二苯乙烯苷通过抑制ROS相关的线粒体和JNK,P38通路对MPTP诱导的帕金森病小鼠模型的神经保护作用

发布时间：2018-07-31 14:18

【摘要】：帕金森病（Parkinson’s disease，PD）是一种以静止性震颤，肌僵直，动作迟缓和姿势反射障碍为主要临床表现的中老年人群中常见的中枢神经系统退行性疾病。其病理特点是中脑炓质致密部(substantia nigra pars compact，SNpc)多巴胺（dopamine，DA）能神经元变性缺失。目前的药物治疗并不能阻止或延缓PD的进程，仅能缓解症状，且长期治疗会出现疗效递减，产生很多副作用。研究表明，PD发病的重要因素有可能是氧化应激或者线粒体功能障碍。还有研究表明MAPK通路在PD的发病中起重要作用。阻断JNK和P38的激活可以抑制MPTP导致的神经毒性。 1-甲基-4苯基-1，2，3，6-四氢吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydropyrid-ine，MPTP）是经典的PD造模药物之一。其代谢产物1-甲基-4苯基吡啶离子（1-methy-4-phenylpyridinium，MPP+）是诱发神经毒性的主要物质。在DA能神经元内，MPP+可以通过抑制线粒体复合物I破坏电子呼吸链，从而产生大量ROS，进一步导致黑质DA能神经元缺失。何首乌的根是一种传统的中草药，具有抗衰老的作用。二苯乙烯苷（2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside，TSG）是其主要活性成分之一。具有很多生物学特性，例如：抗氧化、抗炎、抗动脉硬化等作用。研究发现TSG不仅可以通过抑制ROS保护脑组织，并且可以阻断JNK激活保护大脑不受缺血的损伤，TSG可以降低α突触核蛋白（α-synuclein，α-syn）的过度表达（APP转基因阿尔茨海默病小鼠模型）。还可抑制MPP+对SH-SY5Y细胞的神经毒性作用。我们课题组近期的实验表明，TSG可以通过阻断ROS-NO相关的JNK通路抑制MPP+诱导的PC12细胞凋亡。为了进一步研究TSG在体内的神经保护作用，本次实验的目的如下：1）利用PD小鼠模型，验证TSG的神经保护作用；2）测定这种神经保护作用是否与ROS介导的JNK，P38以及线粒体通路有关。【目的】 1.建立MPTP诱导的PD小鼠模型。 2.观察TSG对MPTP诱导的PD小鼠行为学、形态学及生化方面的影响。 3.探讨TSG是否通过调节ROS介导的JNK，P38和线粒体通路，发挥对MPTP诱导的PD小鼠的神经保护作用。【方法】 1.动物分组与造模：将实验动物随机分为7组，每组10只。A组：正常组，连续7d腹腔注射（i.p.）生理盐水0.1ml/次，1h后给予等量的生理盐水灌胃（i.g.），1次/d，随后的7d，给予生理盐水（i.g.），1次/d；B组：MPTP模型组，连续7d（i.p.）MPTP （生理盐水溶）30mg/kg，1h后给予等量的生理盐水（i.g.），1次/d，随后的7d，给予生理盐水（i.g.），1次/d；C、D组为TSG治疗组，给予MPTP（i.p.）30mg/kg，1h后分别给予TSG（纯度98%）（i.g.）20mg/kg或40mg/kg，1次/d，连续7d，随后的7d停止MPTP注射，只给予TSG（i.g.）20mg/kg或40mg/kg；E、F组，连续7d给予生理盐水（i.p.）0.1ml/次，1h后分别给予TSG（i.g.）20mg/kg或40mg/kg，1次/d，随后的7d只给予TSG（i.g.）20mg/kg或40mg/kg；G组，NAC阻断剂组，给予MPTP（i.p.）30mg/kg，1h后给予NAC（i.g.）200μM/kg，1次/d，连续7d，随后7d停止注射MPTP，只给予NAC200μM/kg （i.g.）。完成给药后1天，利用爬杆和旷场实验，衡量行为学变化。 2.完成行为学检测后，灌注小鼠，迅速取脑，并制作冰冻切片，用免疫荧光染色法检测酪氨酸羟化酶(TH)阳性神经元数目的变化。 3.全波段自动酶标仪检测ROS水平。 4.高效液相色谱法检测纹状体中DA、二羟基苯乙酸（DOPAC）和高香草酸（HVA）的含量。 5. Western Blot检测JNK, p-JNK, P38, p-P38, ERK, p-ERK, bcl-2, bax, cyt c, smac,cleaved-caspase-3, cleaved-caspase-6, cleaved-caspase-9蛋白的表达。【结果】 1.行为学结果显示：在爬杆实验中，给予小鼠MPTP后，与空白对照组比较，爬杆时间（TLa）显著升高（P 0.01）。但在给予TSG治疗后，与模型组相比，TLa明显缩短，而40mg/kgTSG组改善的更为明显（P 0.01）。单纯给予TSG组与空白对照组相比，并没有显著差异（P＞0.05）。给予NAC后，TLa与模型组相比也有明显的改善（Fig.2a, P 0.01）。矿场试验中，与空白对照组比较小鼠的运动速度(V)在给予MPTP后显著降低（P 0.01），在中央停留时间(T)明显延长（Fig.2b, P 0.01），然而经过TSG治疗后，V恢复到4.367cm/s，T下降到11.00s，单独给予TSG组与空白对照组无明显差异（P＞0.05），在给予NAC治疗后，与MPTP组相比，V明显增加，T明显降低（Fig.2c, P 0.05）。 2.免疫荧光染色结果显示：黑质内TH阳性神经元的计数采用双盲法。在荧光显微镜下可观察到与空白对照组相比模型组TH阳性神经元减少幅度约为66.9%（P0.01），然而TSG处理后，TH阳性神经元呈剂量依赖性恢复。而单纯给予TSG组的TH阳性神经元与正常组比较无统计学差异（P＞0.05）。与高剂量组类似，给予NAC治疗后，TH阳性神经元显著升高(Fig.3, P＜0.05)。 3.高效液相结果显示，与空白对照组相比，MPTP处理后纹状体内DA水平下降了82%，DOPAC，HVA水平也相应下降到正常组的15%和19%，但给予TSG治疗后，DA，DOPAC, HVA水平逐渐升高，呈剂量依赖性。单独给予TSG的小鼠纹状体内DA，DOPAC，HVA水平与正常组相比并没有统计学差异（P＞0.05）。而NAC治疗后，DA，DOPAC，HVA水平也显著恢复(Fig.4, P＜0.05)。 4.全波段自动荧光酶标仪检测ROS结果显示，在MPTP处理组中，ROS水平与空白对照组相比，有了明显的升高(P0.01)。不同剂量的TSG (20和40mg/kg)或NAC治疗后，与模型组相比，ROS水平明显下降(P0.01)，且TSG呈剂量依赖性。单独给予TSG后，与正常组比较无统计学意义（P＞0.05）。这说明TSG的保护作用与NAC相似(Fig.5)。 5. Western Blot方法检测结果表明，与MPTP组相比，TSG（20和40mg/kg）干预组可以显著改善JNK,P38以及线粒体通路的激活，但对ERK通路并无影响。给予NAC治疗的小鼠与TSG的效果类似，也抑制了JNK，，P38以及线粒体通路的激活。【结论】 1. TSG对MPTP诱导的PD小鼠具有神经保护作用。 2. TSG可能通过抑制ROS介导的JNK, P38和线粒体通路发挥对MPTP诱导的PD小鼠模型的神经保护作用。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a common central nervous system degenerative disease in middle-aged and elderly people with static tremor, muscle stiffness, motion retardation, and postural reflex disorder. The pathological features of the middle cerebral cortex (substantia nigra pars compact, SNpc) dopamine (dopamine, DA) can be deity The current drug treatment does not prevent or delay the process of PD and can only alleviate the symptoms, and long-term treatment will result in diminishing effect and many side effects. Studies have shown that the important factors in the pathogenesis of PD may be oxidative stress or mitochondrial dysfunction. Further studies have shown that the MAPK pathway plays an important role in the pathogenesis of PD. Blocking the activation of JNK and P38 can inhibit MPTP induced neurotoxicity.
1- methyl -4 phenyl -1,2,3,6- four hydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyrid-ine, MPTP) is one of the classic PD modeling drugs. Its metabolite, 1- methyl -4 phenyl pyridine ion (1-methy-4-phenylpyridinium, MPP+), is the main substance to induce neurotoxicity. In DA neurons, MPP+ can inhibit the mitochondrial complex. Destroying the electron respiratory chain, resulting in a large number of ROS, further leading to the deletion of DA neurons in substantia nigra.
The root of Polygonum multiflorum is a traditional Chinese herbal medicine that has anti aging effect. Two 2,3,5,4 '-tetrahydroxystilbene-2-O- beta -D-glucoside (TSG) is one of its main active components. It has many biological characteristics, such as antioxidant, anti-inflammatory, and anti arteriosclerosis. It is found that TSG can not only inhibit ROS protection. Brain tissue, and can block JNK activation to protect the brain from ischemia injury, and TSG can reduce the overexpression of alpha synuclein (alpha -synuclein, alpha -syn) (APP transgenic Alzheimer's disease model). It also inhibits the neurotoxic effect of MPP+ on SH-SY5Y cells.
Recent experiments in our team showed that TSG could inhibit the apoptosis of PC12 cells induced by MPP+ by blocking the JNK pathway of the ROS-NO. In order to further study the neuroprotective effect of TSG in the body, the aim of this experiment is as follows: 1) use the PD mouse model to verify the neuroprotective effect of TSG; 2) determine whether this neuroprotective effect is possible. It is related to ROS mediated JNK, P38, and mitochondrial pathway.
[Objective]
1. the PD mouse model induced by MPTP was established.
2. to observe the effects of TSG on the behavior, morphology and biochemistry of MPTP induced PD mice.
3. to explore whether TSG regulates ROS mediated JNK, P38 and mitochondrial pathways, and plays a neuroprotective role in MPTP induced PD mice.
[method]
1. animal groups and model building: the experimental animals were randomly divided into 7 groups, 10.A groups in each group: normal group, continuous 7d intraperitoneal injection (i.p.) 0.1ml/ saline, equal amount of normal saline (i.g.), /d, 7d, 1 times / D, 7d, B group: MPTP model group, 1 consecutive (physiological saline solution), 1 H was given the same amount of physiological saline (i.g.), 1 times /d, followed by 7d, given physiological saline (i.g.), 1 times /d, C, D group as TSG treatment group, MPTP (i.p.) 30mg/kg. I.p. (i.p.) 0.1ml/ times, 1h after TSG (i.g.) 20mg/kg or 40mg/kg, /d, then 7d only TSG (i.g.) 20mg/kg. Poles and open field experiments to measure behavioral changes.
2. after completing behavioral tests, the mice were perfused, and the brains were quickly taken and frozen sections were made by immunofluorescence staining.
The number of tyrosine hydroxylase (TH) positive neurons was measured.
The level of ROS was detected by 3. full band automatic enzyme labeling instrument.
4. the contents of DA, two hydroxyphenylacetic acid (DOPAC) and high vanillic acid (HVA) in striatum were detected by HPLC.
5. Western Blot detection JNK, p-JNK, P38, p-P38, ERK, p-ERK, Bcl-2, Bax, Cyt C.
[results]
1. behavioral results showed that in the climbing pole experiment, after the mice were given MPTP, the climbing pole time (TLa) was significantly increased (P 0.01) compared with the blank control group. But after the treatment of TSG, the TLa was significantly shorter than the model group, while the 40mg/kgTSG group improved significantly (P 0.01). There was no significant difference between the group of TSG and the blank control group (P 0.01). P > 0.05). After NAC, TLa was also improved significantly compared with the model group (Fig.2a, P 0.01). In the field test, the speed of movement (V) of mice decreased significantly after MPTP (P 0.01) compared with the blank control group (P 0.01), and the central residence time (T) was significantly prolonged (Fig.2b, P 0.01). There was no significant difference between TSG group and blank control group (P > 0.05). After NAC treatment, compared with MPTP group, V increased and T decreased significantly (Fig. 2c, P 0.05).
2. the results of immunofluorescence staining showed that the count of TH positive neurons in the substantia nigra was double blind. The reduction of TH positive neurons in the model group was about 66.9% (P0.01) compared with the blank control group under the fluorescence microscope. However, after TSG treatment, the TH positive neurons were in a dose-dependent manner. The TH positive neurons in the TSG group were only given the TH positive nerve. Similar to the high dose group, TH positive neurons increased significantly after NAC treatment (Fig. 3, P < 0.05).
3. the results of high performance liquid phase showed that compared with the blank control group, the DA level in the striatum decreased by 82%, and the level of DOPAC and HVA decreased to 15% and 19% in the normal group, but the level of DA, DOPAC, HVA increased gradually and was dose-dependent after the treatment of TSG. The level of DA, DOPAC, HVA level of the mice given to TSG was compared with the normal group. There was no statistical difference (P > 0.05), but after treatment with NAC, the levels of DA, DOPAC and HVA also significantly recovered (Fig.4, P < 0.05).
The results of 4. full band autofluorescence enzyme labeled ROS showed that in the MPTP treatment group, the level of ROS was significantly higher than that in the blank control group (P0.01). After the treatment of TSG (20 and 40mg/kg) or NAC in different doses, the level of ROS decreased significantly (P0.01) compared with the model group, and TSG was dose-dependent. Statistical significance (P > 0.05) indicates that the protective effect of TSG is similar to that of NAC (Fig.5).
The results of 5. Western Blot assay showed that the TSG (20 and 40mg/kg) intervention group could significantly improve the activation of JNK, P38 and mitochondrial pathway, but had no effect on ERK pathway. The mice given NAC therapy were similar to TSG, but also inhibited the activation of JNK, P38, and mitochondrial pathways.
[Conclusion]
1. TSG has neuroprotective effect on MPTP induced PD mice.
2. TSG may play a neuroprotective role in MPTP-induced PD mice by inhibiting ROS-mediated JNK, P38 and mitochondrial pathways.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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