独活抑制阿尔茨海默病Aβ大鼠模型免疫炎性损伤的实验研究

【作者】 张杰；

【导师】 李德新；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2008， 博士

【摘要】 阿尔茨海默病(Alzheimers diseases AD)是发生在早老及老年期、不可逆转的、渐进性的、以精神行为异常为主的脑变性疾病,晚期可危及患者的生命,其发病率和死亡率逐年上升。在本病的病理机制的多种假说中,免疫炎症假说得到了学者的关注。。近年来发现非甾类抗炎药对本病具有一定的治疗作用,进而使本病的本病在中医属痴呆的一种,中医多认为以五脏虚损为本,湿痰瘀为标,本研究以祛风胜湿药独活为本病治疗的切入点,采用非甾体抗炎药为对照,探讨独活在抑制阿尔茨海默病炎性损伤中的作用。目的:本研究旨在探讨独活在抑制阿尔茨海默症Aβ模型大鼠炎性损伤的作用环节,观察独活对模型鼠脑内炎性细胞因子、信号转导通路及凋亡蛋白的影响,探讨祛风胜湿药独活的作用机制为应用祛风除湿法治疗AD提供现代科学依据。方法:采用Aβ蛋白大鼠海马内注射模型,分别予以吲哚美辛、独活、丹参及生理盐水灌胃,四周后,水迷宫实验观察药物对模型鼠学习记忆能力的影响。并以光镜、电镜观察模型鼠脑内神经元形态学的改变;采用免疫组化方法,测得不同组别的大鼠脑内IL-1、IL-6、TNF-a、INOS、NF-κB蛋白的表达差异;以western-blot方法测不同施加因素对大鼠脑内凋亡蛋白Bcl-2、Bax的影响;并通过观察对p38MAPK信号转导通路的影响,来探讨独活的作用机制。结果:给药后除模型组、假手术组外,各组大鼠与给药前比较,逃避潜伏期均有缩短(﹟P<0.05)。说明3种药物对Aβ大鼠的学习记忆能力有改善作用。独活组及西药组的p38 MAPK表达较模型组有所降低,差异显著(P<0.05),独活组、西药组p38 MAPK表达水平较丹参组降低,但差别不显著(P>0.05)。丹参组与模型组比较,p38 MAPK表达有所降低,但差别不显著(p>0.05)。在细胞因子方面,模型组与假手术组比较TNF-α、IL-1、INOS、NF-κB及IL-6有一较高水平的表达,说明大鼠脑内注射Aβ蛋白使大鼠脑中上述细胞因子的分泌明显增加,西药组、独活组、丹参组与假手术组、模型组比较,差异显著(P<0.05),提示三药均能对Aβ大鼠脑中上述指标的表达有很好的抑制作用,但不如假手术组,(丹参组NF-κB的表达同模型组)。丹参组、独活组及西药组比较,差异不显著(P>0.05),说明在两种药物在抑制上述指标的表达上差别不显著(P>0.05)。丹参组在对凋亡蛋白的调控上,丹参及独活均能上调bcl-2的表达,抑制Bax基因表达,提高Bcl-2/Bax的比值,西药吲哚美辛作用弱于此两味药,说明吲哚美辛可以抑制脑内炎性反应,但对抑制凋亡作用不及丹参及独活。结论:1、阿尔茨海默证是以五脏虚损为本,湿痰为标,祛风胜湿药独活可以通过干预阿尔茨海默病的免疫损伤机制,而发挥治疗作用。2、祛风胜湿药之独活可改善Aβ大鼠的学习记忆能力,并通过抑制Aβ大鼠脑中IL-1、IL-6、TNF-A等细胞因子的表达,从而减轻Aβ大鼠脑中炎性反应;对Aβ大鼠脑中核转录因子NF-κb具有调节作用,降低脑中INOS的表达,进而减轻大鼠脑内炎性反应、细胞凋亡、氧化应激等反应,减轻神经元细胞的损害。可通过抑制p38MAPK信号通路的转导及调控凋亡蛋白以减轻脑中的免疫炎性反应,从而抑制细胞凋亡。更多还原

【Abstract】 Alzheimer disease (Alzheimers diseases AD)is the brain’s degenerative disease which occurs in the early period of the old and the elderly age. It is irreversible, progressive, and mainly charactered by the abnormal spirit and behavior.At the late stage, it could endanger the lives of patients. Its rates of incidence and mortality increase year by year. There are several hypotheses on the pathology of this disease. In recent years ,reseachers has found that non-steroidal anti-inflammatory agent has a certain role for the treatment of this disease, thereby the hypothesis called the immune inflammatory disease has come to be the concern of scholars. It belongs to the Dementia in TCM, which thinks that the Ben of this diease is the deficiency of Five Viscera, damp,phlegm and blood stasis as the Biao. Our study put forward to making the herb of dispelling wind and eliminating dampness-Radix angelica tuhuo(Duhuo) as the breakthrough-point, and using non-steroidal anti-inflammatory drugs as the control .Our purpose is to study Radix angelica tuhuo(Duhuo)’s suppression of inflammatory injury of Alzheimer’s disease.Objective: Aim to explore Radix angelica tuhuo(Duhuo)’s inhibition of the Aβrat model’s inflammatory injury of Alzheimer’s disease. By the observation of Radix angelica tuhuo’s action on inflammatory cytokines, the pathway of the signal transduction and apoptosis proteins in the model rat’s brain,we are to explore the mechanism of action of the herb of expelling wind and eliminating dampness- Radix angelica tuhuo .Methods: Model rates injected with Aβprotein in the hippocampus were separately gavaged with indomethacin, Radix angelica Duhuo, Salvia and saline. After four weeks ,1) By water maze test we observed drugs’action on rats’s ability of learning and memory. 2)With light and electron microscopy we observed neurons’s changes in model rats’brain.3) Using immunohisto chemical methods measured expression differences of protein- IL-1, IL-6, TNF-a, INOS, NF-κB in rats’brain of different groups. 4)Western-blot tested different imposed factors’s impact on apoptotic protein Bcl-2 and Bax in the brains of rats, and through observation of the impact on the p38MAPK signal transduction pathway ,we explored the mechanism of action of Radix angelica tuhuo.Results: After administration, in addition to the model group and sham-operation group, the rates of the other groups were compared with the premedication, which had shown the shortened Escape latency ( P <0.05). It illustrated that the three kinds of drugs can improve the ability of learning and memory of rats suffering from AD. The level of p38 MAPK expression of Duhuo Group and WM Group was lower than of model group ,with significant difference ( P <0.05). The level of p38 MAPK expression of Duhuo group and WM group was lower than of Salvia Group, but the difference was not significant ( P> 0.05). When Salvia group was compared with the model group, the level of the expression of p38 MAPK fell, but the difference is not significant ( p> 0.05). In cytokines, TNF-α, IL-1, INOS and IL-6,NF-κB had a relatively higher level of expression in the model group than of the sham-operation group ,which indicated that Aβprotein injected into the rats’brain significantly increased the secretion of above Cytokines . When the WM group, Duhuo group,and Salvia group were compared with sham-operation group and model group, the difference was significant ( P <0.05), which suggested that the three drugs had significant inhibition of expression of the above indicators in AD rats brain, but the effect was worse than of the sham-operation group (the expression of NF-κin Salvia group is the same as the model group). In comparison among Salvia group, Duhuo group and WM group, the difference had not shown significant meanings (P> 0.05), indicating that the effect of the two drugs in inhibiting the expression of the above indicators were not significantly different ( P> 0.05). In the regulation of apoptosis of proteins in Salvia group, Salvia and Duhuo can increase the level of the expression of bcl-2, inhibit Bax genetic expression and improve the ratio of Bcl-2/Bax; western medicine- indomethacin had a weaker role than the two above drugs,which indicates that indomethacin can inhibit the inflammatory reaction in the brain,but had a weaker role in inhibition of apoptosis than Duhuo and Salvia.Conclusion: 1, Alzheimer disease is based on the deficiency of Five Viscera,and damp and phlegm are Biao of this disease.The herb of Dispelling wind and eliminating damp-Duhuo can act by the intervention of immune injury mechanisms of Alzheimer disease. 2, Duhuo can improve the ability of learning and memory of AD rates ,reduce the inflammatory reaction in the brains of AD rates by inhibiting the expression of cytokines- IL-1, IL-6, TNF-A;It can regulate nuclear transcription factor- NF-Κb and reduce the level of expression of INOS in the brain,thereby it can reduce inflammatory reaction,apoptosis, oxidative stress reaction and the injury of neurons in the brain of rates.Reduce immune inflammatory reaction by inhibition of the signal transduction pathway of P38MAPK and regulating the expression of apoptosis protein so as to inhibit cell apoptosis.更多还原