电针对帕金森病模型大鼠黑质肉质网应激IRE1α-ASK1-JNK通路的影响研究

发布时间：2018-07-17 21:10

【摘要】：目的:帕金森病(Parkinson’s Disease,PD),又称为震颤麻痹病,它是中老年人群中好发的一种神经系统退行性病变,其在中老年人群中的发病率仅次于阿尔茨海默病,位居老年神经系统退行性疾病第2位,且有着较高的患病率和致残率。据统计,我国65岁以上人群中PD的总体患病率已达1700/10万,而且其患病率会随着年龄的增加而增高。据国外报道,PD在其发病1至5年的致残率为25%,而发病十年以上的致残率高达80%。PD的临床表现以运动障碍为主,包括静止性震颤、肌强直、运动迟缓以及姿势步态异常等四大临床特征。早期研究发现PD有两大病理特征,一是中脑黑质致密部多巴胺能神经元的变性死亡,二是在残留的神经元胞浆中嗜酸性包涵体—-路易小体(lewy bodies,LB)的形成。国内外大量的研究仍未完全明确PD的病因及发病机制,据悉,遗传、环境毒素、氧化应激、神经系统老化、线粒体功能障碍、免疫反应、兴奋性毒性、炎性反应、蛋白酶体功能障碍等众多因素均参与了PD的发生发展。近年国内外的研究表明,内质网应激(endoplasmic reticulum stress,ERS)也参与了PD的发生发展,并且在PD发病中可能起到至关重要的作用。本研究采用颈背部皮下注射鱼藤酮法制备PD大鼠模型,运用电针“风府”“太冲”穴给予治疗,研究ERS在鱼藤酮诱导的PD模型大鼠中的作用,以及“风府”、“太冲”穴电针对黑质区ERS的影响,以期为针灸临床防治PD开辟新的思路及提供可靠的实验依据。方法:本研究选用SPF级健康雄性SD大鼠120只,随机分为正常组(Normal)、假手术组(sham-operation)、模型组(Model)、电针预治疗组(EA pretreatment)、电针治疗7d组(EA 7d)、电针治疗14d组(EA14d)、电针治疗21d组(EA 21d)、电针治疗28d组(EA 28d),每组15只,采用颈背部皮下注射鱼藤酮法制备PD大鼠模型。假手术组的大鼠仅注射不含鱼藤酮的葵花油。电针治疗组大鼠在造模完成后取“风府”、“太冲”穴进行电针治疗;正常组、模型组、假手术组不做任何治疗;电针预治疗组先对正常大鼠电针治疗7d,然后造模,方法同模型组。电针干预治疗结束后,采用行为学评分法及敞箱实验观测大鼠行为学的变化,采用免疫组化法检测大鼠中脑黑质区α-syn聚集的情况及th阳性神经元数量的变化,并运用rt-pcr法检测大鼠黑质内bipmrna、ire1αmrna、ask1mrna的表达变化,免疫蛋白印迹(westernblot)法检测大鼠中脑黑质区内bip、ire1α、ask1、p-jnk蛋白表达的变化。结果:(1)模型组大鼠表现出明显的pd症候群特征,电针干预后大鼠行为学评分较模型组明显降低(p0.01);(2)模型组大鼠黑质区α-syn的阳性表达较正常组、假手术组显著增高(均p0.01),电针预治疗组和电针治疗7d、14d、21d、28d组大鼠黑质区α-syn的阳性表达较模型组显著降低(均p0.01);(3)模型组大鼠黑质区th的阳性表达较正常组、假手术组显著降低(p0.01),电针预治疗组和电针治疗7d、14d、21d、28d组大鼠黑质区th的阳性表达较模型组显著增高(均p0.01);(4)模型组大鼠黑质区bipmrna、ire1αmrna、ask1mrna的表达较正常组、假手术组显著增高(均p0.01),电针治疗14d组大鼠黑质区bipmrna、ire1αmrna、ask1mrna表达水平较模型组降低(p0.05),电针治疗21d、28d组大鼠黑质区bipmrna、ire1αmrna、ask1mrna表达水平较模型组显著降低(均p0.01);(5)模型组大鼠黑质内bip、ire1α、ask1、p-jnk蛋白表达较正常组和假手术组显著增高(均p0.01),电针预治疗组和电针治疗7d、14d、21d、28d组bip、ire1α、ask1、p-jnk蛋白表达水平较正常组显著降低(均p0.01)。结论:(1)采用颈背部皮下注射鱼藤酮法可成功构建pd大鼠模型,且该造模方法操作简便、经济廉价、成功率较高;(2)鱼藤酮可诱发大鼠中脑黑质区α-syn的聚集,引发内质网应激反应,而长时间的内质网应激会诱发多巴胺能神经元的变性丢失,最终导致PD的发生发展。(3)“风府”、“太冲”穴电针能够明显改善PD模型大鼠的运动行为能力,提高大鼠黑质内TH阳性神经元数量,减少α-syn聚集,降低内质网应激相关蛋白的表达。提示“风府”、“太冲”穴电针可以通过调节PD大鼠内质网应激反应,发挥对多巴胺能神经元的保护作用。(4)“风府”、“太冲”穴电针防治PD的可能作用机制在于,“风府”、“太冲”穴电针治疗能够减少黑质内α-syn聚集,抑制黑质区内质网应激反应,保护多巴胺能神经元免于变性丢失,从而延缓PD的发生发展。
[Abstract]:Objective: Parkinson's disease (Parkinson 's Disease, PD), also known as tremor paralysis, is a good neurodegenerative disease in the middle aged and old age population. The incidence of the disease is second only to Alzheimer's disease, the second in the elderly, with a high incidence of disease and disability. The overall prevalence rate of PD in our country over 65 years old has reached 1700/10 million, and its prevalence will increase with age. According to foreign reports, the rate of disability of PD in its incidence of 1 to 5 years is 25%, and the clinical manifestations of disability over ten years of morbidity as high as 80%.PD are motility disorders, including static tremor, myotonic rigidity, and exercise retardation. Four major clinical features, such as postural gait abnormalities, and other four major clinical features. Early studies have found that PD has two major pathological features, one is the degeneration of dopaminergic neurons in the mesencephalic dense part of the mesencephalon, and two is the form of the eosinophil - Louis corpuscle (Lewy bodies, LB) in the residual cytoplasm of the neurons. A large number of studies at home and abroad have not completely identified the disease of PD A number of factors such as genetic, environmental toxins, oxidative stress, oxidative stress, nervous system aging, mitochondrial dysfunction, immune response, excitotoxicity, inflammatory response, proteasome dysfunction and other factors are involved in the development of PD. In recent years, domestic and foreign studies have shown that endoplasmic reticulum stress (ERS) also participates in the development of endoplasmic reticulum stress (ERS). This study may play a vital role in the development of PD and may play an important role in the pathogenesis of PD. In this study, the rat model of PD rats was prepared by subcutaneous injection of rotenone on the back of the neck. The effect of ERS in the PD model rats induced by rotenone, and the "Feng Fu", "Tai Chong" point electroacupuncture were studied. The effect of ERS in the substantia nigra area is to open up new ideas and provide reliable experimental basis for the clinical prevention and treatment of PD in acupuncture and moxibustion. Methods: 120 healthy male SD rats of grade SPF were selected, randomly divided into normal group (Normal), sham operation group (sham-operation), model group (Model), electroacupuncture pretherapy group (EA pretreatment), electroacupuncture treatment of 7D group (EA 7D), electroacupuncture. Treatment group 14d (EA14d), electroacupuncture treatment group 21d (EA 21d), electroacupuncture treatment of group 28d (EA 28d), 15 rats in each group, using rotenone on the back of the neck to prepare the PD rat model. The rats in the sham operation group were only injected with sunflower oil without rotenone. The rats in the electroacupuncture group were treated with "Feng Fu" and "Tai Chong" point after the model was completed; Group, model group, sham operation group did not do any treatment; Electroacupuncture group first treated normal rats with electroacupuncture treatment 7d, then model group, method and model group. After the treatment of electroacupuncture intervention, the behavior changes of rats were observed by behavioral score and open box test, and immunohistochemical method was used to detect the aggregation of alpha -syn in the substantia nigra area of the rat and t The changes in the number of H positive neurons and the changes in the expression of bipmrna, IRE1 a mRNA and ask1mrna in the substantia nigra of rats were detected by RT-PCR. The changes in the expression of BiP, IRE1 alpha, ASK1, p-JNK protein in the substantia nigra of the rat were detected by immunoblotting (Westernblot). Results: (1) the rats in the model group showed significant PD syndrome characteristics and the prognosis of electroacupuncture. The behavior score of rats was significantly lower than that in the model group (P0.01). (2) the positive expression of alpha -syn in the substantia nigra area of the rat model group was significantly higher than that in the normal group (P0.01). The positive expression of alpha -syn in the substantia nigra area of the group of 7D, 14d, 21d, 28d group was significantly lower than that of the model group (all P0.01), and (3) th in the substantia nigra region of the model group of rats. The positive expression was significantly lower in the sham group than in the normal group (P0.01). The positive expression of th in the substantia nigra area of the group of 7D, 14d, 21d and 28d was significantly higher than that of the model group (P0.01). (4) the expression of bipmrna, IRE1 alpha mRNA, ask1mrna in the model group was significantly higher than that in the normal group, and the sham operation group increased significantly (both P0.01) and electroacupuncture treatment. The expression level of bipmrna, IRE1 alpha mRNA, ask1mrna in the 14d group was lower than that in the model group (P0.05), 21d was treated by electroacupuncture, bipmrna in the substantia nigra area of group 28d, IRE1 alpha mRNA, and the level of ask1mrna expression was significantly lower than that in the model group. (5) the expression of protein in the model group was significantly higher than that in the normal group and the sham operation group. (all P0.01), the expression level of 7D, 14d, 21d, 28d group BiP, IRE1 a, ASK1, p-JNK protein was significantly lower than that of the normal group (P0.01). Conclusion: (1) the use of rotenone by subcutaneous injection of rotenone on the back of the neck can successfully construct the PD rat model, and the method is easy to operate, cheap, and high success rate; (2) rotenone can induce large The aggregation of alpha -syn in the substantia nigra of the rat mesencephalon triggers the endoplasmic reticulum stress response, while prolonged endoplasmic reticulum stress induces degeneration and loss of dopaminergic neurons and eventually leads to the occurrence and development of PD. (3) "Feng Fu", "Tai Chong" point electroacupuncture can obviously improve the transport behavior of PD model rats and improve the TH positive neurons in the substantia nigra of rats. Quantity, reduce the accumulation of alpha -syn and reduce the expression of endoplasmic reticulum stress related proteins. It suggests that the Electroacupuncture of "Feng Fu" and "Tai Chong" point can play a protective effect on dopaminergic neurons by regulating the endoplasmic reticulum stress response of PD rats. (4) the possible mechanism of "Feng Fu" and "Tai Chong" point electroacupuncture on the prevention and control of PD is, "Feng Fu", "Tai Chong" Acupoint electroacupuncture can reduce the accumulation of alpha -syn in the substantia nigra, inhibit the endoplasmic reticulum stress response in the substantia nigra, protect the dopaminergic neurons from degeneration and delay the occurrence and development of PD.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245;R-332

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