瘦素对帕金森病神经损伤的保护和机制初探

发布时间：2018-06-19 00:26

本文选题：瘦素 + 帕金森病　；参考：《中国人民解放军医学院》2014年硕士论文

【摘要】：帕金森病（Parkinson’s diseases, PD）的病理变化为中脑黑质区多巴胺能神经元变性、死亡、缺失，黑质-纹状体多巴胺能系统功能减退，并在残存神经元呈现α-突触核蛋白（α-synuclein）和泛素染色阳性的胞浆包涵体体形成。因此寻找有效的神经保护因子，对帕金森病的发病进程进行干预，进而减轻神经损伤、恢复神经功能，这是目前研究关注的热点。瘦素（Leptin）作为一种能量代谢调节因子，在炎症反应和创伤修复方面也具有重要功能。最近有关其神经保护作用的研究越来越多，但对其作用机制的研究还知之甚少，，尤其是在帕金森病的研究领域。为了解瘦素在帕金森病中的作用及其病理生理机制，本研究拟通过帕金森病患者的血样分析，探讨瘦素对帕金森病的关联性，并建立6-OHDA诱导C57BL/6J小鼠的帕金森动物模型，6-羟基多巴胺（6-hydroxydopamine,6-OHDA）和鱼藤酮（Rotenone）诱导多巴胺能样细胞系SH-SY5Y细胞的帕金森病细胞模型来探讨瘦素对帕金森病神经损伤的保护以及机制。本课题包括以下三个部分工作: 第一部分帕金森病患者血清瘦素及其受体，以及氧化应激的变化目的：探讨血清中的leptin和可溶性瘦素受体（Soluble leptin receptor, sObR）水平，以及氧化和抗氧化指标与帕金森病的关系性；方法：1.检测健康人群（年龄范围平均）人的leptin和sObR水平；2.检测PD患者和对照组（主要老年群体）中的leptin和sObR水平，检测两个群体中的丙二醛（Malondialdehyde, MDA）、超氧化物歧化酶（Superoxide dismutase, SOD）和还原型谷胱甘肽（Glutathione, GSH）的水平；3.分析leptin和sObR水平与帕金森病的相关性，以及与年龄的相关性，与男女性别的相关性；4.分析MDA、GSH以及SOD的水平与帕金森的相关性，以及和leptin与sObR的相关性。结果：1.随着年龄的增加，leptin的水平有所增加但是在40岁左右稳定，sObR变化不大；2.帕金森病患者相对于对照组（老年群体），MDA和sObR水平有所增加，但leptin、SOD和GSH水平有所下降（P0.05）；3.女性的leptin水平高于男性，sObR水平低于男性（P0.05）；4. GSH和SOD与leptin水平呈正相关，与sObR呈负相关；MDA与leptin水平呈负相关，与sObR呈正相关。结论：1.帕金森患者中，血清中氧化应激水平高，leptin水平低，sObR水平高；2.保持较高的血清leptin水平，有降低患帕金森病风险的可能性。第二部分瘦素对帕金森病动物模型黑质区域的保护目的：在建立的PD动物模型基础上，观察leptin对于黑质区域损伤的保护，以及下游氧化应激水平的变化。方法：①建立6-OHDA诱导的右侧损伤的PD动物模型，假手术组（对照组），模型组，模型+leptin组，模型+leptin+nanobody组，通过皮下注射阿扑吗啡，通过向左侧旋转次数鉴定动物模型是否建立成功；②免疫组化检测各组中脑黑质区多巴胺能神经元特异性蛋白酪氨酸羟化酶的变化；③收集各组血清，检测MDA、GSH和SOD的变化。结果：1.阿扑吗啡皮下注射后，模型组相对于对照组，左侧旋转次数显著增加，模型+leptin组旋转次数减少，加入nanobody后，旋转次数右显著增加；2.模型组及模型+leptin+nanobody组右侧黑质区域多巴胺能相对于左侧明显减少，模型+leptin组右侧黑质区域多巴胺能相对于左侧有所减少，但较模型组和加入nanobody组有所增加。3.各组血清中，模型组相对于对照组MDA有所增加，GSH和SOD有所减少，模型+leptin组相对于模型组和leptin+nanobody组，MDA有所减少，GSH和SOD有所增加。结论:1.外源性Leptin能够减少PD的严重程度，能够减少多巴胺能神经元的丢失；2. Leptin在PD动物模型中，可显著降低血清中MDA水平，升高SOD和GSH水平,在阻断脂质过氧化,增加抗氧化性，维持机体氧化平衡的稳定具有积极作用。第三部分瘦素减轻6-羟基多巴胺和鱼藤酮诱导的SH-SY5Y细胞损伤的机制研究目的:通过6-OHDA和rotenone诱导SH-SY5Y的帕金森病细胞模型，对leptin的神经保护作用及机制进行探讨。方法:6-OHDA和rotenone去诱导SH-SY5Y。①CCK-8和LDH分别检测不同浓度6-OHDA和rotenone诱导SH-SY5Y细胞的存活率和损伤率;②CCK-8、LDH、台盼蓝和流式细胞术分别检测leptin对于100μmol/L6-OHDA和5μmol/Lrotenone诱导SH-SY5Y细胞的存活、损伤、死亡以及凋亡的影响，以及其受体阻断剂nanobody的影响。③荧光探针检测，leptin对于6-OHDA和rotenone损伤导致ATP和ROS变化的影响，以及，免疫荧光和Westernblot检测和cleaved caspase-3的变化。④Nanobody加入后，检测相关细胞裂解液中的SOD、MDA和GSH这些氧化性和抗氧化性指标的变化。⑤CCK-8检测，AG490、nanobody、LY294002、compound C、U0126、ZnPP，对于leptin对于6-OHDA和rotenone损伤减轻的影响，并比较LiCl和CoPP对细胞的保护。⑥Leptin模型保护加入LY294002、compound C和U0126后，Western blot检测凋亡相关蛋白Bcl-2、Bax，以及相关通路分子p-Akt，p-ERK1/2，p-AMPK，p-GSK3β，HO-1，Western blot及RT-PCR检测帕金森相关蛋白，RT-PCR检测α-synuclein的mRNA水平。Westernblot检测LiCl和CoPP对于模型组Bcl-2、Bax以及HO-1的表达影响。结果:1. Leptin提高了SH-SY5Y细胞的存活率，降低了损伤率及凋亡率；2.相应的阻断剂加入后，leptin的保护作用均有所减弱；3. Leptin提高了ATP的增加，降低了ROS的水平。4. Nanobody加入，逆转了leptin引起的MDA的降低，SOD和GSH的升高；5. Leptin提高了Bcl-2和HO-1的表达，降低了cleaved caspase-3、Bax和α-synuclein的表达，并且提高了p-Akt、p-ERK1/2、p-AMPK以及p-GSK3β的水平，阻断剂的加入部分逆转了leptin的影响，相应蛋白的阻断剂都降低了p-GSK3β的水平，并降低了leptin提高的Bcl-2和HO-1的表达，以及提高leptin降低的Bax水平。结论：1.在建立帕金森的细胞模型基础上，leptin减轻了损伤；2. Leptin对于细胞模型的保护，分别通过Akt、ERK以及AMPK途径，汇聚于GSK-3β，进而参与下游的调节，其下游的调节保护，主要是提高HO-1降低ROS和MDA的水平，提高SOD和GSH的水平以及能量ATP的水平，并下调凋亡的水平，进而降低损伤。
[Abstract]:The pathological changes of Parkinson's disease (Parkinson 's diseases, PD) are the degeneration, death, deletion, degeneration of the nigrostriatal dopaminergic system in the mesencephalic substantia nigra, and the formation of an alpha synuclein (alpha -synuclein) and a cytoplasmic inclusion body with positive ubiquitin staining in the remaining neurons. Protective factors, which intervene in the pathogenesis of Parkinson's disease, reduce nerve injury and restore nerve function, are the focus of current research.
Leptin (Leptin), as an energy metabolic regulator, also plays an important role in inflammatory response and wound repair. Recently, more and more studies have been made on its neuroprotective effects, but little is known about its mechanism of action, especially in the field of Parkinson's disease. To understand the role of leptin in Parkinson's disease and to understand the role of leptin in Parkinson's disease, Pathophysiological mechanism, this study intends to explore the relevance of leptin to Parkinson's disease by analysis of blood samples from patients with Parkinson's disease, and to establish a Parkinson animal model of C57BL/6J mice induced by 6-OHDA, 6- hydroxyl dopamine (6-hydroxydopamine, 6-OHDA) and rotenone (Rotenone) to induce the Parkinson disease of the dopaminergic cell line SH-SY5Y cells. Cell model is used to investigate the protection and mechanism of leptin on Parkinson's disease.
This topic includes the following three parts:
Part 1 Changes of serum leptin and its receptors and oxidative stress in patients with Parkinson's disease
Objective: To investigate the levels of leptin and Soluble leptin receptor (sObR) in serum and the relationship between oxidation and antioxidant indicators and Parkinson's disease. Methods: 1. to detect leptin and sObR levels in healthy people (average age range); and 2. to detect leptin and sObR in PD patients and control groups (the main elderly group). Levels, the levels of Malondialdehyde, MDA, Superoxide dismutase, SOD and Glutathione, GSH in two groups; 3. analysis of the correlation between leptin and sObR levels and Parkinson's disease, the correlation with age, the correlation with sex and sex; 4. analysis of MDA, GSH, and The correlation between the level of SOD and Parkinson, as well as the correlation with leptin and sObR. Results: 1. with the increase of age, the level of leptin increased but the level of sObR was stable at the age of 40. The levels of MDA and sObR in 2. Parkinson's patients were increased relative to the control group (old group), but leptin, SOD and GSH decreased (P). 0.05): the level of leptin in 3. women was higher than that of men, and the level of sObR was lower than that of men (P0.05); 4. GSH and SOD were positively correlated with leptin, and negatively correlated with sObR; MDA and leptin were negatively correlated with sObR. Conclusion: in 1. Parkinson patients, the level of oxidative stress was high, leptin was low, sObR level was high, and 2. remained high. Serum leptin level has the potential to reduce the risk of Parkinson's disease.
The second part of leptin protects the substantia nigra in Parkinson's disease animal models.
Objective: on the basis of the established PD animal model, we observed the protection of leptin in the damage of the substantia nigra and the changes in the level of oxidative stress in the lower reaches. Methods: (1) the establishment of a PD animal model of the right injury induced by 6-OHDA, the sham operation group (control group), the model group, the model +leptin group, the model +leptin+nanobody group, and the subcutaneous injection of opiapine. The changes of dopaminergic neuron specific tyrosine hydroxylase of dopaminergic neurons in the substantia nigra of each group were detected by immunohistochemistry. (3) the changes in MDA, GSH and SOD were detected in each group of serum. Results: after subcutaneous injection of 1. apomorphine, the model group was compared with the control group, left The number of lateral rotation increased significantly, and the number of rotation in model +leptin group decreased. After adding nanobody, the number of rotations increased significantly. 2. model group and model +leptin+nanobody group were significantly reduced to the left side of the right substantia nigra compared to the left side, and the dopamine in the right substantia nigra region of model +leptin group decreased compared with the left side, but the model group was more than the model group. And the addition of nanobody group increased the serum of.3. in each group, the MDA of model group was increased compared with the control group, GSH and SOD decreased. The MDA decreased in model +leptin group and leptin+nanobody group, and GSH and SOD increased. Conclusion: 1. exogenous Leptin can reduce the severity of PD, and can reduce dopaminergic neurons. The loss of 2. Leptin in the PD animal model can significantly reduce the level of MDA in the serum, increase the level of SOD and GSH, and play an active role in blocking lipid peroxidation, increasing antioxidant activity, and maintaining the stability of the body's oxidative balance.
The third part is the mechanism of leptin alleviated 6- hydroxydopamine and rotenone induced SH-SY5Y cell injury.
Objective: to induce the Parkinson's disease cell model of SH-SY5Y by 6-OHDA and rotenone, the neuroprotective effect and mechanism of leptin were discussed. Methods: 6-OHDA and rotenone were used to induce SH-SY5Y. (CCK-8 and LDH) to detect the survival rate and damage rate of SH-SY5Y cells with different concentrations of 6-OHDA and rotenone, respectively. The effects of leptin on the survival, damage, death and apoptosis of SH-SY5Y cells induced by 100 and 5 mu mol/Lrotenone, and the effect of its receptor blocker nanobody. (3) fluorescence probe detection, the effect of leptin on ATP and ROS changes caused by 6-OHDA and rotenone damage, as well as immunofluorescence and Westernblot detection And cleaved caspase-3 changes. (4) after Nanobody, the changes in the oxidative and antioxidant properties of SOD, MDA and GSH in the related cell lysates were detected. 5. CCK-8 detection, AG490, nanobody, LY294002, compound C. 6. After the Leptin model is protected by LY294002, compound C and U0126, Western blot is used to detect apoptosis related proteins Bcl-2, Bax, and the associated pathway molecules p-Akt, p-ERK1/2, p-AMPK, and the detection of Parkinson related proteins. 2, Bax and HO-1 expression. Results: 1. Leptin increased the survival rate of SH-SY5Y cells and reduced the rate of damage and apoptosis; 2. the protective effect of leptin was weakened after the addition of the corresponding blockers; 3. Leptin increased the increase of ATP, reduced the.4. Nanobody addition of ROS, and reversed the MDA reduction caused by leptin. The increase of SH; 5. Leptin increased the expression of Bcl-2 and HO-1, reduced the expression of cleaved Caspase-3, Bax and alpha -synuclein, and increased the level of p-Akt, p-ERK1/2, p-AMPK and p-GSK3 beta. The expression of Bcl-2 and HO-1, as well as the Bax level reduced by leptin. Conclusion: 1. on the basis of the establishment of Parkinson's cell model, leptin reduces the damage; 2. Leptin protects the cell model by Akt, ERK and AMPK pathway, which converges to GSK-3 beta, and then participates in the regulation of the lower reaches, and the downstream regulation and protection is mainly to improve HO-1. Reduce the level of ROS and MDA, increase the level of SOD and GSH, and the level of energy ATP, and down regulate the level of apoptosis, thereby reducing the injury.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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