電針對(duì)帕金森病模型大鼠黑質(zhì)肉質(zhì)網(wǎng)應(yīng)激IRE1α-ASK1-JNK通路的影響研究
發(fā)布時(shí)間：2018-07-17 21:10
【摘要】：目的:帕金森病(Parkinson’s Disease,PD),又稱(chēng)為震顫麻痹病,它是中老年人群中好發(fā)的一種神經(jīng)系統(tǒng)退行性病變,其在中老年人群中的發(fā)病率僅次于阿爾茨海默病,位居老年神經(jīng)系統(tǒng)退行性疾病第2位,且有著較高的患病率和致殘率。據(jù)統(tǒng)計(jì),我國(guó)65歲以上人群中PD的總體患病率已達(dá)1700/10萬(wàn),而且其患病率會(huì)隨著年齡的增加而增高。據(jù)國(guó)外報(bào)道,PD在其發(fā)病1至5年的致殘率為25%,而發(fā)病十年以上的致殘率高達(dá)80%。PD的臨床表現(xiàn)以運(yùn)動(dòng)障礙為主,包括靜止性震顫、肌強(qiáng)直、運(yùn)動(dòng)遲緩以及姿勢(shì)步態(tài)異常等四大臨床特征。早期研究發(fā)現(xiàn)PD有兩大病理特征,一是中腦黑質(zhì)致密部多巴胺能神經(jīng)元的變性死亡,二是在殘留的神經(jīng)元胞漿中嗜酸性包涵體—-路易小體(lewy bodies,LB)的形成。國(guó)內(nèi)外大量的研究仍未完全明確PD的病因及發(fā)病機(jī)制,據(jù)悉,遺傳、環(huán)境毒素、氧化應(yīng)激、神經(jīng)系統(tǒng)老化、線(xiàn)粒體功能障礙、免疫反應(yīng)、興奮性毒性、炎性反應(yīng)、蛋白酶體功能障礙等眾多因素均參與了PD的發(fā)生發(fā)展。近年國(guó)內(nèi)外的研究表明,內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERS)也參與了PD的發(fā)生發(fā)展,并且在PD發(fā)病中可能起到至關(guān)重要的作用。本研究采用頸背部皮下注射魚(yú)藤酮法制備PD大鼠模型,運(yùn)用電針“風(fēng)府”“太沖”穴給予治療,研究ERS在魚(yú)藤酮誘導(dǎo)的PD模型大鼠中的作用,以及“風(fēng)府”、“太沖”穴電針對(duì)黑質(zhì)區(qū)ERS的影響,以期為針灸臨床防治PD開(kāi)辟新的思路及提供可靠的實(shí)驗(yàn)依據(jù)。方法:本研究選用SPF級(jí)健康雄性SD大鼠120只,隨機(jī)分為正常組(Normal)、假手術(shù)組(sham-operation)、模型組(Model)、電針預(yù)治療組(EA pretreatment)、電針治療7d組(EA 7d)、電針治療14d組(EA14d)、電針治療21d組(EA 21d)、電針治療28d組(EA 28d),每組15只,采用頸背部皮下注射魚(yú)藤酮法制備PD大鼠模型。假手術(shù)組的大鼠僅注射不含魚(yú)藤酮的葵花油。電針治療組大鼠在造模完成后取“風(fēng)府”、“太沖”穴進(jìn)行電針治療;正常組、模型組、假手術(shù)組不做任何治療;電針預(yù)治療組先對(duì)正常大鼠電針治療7d,然后造模,方法同模型組。電針干預(yù)治療結(jié)束后,采用行為學(xué)評(píng)分法及敞箱實(shí)驗(yàn)觀(guān)測(cè)大鼠行為學(xué)的變化,采用免疫組化法檢測(cè)大鼠中腦黑質(zhì)區(qū)α-syn聚集的情況及th陽(yáng)性神經(jīng)元數(shù)量的變化,并運(yùn)用rt-pcr法檢測(cè)大鼠黑質(zhì)內(nèi)bipmrna、ire1αmrna、ask1mrna的表達(dá)變化,免疫蛋白印跡(westernblot)法檢測(cè)大鼠中腦黑質(zhì)區(qū)內(nèi)bip、ire1α、ask1、p-jnk蛋白表達(dá)的變化。結(jié)果:(1)模型組大鼠表現(xiàn)出明顯的pd癥候群特征,電針干預(yù)后大鼠行為學(xué)評(píng)分較模型組明顯降低(p0.01);(2)模型組大鼠黑質(zhì)區(qū)α-syn的陽(yáng)性表達(dá)較正常組、假手術(shù)組顯著增高(均p0.01),電針預(yù)治療組和電針治療7d、14d、21d、28d組大鼠黑質(zhì)區(qū)α-syn的陽(yáng)性表達(dá)較模型組顯著降低(均p0.01);(3)模型組大鼠黑質(zhì)區(qū)th的陽(yáng)性表達(dá)較正常組、假手術(shù)組顯著降低(p0.01),電針預(yù)治療組和電針治療7d、14d、21d、28d組大鼠黑質(zhì)區(qū)th的陽(yáng)性表達(dá)較模型組顯著增高(均p0.01);(4)模型組大鼠黑質(zhì)區(qū)bipmrna、ire1αmrna、ask1mrna的表達(dá)較正常組、假手術(shù)組顯著增高(均p0.01),電針治療14d組大鼠黑質(zhì)區(qū)bipmrna、ire1αmrna、ask1mrna表達(dá)水平較模型組降低(p0.05),電針治療21d、28d組大鼠黑質(zhì)區(qū)bipmrna、ire1αmrna、ask1mrna表達(dá)水平較模型組顯著降低(均p0.01);(5)模型組大鼠黑質(zhì)內(nèi)bip、ire1α、ask1、p-jnk蛋白表達(dá)較正常組和假手術(shù)組顯著增高(均p0.01),電針預(yù)治療組和電針治療7d、14d、21d、28d組bip、ire1α、ask1、p-jnk蛋白表達(dá)水平較正常組顯著降低(均p0.01)。結(jié)論:(1)采用頸背部皮下注射魚(yú)藤酮法可成功構(gòu)建pd大鼠模型,且該造模方法操作簡(jiǎn)便、經(jīng)濟(jì)廉價(jià)、成功率較高;(2)魚(yú)藤酮可誘發(fā)大鼠中腦黑質(zhì)區(qū)α-syn的聚集,引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),而長(zhǎng)時(shí)間的內(nèi)質(zhì)網(wǎng)應(yīng)激會(huì)誘發(fā)多巴胺能神經(jīng)元的變性丟失,最終導(dǎo)致PD的發(fā)生發(fā)展。(3)“風(fēng)府”、“太沖”穴電針能夠明顯改善PD模型大鼠的運(yùn)動(dòng)行為能力,提高大鼠黑質(zhì)內(nèi)TH陽(yáng)性神經(jīng)元數(shù)量,減少α-syn聚集,降低內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白的表達(dá)。提示“風(fēng)府”、“太沖”穴電針可以通過(guò)調(diào)節(jié)PD大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),發(fā)揮對(duì)多巴胺能神經(jīng)元的保護(hù)作用。(4)“風(fēng)府”、“太沖”穴電針?lè)乐蜳D的可能作用機(jī)制在于,“風(fēng)府”、“太沖”穴電針治療能夠減少黑質(zhì)內(nèi)α-syn聚集,抑制黑質(zhì)區(qū)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),保護(hù)多巴胺能神經(jīng)元免于變性丟失,從而延緩PD的發(fā)生發(fā)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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R245;R-332
本文編號(hào)：2130944
[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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R245;R-332
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相關(guān)博士學(xué)位論文 前1條
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