马钱苷对MPTP诱导帕金森病小鼠模型的神经保护作用
发布时间：2018-09-07 16:46
【摘要】：帕金森病(Parkinson’s disease,PD)是仅次于阿尔茨海默疾病的第二大神经退行性疾病,主要临床表现为静止性震颤,行动迟缓为主的运动障碍。目前,对于帕金森病病因的了解并不完全清楚,较多的研究是从环境、基因和衰老方面对其致病原因进行机制探讨,比如炎症、氧化应激,内质网应激,自噬等方面。然而对其治疗手段研究的进展较为缓慢,且关于治疗方面的探索力度仍不够,很多具有潜在作用的单体或单体群有待开发,包括天然产物里面的单体及单体群。马钱苷(Loganin,Log)是众多化合物中的一种,为山茱萸(Cornus officinalis sieb.et zuce.)的主要成分之一,已有文献报道马钱苷可调节大鼠机体免疫反应;可减轻氧化应激损伤;并具有神经营养作用,从而提高细胞的生存能力;改善糖尿病大鼠空间记忆能力。在这里我们选取8周龄左右雄性C57小鼠为实验动物,采用1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)诱导的小鼠帕金森病模型;将给药组分为马钱苷预防给药组(Log+MPTP)和治疗给药组(MPTP+Log),并设置预防对照组(Saline+MPTP)、治疗对照组(MPTP+Saline)和空白对照组(Saline)。给药结束后用爬杆试验进行行为学检测;用高效液相色谱法(HPLC)检测小鼠纹状体内多巴胺和3,4-二羟基苯乙酸(DOPAC)的含量变化;同时用western blot、ELISA检测纹状体内炎症因子caspase-3、TNF-α,上游调控因子NFκB,p38、c-Abl,以及与自噬相关的LC3-II、Drp1表达变化;并结合免疫荧光染色法检测胶质细胞和caspase-3、c-Abl、LC3-II脑内纹状体与黑质致密部的分布情况;同时采用PC12细胞建立PD模型后给药,用吖啶橙染色后观察酸性自噬泡的水平,以评价马钱苷对自噬的作用情况。实验结果表明,马钱苷治疗给药组可抑制MPTP诱导的PD小鼠纹状体内多巴胺和TH表达的下降和多巴胺能神经元的丢失,且爬杆总运动时间(TLA)下降;但马钱苷预防给药组未见差异。DOPAC的含量在各组间未见差异。经过马钱苷给药后,小鼠黑质内小胶质细胞和星形胶质细胞的活性较其相应对照组有所抑制;caspase-3、TNF-α及其上游调控因子NFκB,p38和c-Abl的表达较其相应对照组均有下降其。提示马钱苷可能通过抑制炎症及c-Abl-p38-NFκB信号通路实现保护作用。LC3-II,Drp1表达在马钱苷给药后较其对照组也有下降。且细胞实验显示马钱苷给药组与其相应的对照组相比,细胞内酸性自噬泡水平有所下降,提示马钱苷也可能对细胞自噬产生影响。以上研究表明,马钱苷对MPTP诱导的PD小鼠模型具有神经保护的作用,可能是通过抑制胶质细胞的聚集及激活、抑制炎症因子的表达、并可能通过c-Abl-p38-NFκB通路来抑制细胞死亡,从而改善行为学表现。同时马钱苷也有可能通过影响细胞自噬来增强细胞的生存能力。这些结果为临床治疗帕金森病提供了新的思路。
[Abstract]:Parkinson's disease (Parkinson's disease,PD) is the second most common neurodegenerative disease after Alzheimer's disease. At present, the etiology of Parkinson's disease is not fully understood. Many researches have been done on the pathogenesis of Parkinson's disease from the aspects of environment, gene and aging, such as inflammation, oxidative stress, endoplasmic reticulum stress, autophagy and so on. However, the research on the therapeutic methods is slow, and the research on the treatment is not enough. Many potential monomers or groups of monomers, including those in natural products, need to be developed. Loganin,Log is one of a variety of compounds, which is the (Cornus officinalis sieb.et zuce. of Cornus officinalis. One of the main components has been reported in the literature can regulate the immune response of rats, reduce oxidative stress injury, and neurotrophic effect, thereby improve the viability of cells, improve the ability of spatial memory in diabetic rats. In this paper, we selected male C57 mice about 8 weeks old as experimental animals and used 1-methyl-4-phenyl-1-trihydropyridine 6-tetrahydropyridine (MPTP) to induce the model of Parkinson's disease in mice. The drug groups were divided into two groups: (Log MPTP) in the preventive administration group and (MPTP Log), in the treatment group and (Saline MPTP), in the preventive control group and (Saline). In the blank control group. At the end of administration, the behavioral tests were carried out by climbing pole test, and the contents of dopamine and (DOPAC) in the striatum of mice were detected by HPLC (HPLC), and the changes of dopamine and 3-dihydroxyphenylacetate (DOPAC) in the striatum of mice were determined by HPLC. At the same time, western blot,ELISA was used to detect the expression of inflammatory factor caspase-3,TNF- 伪, upstream regulatory factor NF 魏 B, p38 c-Abland the expression of LC3-II,Drp1 associated with autophagy, and the distribution of glial cells and the substantia nigra compact region in caspase-3,c-Abl,LC3-II brain was detected by immunofluorescence staining. At the same time, PC12 cells were used to establish the PD model and the acridine orange staining was used to observe the level of acidic autophagy to evaluate the effect of marganin on autophagy. The results showed that the treatment group could inhibit the decrease of dopamine and TH expression and the loss of dopaminergic neurons in the striatum of PD mice induced by MPTP, and the decrease of total climbing time (TLA). However, there was no difference in the content of DOPAC between the two groups. The activities of microglia and astrocytes in the substantia nigra of mice were inhibited by the administration of marganin, and the expression of caspase-3 tNF- 伪 and its upstream regulatory factor, NF 魏 B, p38 and c-Abl, decreased compared with those of the corresponding control group. These results suggest that the expression of rhiganoside may be inhibited by inhibiting inflammation and c-Abl-p38-NF 魏 B signaling pathway. The expression of LC3-IIp1 may also decrease compared with that of the control group. Compared with the control group, the intracellular acidic autophagy level in the group treated with marganin decreased, which suggested that it might also have an effect on autophagy. These results suggest that the PD mice model induced by MPTP has neuroprotective effect of marganin, which may inhibit the accumulation and activation of glial cells, inhibit the expression of inflammatory factors, and inhibit cell death through the c-Abl-p38-NF 魏 B pathway. In order to improve behavioral performance. At the same time, it is possible to increase the viability of the cells by affecting autophagy. These results provide new ideas for clinical treatment of Parkinson's disease.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5;R-332
[Abstract]:Parkinson's disease (Parkinson's disease,PD) is the second most common neurodegenerative disease after Alzheimer's disease. At present, the etiology of Parkinson's disease is not fully understood. Many researches have been done on the pathogenesis of Parkinson's disease from the aspects of environment, gene and aging, such as inflammation, oxidative stress, endoplasmic reticulum stress, autophagy and so on. However, the research on the therapeutic methods is slow, and the research on the treatment is not enough. Many potential monomers or groups of monomers, including those in natural products, need to be developed. Loganin,Log is one of a variety of compounds, which is the (Cornus officinalis sieb.et zuce. of Cornus officinalis. One of the main components has been reported in the literature can regulate the immune response of rats, reduce oxidative stress injury, and neurotrophic effect, thereby improve the viability of cells, improve the ability of spatial memory in diabetic rats. In this paper, we selected male C57 mice about 8 weeks old as experimental animals and used 1-methyl-4-phenyl-1-trihydropyridine 6-tetrahydropyridine (MPTP) to induce the model of Parkinson's disease in mice. The drug groups were divided into two groups: (Log MPTP) in the preventive administration group and (MPTP Log), in the treatment group and (Saline MPTP), in the preventive control group and (Saline). In the blank control group. At the end of administration, the behavioral tests were carried out by climbing pole test, and the contents of dopamine and (DOPAC) in the striatum of mice were detected by HPLC (HPLC), and the changes of dopamine and 3-dihydroxyphenylacetate (DOPAC) in the striatum of mice were determined by HPLC. At the same time, western blot,ELISA was used to detect the expression of inflammatory factor caspase-3,TNF- 伪, upstream regulatory factor NF 魏 B, p38 c-Abland the expression of LC3-II,Drp1 associated with autophagy, and the distribution of glial cells and the substantia nigra compact region in caspase-3,c-Abl,LC3-II brain was detected by immunofluorescence staining. At the same time, PC12 cells were used to establish the PD model and the acridine orange staining was used to observe the level of acidic autophagy to evaluate the effect of marganin on autophagy. The results showed that the treatment group could inhibit the decrease of dopamine and TH expression and the loss of dopaminergic neurons in the striatum of PD mice induced by MPTP, and the decrease of total climbing time (TLA). However, there was no difference in the content of DOPAC between the two groups. The activities of microglia and astrocytes in the substantia nigra of mice were inhibited by the administration of marganin, and the expression of caspase-3 tNF- 伪 and its upstream regulatory factor, NF 魏 B, p38 and c-Abl, decreased compared with those of the corresponding control group. These results suggest that the expression of rhiganoside may be inhibited by inhibiting inflammation and c-Abl-p38-NF 魏 B signaling pathway. The expression of LC3-IIp1 may also decrease compared with that of the control group. Compared with the control group, the intracellular acidic autophagy level in the group treated with marganin decreased, which suggested that it might also have an effect on autophagy. These results suggest that the PD mice model induced by MPTP has neuroprotective effect of marganin, which may inhibit the accumulation and activation of glial cells, inhibit the expression of inflammatory factors, and inhibit cell death through the c-Abl-p38-NF 魏 B pathway. In order to improve behavioral performance. At the same time, it is possible to increase the viability of the cells by affecting autophagy. These results provide new ideas for clinical treatment of Parkinson's disease.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5;R-332
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