人参皂苷Rg3对鱼藤酮诱导的PD模型多巴胺能神经元的保护作用及其机制研究

发布时间：2018-04-11 23:07

  本文选题：帕金森病 + 人参皂苷Rg3　； 参考：《扬州大学》2013年硕士论文

【摘要】：帕金森病(Parkinson's disease,PD)是一种常见于中老年人的神经系统退行性疾病,其主要病理特征为黑质(Substantia Nigra,SN)致密部多巴胺(dopamine,DA)能神经元的进行性变性死亡和路易氏小体(Lewy body,LB)的形成,引起黑质纹状体通路中DA水平的降低,最终导致整个基底神经节环路功能的改变。患者大多表现为运动迟缓、肌肉僵直、震颤和步态不稳等。PD发病机制复杂,一般认为可能与氧化应激、线粒体损伤、神经免疫炎症和神经兴奋性毒性有关。 人参皂苷Rg3(ginsenoside Rg3,以下简称Rg3)是中国传统中药人参的有效成分,Rg3能预防和治疗癌症、改善心脏血管功能、抗血小板凝集、保护脑神经细胞和提高机体免疫力。本课题组前期研究表明,Rg3能减轻鱼藤酮纳米脂质载体(R-NLC)诱导的PD模型大鼠行为运动障碍,并能有效抑制黑质DA神经元凋亡。进一步研究发现,这一作用与Rg3减弱氧化应激反应、上调Bcl-2和Bax的比值以及抑制‘caspase通路激活有关。 作为上述工作的继续,本实验采用鱼藤酮诱导PC12细胞损伤模型,用R-NLC制备PD大鼠模型,从体内、体外两条途径,进一步确证人参皂苷Rg3对鱼藤酮诱导的多巴胺能神经元损伤的保护作用,着重探讨p38MAPK-iNOS-NO通路在其中的作用,以进一步探讨Rg3保护DA能神经元的分子生物学机制。主要结果如下： 1.人参皂苷Rg3对鱼藤酮诱导的PD模型DA能神经元的保护作用 方法：PCl2细胞分组：(1)对照组：0.1%DMSO;(2)模型组：1μM鱼藤酮；(3)1μM鱼藤酮+0.3125μRg3；(4)1μM鱼藤酮+0.625μM Rg3;(5)1μM鱼藤酮+1.25μM Rg3；(6)1μM鱼藤酮+2.5μM Rg3；(7)1μM鱼藤酮+5μM Rg3。Rg3溶液在鱼藤酮前4小时加入,用MTT法测定24h各组细胞OD值,计算细胞存活率,流式细胞术测定细胞凋亡率。 动物分组：(1)对照组：sc空白NLC+ig0.5%CMC-Na；(2)模型组：sc R-NLC+ig0.5%CMC-Na；(3)阳性药物组：sc R-NLC+ig司来吉兰(11mg/kg)；(4)Rg3低剂量组：sc R-NLC+igRg3(3mg/kg)；(5)Rg3中剂量组：sc R-NLC+igRg3(6mg/kg)；(6)Rg3高剂量组：sc R-NLC+igRg3(12mg/kg).大鼠皮下注射(sc)R-NLC,首剂量0.5mg/kg,第二次0.8mg/kg,此后每次1mg/kg,全程2天一次,共28天。其它药物提前3天灌胃给药,每天一次,共31天。末次注射给药后24h,分离大鼠黑质和纹状体。以评分法考察大鼠行为学表现、HE染色观察黑质细胞形态,高效液相-电化学法测定纹状体DA含量。 结果：(1)对PC12细胞损伤的影响：模型组OD值为0.340±0.007,细胞存活率为69.5%,0.3125、0.625、1.25、2.5、5μMRg3组OD值分别为0.354±0.011(P0.05),0.388±0.018(P0.01),0.423±0.009(P0.01),0.441±0.007(P0.01)和0.416±0.007(P0.01),细胞存活率分别为72.4%,79.3%,86.5%,90.3%和85.1%,与模型组比较有显著升高。 (2)对PC12细胞凋亡的影响：鱼藤酮使PC12细胞出现早期和晚期凋亡,总凋亡率达71.1%(P0.01)。1.25,2.5μMRg3预处理后,细胞凋亡率下降为47.9%和28.1%(P0.01)。 (3)对PD大鼠外观行为和DA能神经元的影响：①行为学评分：与模型组(3.83±3.13)评分比较,低、中、高剂量Rg3干预后,评分分别为1.57±0.98、1.57±0.98和1.43±1.13,呈显著性降低(P0.05)。②黑质HE染色：与对照组相比,模型组大鼠黑质神经元出现核固缩,胞体缩小,胞内尼氏体明显减少；Rg3干预后能明显改善上述变化。③纹状体DA含量：模型组纹状体DA含量为0.57±0.11nmol/g,较对照组(14.15±3.68nmol/g)显著降低(P0.01)。低、中、高剂量Rg3组DA含量分别为1.38±0.79nmol/g (P0.05)、1.15±0.34nmol/g (P0.01)和9.49±6.93nmol/g (P0.01),与模型组比较,呈显著升高。 结论：人参皂苷Rg3可抑制体内、外鱼藤酮诱导的PD模型DA能神经元损伤,并能抑制鱼藤酮诱导的PC12细胞凋亡。 2.人参皂苷Rg3对鱼藤酮诱导的PD模型DA能神经元iNOS活力和NO含量的影响 方法：PC12细胞分组：(1)对照组：0.1%DMSO;(2)模型组：1μM鱼藤酮；(3)1μM鱼藤酮+1.25μM Rg3；(4)1μM鱼藤酮+2.5μM Rg3；大鼠分组和给药方法同第一部分。比色法测定细胞和大鼠黑质iNOS活力和NO含量。 结果：(1)细胞iNOS活力和NO含量：模型组iNOS活力和NO含量分别为0.692±0.030U/mL和7.67±1.45μM,与对照组(0.431±0.051U/mL和2.90±0.53μM)比较,呈显著升高(P0.01)；1.25、2.51μM Rg3组iNOS活力分别为0.636±0.031U/mL和0.643±0.009U/mL,NO含量分别为4.84±1.20μM和4.91±1.72μM,与模型组比较,呈显著下降(P0.05或0.01)。 (2)大鼠黑质iNOS活力和NO含量：模型组黑质iNOS活力和NO含量分别为0.223±0.044U/mgprot和8.06±2.35μmol/gprot,与对照组(0.142±0.016U/mgprot和4.57±1.30μmol/gprot)比较,呈显著升高(P0.01)；低、中、高剂量Rg3组黑质iNOS活力分别为0.140±0.033U/mgprot,0.132±0.023U/mgprot和0.145±0.035U/mgprot,NO含量分别为5.26±1.18μmol/gprot,4.41±1.12μmol/gprot和5.40±1.03μmol/gprot,与模型组比较,呈显著下降(低、高剂量组P0.05,中剂量组P0.01)。 结论：人参皂苷Rg3能显著抑制体内、外鱼藤酮诱导的iNOS活力增加和NO产生,提示Rg3可能通过抑制NO的生成来保护DA能神经元。 3.人参皂苷Rg3对鱼藤酮诱导的PD模型DA能神经元p38MAPK与NO的关系研究 方法：(1)体外实验：分组：(1)对照组：0.1%DMSO；(2)模型组：1μM鱼藤酮；(3)1μM鱼藤酮+10μM SB203580; MTT法测定细胞存活率,比色法测定iNOS活力和NO含量。 (2)体内实验：大鼠分组和给药方法同第一部分。末次给药后24h,Western blot法测定黑质中p-p38MAPK蛋白表达；免疫组化法测定p-p38MAPK和iNOS的表达,并对其进行非线性相关分析。 结果：(1)p38MAPK抑制剂SB203580对细胞存活率、iNOS活力和NO含量的影响：SB203580组细胞存活率为81.0%,较模型组(69.0%)显著升高(P0.05)。模型组iNOS活力和NO含量分别为0.692±0.030U/mL和7.67±1.45μM, SB203580干预后,分别下降为0.634±0.014U/mL和5.27±1.66μM (P0.05)。提示鱼藤酮引起PD损伤的机制与p38MAPK有关,p38MAPK通路可能调控了iNOS活力和NO水平。 (2)对p-p38MAPK表达的影响：与对照组相比,模型组黑质中p-p38MAPK表达水平显著增强,Rg3预处理后,p-p38MAPK表达水平显著降低,提示Rg3抗PD大鼠黑质DA能神经元凋亡的作用机制可能与下调p-p38MAPK水平有关。 (3)p-p38MAPK和iNOS表达量的相关性分析：与对照组相比,模型组黑质中p-p38MAPK和iNOS阳性细胞百分数均有显著升高；Rg3预处理后,两种蛋白阳性细胞百分数都呈明显降低。对两种蛋白阳性细胞百分数进行相关性分析,表明p-p38MAPK与iNOS呈正相关。提示Rg3可能是通过抑制p38MAPK的磷酸化,从而减少iNOS的活化。 结论：Rg3可能是通过抑制p38MAPK的磷酸化,继而降低iNOS活化水平、减少NO生成,而产生抗PD作用的。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is one of the most common in the elderly neurodegenerative disease, the main pathological features of substantia nigra (Substantia Nigra, SN) compacta dopamine (dopamine, DA) neurons degeneration and death of Lewis corpuscles (Lewy body, LB) formed by reduce the level of DA in the nigrostriatal pathway, the basal ganglia circuits function change. Most patients showed muscle rigidity, bradykinesia, tremor and gait instability.PD pathogenesis is complex, generally considered possible mitochondrial oxidative stress, injury, inflammation and immune related neural excitotoxicity.
Ginsenoside Rg3 (ginsenoside Rg3, hereinafter referred to as Rg3) is the active ingredient of traditional Chinese medicine China ginseng, Rg3 can prevent and treat cancer, improve cardiovascular function, platelet aggregation, protect brain cells and improve immunity. Ourprevious studies showed that Rg3 can reduce the rotenone nanostructured lipid carrier (R-NLC) behavior of PD model rats induced dyskinesia, and can inhibit the apoptosis of substantia nigra DA neurons. Further studies showed that the effect of Rg3 and decreased oxidative stress, upregulation of Bcl-2 and the ratio of Bax and inhibit the activation of caspase. "
As a continuation of the above work, this experiment used rotenone induced PC12 cell injury model, model, PD rats were prepared with R-NLC from the body, in two ways, further confirmed the protective effect of ginsenoside Rg3 on neuronal injury induced by rotenone dopamine, focused on the p38MAPK-iNOS-NO pathway in the role in molecular biology mechanism to further explore the Rg3 protect DA neurons. The main results are as follows:
Protective effect of 1. ginsenoside Rg3 on DA neurons of rotenone induced PD model
Methods: PCl2 cell groups: (1) control group: 0.1%DMSO (2); model group: 1 M rotenone; (3) 1 M rotenone +0.3125 Rg3; (4) 1 M +0.625 M Rg3 rotenone; (5) 1 M +1.25 M Rg3 rotenone; (6) 1 M +2.5 M Rg3 rotenone; (7) 1 M +5 M rotenone solution Rg3.Rg3 to 4 hours before the determination of 24h in rotenone, each cell OD value by MTT method, the cell survival rate, cell apoptosis was detected by flow cytometry.
Animal grouping: (1) blank control group: SC NLC+ig0.5%CMC-Na (2); model group: SC R-NLC+ig0.5%CMC-Na; (3) positive drug group: SC R-NLC+ig selegiline (11mg/kg); (4) low dose Rg3 group: SC R-NLC+igRg3 (3mg/kg); (5) Rg3 middle dose group: SC R-NLC (+igRg3 6mg/kg (6) Rg3); high dose group: SC R-NLC+igRg3 (12mg/kg). Rats by subcutaneous injection of R-NLC (SC), the first dose of 0.5mg/kg, second 0.8mg/kg, then 1mg/kg every time, the entire 2 days time, a total of 28 days to 3 days in advance. Other drugs administered orally, once a day, a total of 31 day. The last injection after administration of 24h, the separation of the substantia nigra and striatum in rats. To score method to study the behavior of rats, to observe the morphology of nigral cells stained with HE, DA content in striatum was determined by HPLC.
Results: (1) effect on PC12 cell injury model: od group is 0.340 + 0.007, the survival rate of cells was 69.5% 0.3125,0.625,1.25,2.5,5, the OD value of MRg3 group were 0.354 + 0.011 (P0.05), 0.388 + 0.018, 0.423 + 0.009 (P0.01) (P0.01), 0.441 + 0.007 and 0.416 + 0.007 (P0.01) (P0.01), cell survival rates were 72.4%, 79.3%, 86.5%, 90.3% and 85.1%, compared with the model group increased significantly.
(2) the effect of rotenone on the apoptosis of PC12 cells: rotenone induced early and late apoptosis of PC12 cells, and the total apoptosis rate reached 71.1% (P0.01). After pretreatment with.1.25,2.5 MRg3, the apoptosis rate decreased to 47.9% and 28.1% (P0.01), respectively.
(3) influence on the appearance and behavior of PD rats and DA neurons: scores of praxiology: model group (3.83 + 3.13) score, low, high dose intervention of Rg3 scores were 1.57 + 0.98,1.57 + 0.98 and 1.43 + 1.13, was significantly decreased (P0.05). The substantia nigra HE staining: compared with the control group, the model group of rat substantia nigra neurons karyopyknosis, cell body shrinkage, cytoplasmic Nissl body reduced obviously; after Rg3 intervention can significantly improve the change. The content of striatal DA: model group DA in striatum was 0.57 + 0.11nmol/g, 14.15 + 3.68nmol/g (compared with the control group) decreased significantly (P0.01). The low and high dose of Rg3 group DA was 1.38 + 0.79nmol/g (P0.05), 0.34nmol/g (P0.01) 1.15 + and 9.49 + 6.93nmol/g (P0.01), compared with the model group was significantly increased.
Conclusion: ginsenoside Rg3 can inhibit the injury of DA neurons in PD model induced by rotenone and inhibit the apoptosis of PC12 cells induced by rotenone.
Effect of 2. ginsenoside Rg3 on iNOS activity and NO content of DA neurons in PD model induced by rotenone
Methods: PC12 cells were divided into: (1) control group: 0.1%DMSO; (2) model group: 1 M M rotenone; (3) 1 micron M rotenone +1.25 M M; (4) 1, M, rotenone +2.5, small M Rg3; rats were divided into groups and administrated with the first part.
Results: (1) iNOS activity and NO content of cells: iNOS activity and NO content in model group were 0.692 + 0.030U/mL and 7.67 + 1.45 M, and the control group (0.431 + 0.051U/mL and 2.90 + 0.53 M), was significantly increased (P0.01); 1.25,2.51 M group Rg3 iNOS activity were 0.636 + 0.031U/mL and 0.643 + 0.009U/mL, the content of NO was 4.84 + 1.20 M and 4.91 + 1.72 M, compared with the model group was significantly decreased (P0.05 or 0.01).
(2) the substantia nigra iNOS activity and NO content in rats: the substantia nigra iNOS activity and NO content were 0.223 + 0.044U/mgprot and 8.06 + 2.35 mol/gprot, and the control group (0.142 + 0.016U/mgprot and 4.57 + 1.30 mol/gprot), was significantly increased (P0.01); low, high dose Rg3 group of substantia nigra iNOS activity were 0.140 + 0.033U/mgprot, 0.132 + 0.023U/mgprot and 0.145 + 0.035U/mgprot, the content of NO was 5.26 + 1.18 mol/gprot 4.41 + 1.12 mol/gprot and 5.40 + 1.03 mol/gprot, compared with the model group was significantly decreased (low, high dose group, middle dose group P0.05, P0.01).
Conclusion: ginsenoside Rg3 can significantly inhibit rotenone induced iNOS activity and NO production, suggesting that Rg3 may protect DA neurons by inhibiting the generation of NO.
3. ginsenoside Rg3 on the relationship between p38MAPK and NO in DA neurons of rotenone induced PD model
Methods: (1) in vitro experiments: grouped: (1) control group: 0.1%DMSO; (2) model group: 1 M rotenone; (3) 1 M M rotenone +10 M SB203580; MTT method was used to measure cell viability, and iNOS activity and NO content were determined by colorimetry.
(2) in vivo experiments: the rats were divided into groups. The rats were divided into groups. The same method was given in the first part. After the last administration, the expression of p-p38MAPK protein in substantia nigra was detected by 24h Western blot method. The expression of p-p38MAPK and iNOS was detected by immunohistochemistry, and the correlation analysis between them was made.
Results: (1) p38MAPK inhibitor SB203580 on cell viability, iNOS activity and NO content: SB203580 group cell survival rate was 81%, compared with the model group (69%) was significantly increased (P0.05). INOS activity and NO content in model group were 0.692 + 0.030U/mL and 7.67 + 1.45 M and the intervention of SB203580 decreased to 0.634 + 0.014U/mL and 5.27 + 1.66 M (P0.05). It is suggested that rotenone induced PD damage mechanism associated with p38MAPK, p38MAPK pathway may regulate the activity of iNOS and NO levels.
(2) effects on the expression of p-p38MAPK: compared with the control group, model group, the expression level of p-p38MAPK in the substantia nigra was significantly enhanced after pretreatment with Rg3, the expression level of p-p38MAPK was significantly decreased, suggesting that Rg3 anti PD rat nigral DA neuron apoptosis mechanism may be related to the down-regulation of p-p38MAPK level.
(3) analysis the correlation between the expression of p-p38MAPK and iNOS: compared with the control group, the model group in the substantia nigra of p-p38MAPK and iNOS positive cell percentage increased significantly; after Rg3 pretreatment, two protein positive cell percentage were significantly reduced. The correlation analysis of two protein positive cells showed a positive percentage, p-p38MAPK iNOS. Suggesting that Rg3 may be through inhibiting the phosphorylation of p38MAPK, thereby reducing the activation of iNOS.
Conclusion: Rg3 may inhibit the phosphorylation of p38MAPK, then reduce the activation level of iNOS, reduce the formation of NO, and produce anti PD effect.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R-332;R285.5

【参考文献】

相关期刊论文 前10条

1 梅少林;袁红艳;曹湘博;常雅萍;李平亚;刘金萍;;人参皂苷Rg3对HSE模型鼠的治疗作用及免疫调节效应[J];吉林大学学报(医学版);2007年01期

2 马晓凯;范凯;王滨;;一氧化氮与帕金森病[J];大连医科大学学报;2007年01期

3 李云鹏,董兆君,王仕丽,蔡颖,李奇慧,乐卫东;重复注射鱼藤酮对中枢多巴胺神经元的毒性作用[J];第三军医大学学报;2003年13期

4 赵黔鲁,柴锡庆;鱼藤酮致PC12细胞线粒体功能及超微结构的影响[J];第三军医大学学报;2005年07期

5 董兆君;鱼藤酮的多巴胺神经元毒性与帕金森病[J];国外医学.神经病学神经外科学分册;2001年05期

6 胡丹;孙圣刚;;鱼藤酮对大鼠嗜铬细胞瘤细胞PC12的毒性作用[J];神经损伤与功能重建;2008年05期

7 王铭维,孙秀巧,顾平,王彦永,刘力,崔冬生;鱼藤酮对PC12细胞的毒性作用[J];环境与健康杂志;2005年06期

8 杨海东;姜宏;宋宁;王俊;谢俊霞;;人参皂苷Rg_1对MPTP致帕金森病模型小鼠多巴胺能神经元保护作用研究[J];解放军药学学报;2007年01期

9 邓宝;王彦永;王铭维;;鱼藤酮与帕金森病动物模型[J];脑与神经疾病杂志;2006年02期

10 黄晓峰;尹文;王文勇;;鱼藤酮诱导的多巴胺能神经细胞死亡与免疫[J];细胞与分子免疫学杂志;2010年10期

，

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