研究进展[J];中草药;2009年05期
9 单悌超;任玉娇;于学慧;袁彦平;;帕金森病模型研究进展[J];中国老年学杂志;2009年09期
10 Stig Bengmark;刘青;;植物源保护剂姜黄素的研究进展[J];现代药物与临床;2009年01期
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