FJ1的抗炎症和抗帕金森症作用及机制研究

发布时间：2018-05-01 00:06

本文选题：FJ1 + 炎症　；参考：《山东大学》2014年硕士论文

【摘要】：3β-当归酰氧基-8p,10p-二羟基佛术烯-7(11)-烯-12,8α-内酯(FJ1)是从传统中药大吴风草中提取的活性化合物。大吴风草为多年生草本菊科植物,其药用全株叫做莲蓬草,主要分布于中国的东南部和南部、日本、韩国[1],对于例如感冒、咳嗽、炎症等多种疾病均有良好疗效,因此推测其活性提取物具有抗炎症作用。课题中研究的FJ1是本实验室由大吴风草中提取的多种活性产物经筛选后确定的考察对象。炎症是机体对由微生物感染和其他伤害性刺激造成的组织损伤的响应,是一种十分常见同时也非常重要的防御机制,然而,低分辨率和不受控制的炎症反应会导致一种与包括癌症在内的多种人类疾病有关的慢性炎症状态。本课题旨在验证FJ1的抗验证活性并阐明其作用机制。实验采用脂多糖(LPS, Lipopolysaccharide)诱导小鼠单核巨噬细胞白血病细胞RAW264.7构建炎症细胞模型。MTT实验检测FJ1的细胞毒活性以及对LPS损伤细胞的保护活性,结果表明,二者均具有时间和剂量依赖性,细胞毒在安全范围内,FJ1能够提高保护组细胞活性。利用一氧化氮试剂盒测定NO的产生量,结果显示FJ1明显抑制LPS诱导RAW264.7产生NO量,且呈剂量依赖性。流式细胞技术检测细胞活性氧水平,结果与上述结论相符。蛋白免疫印迹法考察ERK, p38, JNK, p-ERK, p-p38, p-JNK[9], NF-κB, IκB-α, p-1κB-α[10]的蛋白表达水平变化,结果表明,ERK, p38, JNK蛋白表达水平没有明显变化,p-ERK, p-p38, p-JNK, NF-κB, p-1κB-a[12]的蛋白表达水平随着FJ1浓度升高而降低,而1κB-α蛋白表达水平则随着FJ1浓度升高而增加。RT-PCR检测炎症相关基因iNOS,COX-2, TNF-a的RNA表达水平,结果发现,三者均随着FJ1浓度的升高而显著降低。综上所述,FJ1对炎症细胞模型具有显著的保护作用,提示FJ1的潜在抗炎症活性可能成为我国传统中药对世界医药发展的又一贡献。帕金森病(Parkinson's disease, PD)是一种常见的神经系统变性疾病,其临床表现主要包括静止性震颤、运动迟缓、肌强直和姿势步态障碍,同时患者可伴有抑郁、便秘和睡眠障碍等非运动症状,给患者及其家属带来巨大的痛苦。帕金森症与氧化应激有着密切的关联,FJ1在抗炎症活性探讨中表现出来的抗氧化活性提示我们其潜在的抗帕金森症活性。经典的帕金森症细胞模型是由1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导褐家鼠肾上腺嗜铬细胞瘤细胞PC12细胞株构建而成,百草枯(PQ)是一种常见的农业毒素,其结构与MPTP的活性部分MPP+相近,因此近年来多被用于构建新型帕金森症模型,本研究采用此种模型,也是本实验室首次自行构建此模型,因此需要对其各项指标进行检测。MTT实验检测PQ的细胞毒活性,结果显示PQ抑制PC12增殖,且具有明显的剂量和时间依赖性。流式细胞技术测定细胞线粒体膜电势[24]以及细胞凋亡,结果表明高浓度PQ使细胞膜电势下降,诱导PC12凋亡。上述结果均与已报道的MPTP以及其他实验室构建该模型的数据相符,可用作后续研究中的神经细胞保护实验。在FJ1对PC12的保护活性研究中,MTT实验检测FJ1对PQ诱导PC12细胞活性的影响,结果表明FJ1对受损细胞具有保护作用且具有时间和剂量依赖性。流式细胞技术检测细胞活性氧水平[26],细胞线粒体膜电势以及细胞凋亡,结果显示FJ1能够抑制由PQ导致的ROS和凋亡的增长,提高MMP水平。蛋白免疫印迹法考察ERK, p38, JNK, p-ERK, p-p38, p-JNK, Bcl-2, Bax的蛋白表达水平变化,结果表明,ERK, p38, JNK蛋白表达水平没有明显变化,p-ERK, p-p38, p-JNK, Bc1-2的蛋白表达水平随着FJ1浓度升高而降低,而Bax蛋白表达水平则随着FJ1浓度升高而增加。本论文首次研究了本实验室提取的大吴风草活性提取物FJ1的抗炎症以及抗帕金森症活性,为其临床治疗炎症相关疾病和帕金森症等神经退行性疾病提供实验依据。此外,有研究表明,近年来帕金森症多发与PQ的广泛应用存在一定关联,本实验采用PQ作为损伤因素,筛选并验证FJ1对神经细胞的保护作用,使得本研究具有环境学与流行病学的双重意义。
[Abstract]:3 beta - angelicoyl -8p, 10p- two hydroxybuddle -7 (11) - -12,8 alpha - lactone (FJ1) is an active compound extracted from the traditional Chinese herbal medicine. It is a perennial herb of perennial herb. The whole medicinal plant is called lieneronium. It is mainly distributed in Southeast and south of China, Japan, and [1] in Korea, for example, cold, coughing, and inflammation. So many kinds of diseases have good curative effect, so it is speculated that the active extract has the effect of anti-inflammatory. The study of FJ1 is the subject of screening of various active products extracted from our laboratory.
Inflammation is the body's response to tissue damage caused by microbial infection and other nociceptive stimuli. It is a very common and very important defense mechanism. However, low resolution and uncontrolled inflammatory response can lead to a chronic inflammatory state associated with a variety of human diseases, including cancer. Verify the anti validation activity of FJ1 and clarify its mechanism. The experiment used LPS (Lipopolysaccharide) to induce murine mononuclear macrophage leukemia cell RAW264.7 to construct an inflammatory cell model.MTT test to detect the cytotoxic activity of FJ1 and the protective activity to LPS damaged cells. The results showed that all of the two had time and dose dependence. In a safe range, FJ1 could improve the cell activity of the protective group. The production of NO was measured by the nitric oxide kit. The results showed that FJ1 obviously inhibited the NO amount produced by RAW264.7 induced by LPS, and was dose-dependent. Flow cytometry was used to detect the level of reactive oxygen species in cells. The results were in accordance with the above conclusion. Protein immunoblotting was in accordance with the results of Western blot. ERK, p38, JNK, p-ERK, p-p38, p-JNK[9], NF- kappa B, I kappa B- alpha, and P-1 kappa B- alpha protein expression level. The increase of.RT-PCR detected the RNA expression level of inflammation related genes iNOS, COX-2 and TNF-a. The results showed that the three were significantly decreased with the increase of FJ1 concentration. To sum up, FJ1 has a significant protective effect on the inflammatory cell model, suggesting that the potential anti-inflammatory activity of FJ1 may become a traditional Chinese medicine for the development of the world medicine. A contribution.
Parkinson's disease (Parkinson's disease, PD) is a common degenerative disease of the nervous system. Its clinical manifestations include static tremor, slow motion, myotonic and postural gait disorders, and patients with depression, constipation and sleep disorders, and other non motor symptoms, bring great pain to the patients and their families. Parkinson's disease and oxidation Stress has a close association, and FJ1's anti - inflammatory activity in the anti - inflammatory activity suggests its potential anti Parkinson activity. The classic Parkinson's disease cell model is constructed by 1- methyl -4- phenyl -1,2,3,6- four hydropyridine (MPTP) induced Rattus norvegicus adrenal chromaffin cells PC12 cell lines, and paraquat (P). Q) is a common agricultural toxin, its structure is similar to the active part of the MPTP, so it has been used to construct a new Parkinson's disease model in recent years. This study is the first time that this model is used to construct this model in our laboratory. So it is necessary to detect the cytotoxic activity of PQ by testing its various indexes by.MTT test, and the result shows P. Q inhibited the proliferation of PC12 and had a significant dose and time dependence. Flow cytometry was used to determine the mitochondrial membrane potential [24] and apoptosis. The results showed that the high concentration of PQ reduced the cell membrane potential and induced the apoptosis of PC12. The results were all consistent with the reported data of MPTP and other laboratory construction of the model, which could be used as a follow-up. In the study of neural cell protection experiments. In the study of the protective activity of FJ1 to PC12, MTT test detected the effect of FJ1 on the activity of PC12 cells induced by PQ. The results showed that FJ1 had protective effects on damaged cells and had time and dose dependence. Flow cytometry was used to detect the level of reactive oxygen species ([26]), cell mitochondrial membrane potential and cells of cells. The results showed that FJ1 could inhibit the growth of ROS and apoptosis caused by PQ and increase the level of MMP. The protein expression level of ERK, p38, JNK, p-ERK, p-p38, p-JNK, Bcl-2, Bax protein expression level was examined by Western blot. With the increase of FJ1 concentration, the expression level of Bax increased with the increase of FJ1 concentration.
In this paper, we first studied the anti inflammatory and anti Parkinson activity of FJ1 extracted from our laboratory, and provide experimental basis for its clinical treatment of inflammation related diseases and neurodegenerative diseases such as Parkinson's disease. In addition, some studies have shown that in recent years, many cases of Parkinson's disease are associated with the extensive use of PQ. In this study, PQ was used as a damage factor to screen and verify the protective effect of FJ1 on neurons. This study has the dual meaning of environmental science and epidemiology.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

【参考文献】