二苯乙烯苷通過(guò)抑制ROS相關(guān)的線(xiàn)粒體和JNK,P38通路對(duì)MPTP誘導(dǎo)的帕金森病小鼠模型的神經(jīng)保護(hù)作用
發(fā)布時(shí)間：2018-07-31 14:18
【摘要】：帕金森病（Parkinson’s disease，PD）是一種以靜止性震顫，肌僵直，動(dòng)作遲緩和姿勢(shì)反射障礙為主要臨床表現(xiàn)的中老年人群中常見(jiàn)的中樞神經(jīng)系統(tǒng)退行性疾病。其病理特點(diǎn)是中腦炓質(zhì)致密部(substantia nigra pars compact，SNpc)多巴胺（dopamine，DA）能神經(jīng)元變性缺失。目前的藥物治療并不能阻止或延緩PD的進(jìn)程，僅能緩解癥狀，且長(zhǎng)期治療會(huì)出現(xiàn)療效遞減，產(chǎn)生很多副作用。研究表明，PD發(fā)病的重要因素有可能是氧化應(yīng)激或者線(xiàn)粒體功能障礙。還有研究表明MAPK通路在PD的發(fā)病中起重要作用。阻斷JNK和P38的激活可以抑制MPTP導(dǎo)致的神經(jīng)毒性。 1-甲基-4苯基-1，2，3，6-四氫吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydropyrid-ine，MPTP）是經(jīng)典的PD造模藥物之一。其代謝產(chǎn)物1-甲基-4苯基吡啶離子（1-methy-4-phenylpyridinium，MPP+）是誘發(fā)神經(jīng)毒性的主要物質(zhì)。在DA能神經(jīng)元內(nèi)，MPP+可以通過(guò)抑制線(xiàn)粒體復(fù)合物I破壞電子呼吸鏈，從而產(chǎn)生大量ROS，進(jìn)一步導(dǎo)致黑質(zhì)DA能神經(jīng)元缺失。 何首烏的根是一種傳統(tǒng)的中草藥，具有抗衰老的作用。二苯乙烯苷（2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside，TSG）是其主要活性成分之一。具有很多生物學(xué)特性，例如：抗氧化、抗炎、抗動(dòng)脈硬化等作用。研究發(fā)現(xiàn)TSG不僅可以通過(guò)抑制ROS保護(hù)腦組織，并且可以阻斷JNK激活保護(hù)大腦不受缺血的損傷，TSG可以降低α突觸核蛋白（α-synuclein，α-syn）的過(guò)度表達(dá)（APP轉(zhuǎn)基因阿爾茨海默病小鼠模型）。還可抑制MPP+對(duì)SH-SY5Y細(xì)胞的神經(jīng)毒性作用。 我們課題組近期的實(shí)驗(yàn)表明，TSG可以通過(guò)阻斷ROS-NO相關(guān)的JNK通路抑制MPP+誘導(dǎo)的PC12細(xì)胞凋亡。為了進(jìn)一步研究TSG在體內(nèi)的神經(jīng)保護(hù)作用，本次實(shí)驗(yàn)的目的如下：1）利用PD小鼠模型，驗(yàn)證TSG的神經(jīng)保護(hù)作用；2）測(cè)定這種神經(jīng)保護(hù)作用是否與ROS介導(dǎo)的JNK，P38以及線(xiàn)粒體通路有關(guān)。 【目的】 1.建立MPTP誘導(dǎo)的PD小鼠模型。 2.觀察TSG對(duì)MPTP誘導(dǎo)的PD小鼠行為學(xué)、形態(tài)學(xué)及生化方面的影響。 3.探討TSG是否通過(guò)調(diào)節(jié)ROS介導(dǎo)的JNK，P38和線(xiàn)粒體通路，發(fā)揮對(duì)MPTP誘導(dǎo)的PD小鼠的神經(jīng)保護(hù)作用。 【方法】 1.動(dòng)物分組與造模：將實(shí)驗(yàn)動(dòng)物隨機(jī)分為7組，每組10只。A組：正常組，連續(xù)7d腹腔注射（i.p.）生理鹽水0.1ml/次，1h后給予等量的生理鹽水灌胃（i.g.），1次/d，隨后的7d，給予生理鹽水（i.g.），1次/d；B組：MPTP模型組，連續(xù)7d（i.p.）MPTP （生理鹽水溶）30mg/kg，1h后給予等量的生理鹽水（i.g.），1次/d，隨后的7d，給予生理鹽水（i.g.），1次/d；C、D組為T(mén)SG治療組，給予MPTP（i.p.）30mg/kg，1h后分別給予TSG（純度98%）（i.g.）20mg/kg或40mg/kg，1次/d，連續(xù)7d，隨后的7d停止MPTP注射，只給予TSG（i.g.）20mg/kg或40mg/kg；E、F組，連續(xù)7d給予生理鹽水（i.p.）0.1ml/次，1h后分別給予TSG（i.g.）20mg/kg或40mg/kg，1次/d，隨后的7d只給予TSG（i.g.）20mg/kg或40mg/kg；G組，NAC阻斷劑組，給予MPTP（i.p.）30mg/kg，1h后給予NAC（i.g.）200μM/kg，1次/d，連續(xù)7d，隨后7d停止注射MPTP，只給予NAC200μM/kg （i.g.）。完成給藥后1天，利用爬桿和曠場(chǎng)實(shí)驗(yàn)，衡量行為學(xué)變化。 2.完成行為學(xué)檢測(cè)后，灌注小鼠，迅速取腦，并制作冰凍切片，用免疫熒光染色法檢 測(cè)酪氨酸羥化酶(TH)陽(yáng)性神經(jīng)元數(shù)目的變化。 3.全波段自動(dòng)酶標(biāo)儀檢測(cè)ROS水平。 4.高效液相色譜法檢測(cè)紋狀體中DA、二羥基苯乙酸（DOPAC）和高香草酸（HVA）的含量。 5. Western Blot檢測(cè)JNK, p-JNK, P38, p-P38, ERK, p-ERK, bcl-2, bax, cyt c, smac,cleaved-caspase-3, cleaved-caspase-6, cleaved-caspase-9蛋白的表達(dá)。 【結(jié)果】 1.行為學(xué)結(jié)果顯示：在爬桿實(shí)驗(yàn)中，給予小鼠MPTP后，與空白對(duì)照組比較，爬桿時(shí)間（TLa）顯著升高（P 0.01）。但在給予TSG治療后，與模型組相比，TLa明顯縮短，而40mg/kgTSG組改善的更為明顯（P 0.01）。單純給予TSG組與空白對(duì)照組相比，并沒(méi)有顯著差異（P＞0.05）。給予NAC后，TLa與模型組相比也有明顯的改善（Fig.2a, P 0.01）。礦場(chǎng)試驗(yàn)中，與空白對(duì)照組比較小鼠的運(yùn)動(dòng)速度(V)在給予MPTP后顯著降低（P 0.01），在中央停留時(shí)間(T)明顯延長(zhǎng)（Fig.2b, P 0.01），然而經(jīng)過(guò)TSG治療后，V恢復(fù)到4.367cm/s，T下降到11.00s，單獨(dú)給予TSG組與空白對(duì)照組無(wú)明顯差異（P＞0.05），在給予NAC治療后，與MPTP組相比，V明顯增加，T明顯降低（Fig.2c, P 0.05）。 2.免疫熒光染色結(jié)果顯示：黑質(zhì)內(nèi)TH陽(yáng)性神經(jīng)元的計(jì)數(shù)采用雙盲法。在熒光顯微鏡下可觀察到與空白對(duì)照組相比模型組TH陽(yáng)性神經(jīng)元減少幅度約為66.9%（P0.01），然而TSG處理后，TH陽(yáng)性神經(jīng)元呈劑量依賴(lài)性恢復(fù)。而單純給予TSG組的TH陽(yáng)性神經(jīng)元與正常組比較無(wú)統(tǒng)計(jì)學(xué)差異（P＞0.05）。與高劑量組類(lèi)似，給予NAC治療后，TH陽(yáng)性神經(jīng)元顯著升高(Fig.3, P＜0.05)。 3.高效液相結(jié)果顯示，與空白對(duì)照組相比，MPTP處理后紋狀體內(nèi)DA水平下降了82%，DOPAC，HVA水平也相應(yīng)下降到正常組的15%和19%，但給予TSG治療后，DA，DOPAC, HVA水平逐漸升高，呈劑量依賴(lài)性。單獨(dú)給予TSG的小鼠紋狀體內(nèi)DA，DOPAC，HVA水平與正常組相比并沒(méi)有統(tǒng)計(jì)學(xué)差異（P＞0.05）。而NAC治療后，DA，DOPAC，HVA水平也顯著恢復(fù)(Fig.4, P＜0.05)。 4.全波段自動(dòng)熒光酶標(biāo)儀檢測(cè)ROS結(jié)果顯示，在MPTP處理組中，ROS水平與空白對(duì)照組相比，有了明顯的升高(P0.01)。不同劑量的TSG (20和40mg/kg)或NAC治療后，與模型組相比，ROS水平明顯下降(P0.01)，且TSG呈劑量依賴(lài)性。單獨(dú)給予TSG后，與正常組比較無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。這說(shuō)明TSG的保護(hù)作用與NAC相似(Fig.5)。 5. Western Blot方法檢測(cè)結(jié)果表明，與MPTP組相比，TSG（20和40mg/kg）干預(yù)組可以顯著改善JNK,P38以及線(xiàn)粒體通路的激活，但對(duì)ERK通路并無(wú)影響。給予NAC治療的小鼠與TSG的效果類(lèi)似，也抑制了JNK，，P38以及線(xiàn)粒體通路的激活。 【結(jié)論】 1. TSG對(duì)MPTP誘導(dǎo)的PD小鼠具有神經(jīng)保護(hù)作用。 2. TSG可能通過(guò)抑制ROS介導(dǎo)的JNK, P38和線(xiàn)粒體通路發(fā)揮對(duì)MPTP誘導(dǎo)的PD小鼠模型的神經(jīng)保護(hù)作用。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a common central nervous system degenerative disease in middle-aged and elderly people with static tremor, muscle stiffness, motion retardation, and postural reflex disorder. The pathological features of the middle cerebral cortex (substantia nigra pars compact, SNpc) dopamine (dopamine, DA) can be deity The current drug treatment does not prevent or delay the process of PD and can only alleviate the symptoms, and long-term treatment will result in diminishing effect and many side effects. Studies have shown that the important factors in the pathogenesis of PD may be oxidative stress or mitochondrial dysfunction. Further studies have shown that the MAPK pathway plays an important role in the pathogenesis of PD. Blocking the activation of JNK and P38 can inhibit MPTP induced neurotoxicity.
1- methyl -4 phenyl -1,2,3,6- four hydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyrid-ine, MPTP) is one of the classic PD modeling drugs. Its metabolite, 1- methyl -4 phenyl pyridine ion (1-methy-4-phenylpyridinium, MPP+), is the main substance to induce neurotoxicity. In DA neurons, MPP+ can inhibit the mitochondrial complex. Destroying the electron respiratory chain, resulting in a large number of ROS, further leading to the deletion of DA neurons in substantia nigra.
The root of Polygonum multiflorum is a traditional Chinese herbal medicine that has anti aging effect. Two 2,3,5,4 '-tetrahydroxystilbene-2-O- beta -D-glucoside (TSG) is one of its main active components. It has many biological characteristics, such as antioxidant, anti-inflammatory, and anti arteriosclerosis. It is found that TSG can not only inhibit ROS protection. Brain tissue, and can block JNK activation to protect the brain from ischemia injury, and TSG can reduce the overexpression of alpha synuclein (alpha -synuclein, alpha -syn) (APP transgenic Alzheimer's disease model). It also inhibits the neurotoxic effect of MPP+ on SH-SY5Y cells.
Recent experiments in our team showed that TSG could inhibit the apoptosis of PC12 cells induced by MPP+ by blocking the JNK pathway of the ROS-NO. In order to further study the neuroprotective effect of TSG in the body, the aim of this experiment is as follows: 1) use the PD mouse model to verify the neuroprotective effect of TSG; 2) determine whether this neuroprotective effect is possible. It is related to ROS mediated JNK, P38, and mitochondrial pathway.
[Objective]
1. the PD mouse model induced by MPTP was established.
2. to observe the effects of TSG on the behavior, morphology and biochemistry of MPTP induced PD mice.
3. to explore whether TSG regulates ROS mediated JNK, P38 and mitochondrial pathways, and plays a neuroprotective role in MPTP induced PD mice.
[method]
1. animal groups and model building: the experimental animals were randomly divided into 7 groups, 10.A groups in each group: normal group, continuous 7d intraperitoneal injection (i.p.) 0.1ml/ saline, equal amount of normal saline (i.g.), /d, 7d, 1 times / D, 7d, B group: MPTP model group, 1 consecutive (physiological saline solution), 1 H was given the same amount of physiological saline (i.g.), 1 times /d, followed by 7d, given physiological saline (i.g.), 1 times /d, C, D group as TSG treatment group, MPTP (i.p.) 30mg/kg. I.p. (i.p.) 0.1ml/ times, 1h after TSG (i.g.) 20mg/kg or 40mg/kg, /d, then 7d only TSG (i.g.) 20mg/kg. Poles and open field experiments to measure behavioral changes.
2. after completing behavioral tests, the mice were perfused, and the brains were quickly taken and frozen sections were made by immunofluorescence staining.
The number of tyrosine hydroxylase (TH) positive neurons was measured.
The level of ROS was detected by 3. full band automatic enzyme labeling instrument.
4. the contents of DA, two hydroxyphenylacetic acid (DOPAC) and high vanillic acid (HVA) in striatum were detected by HPLC.
5. Western Blot detection JNK, p-JNK, P38, p-P38, ERK, p-ERK, Bcl-2, Bax, Cyt C.
[results]
1. behavioral results showed that in the climbing pole experiment, after the mice were given MPTP, the climbing pole time (TLa) was significantly increased (P 0.01) compared with the blank control group. But after the treatment of TSG, the TLa was significantly shorter than the model group, while the 40mg/kgTSG group improved significantly (P 0.01). There was no significant difference between the group of TSG and the blank control group (P 0.01). P > 0.05). After NAC, TLa was also improved significantly compared with the model group (Fig.2a, P 0.01). In the field test, the speed of movement (V) of mice decreased significantly after MPTP (P 0.01) compared with the blank control group (P 0.01), and the central residence time (T) was significantly prolonged (Fig.2b, P 0.01). There was no significant difference between TSG group and blank control group (P > 0.05). After NAC treatment, compared with MPTP group, V increased and T decreased significantly (Fig. 2c, P 0.05).
2. the results of immunofluorescence staining showed that the count of TH positive neurons in the substantia nigra was double blind. The reduction of TH positive neurons in the model group was about 66.9% (P0.01) compared with the blank control group under the fluorescence microscope. However, after TSG treatment, the TH positive neurons were in a dose-dependent manner. The TH positive neurons in the TSG group were only given the TH positive nerve. Similar to the high dose group, TH positive neurons increased significantly after NAC treatment (Fig. 3, P < 0.05).
3. the results of high performance liquid phase showed that compared with the blank control group, the DA level in the striatum decreased by 82%, and the level of DOPAC and HVA decreased to 15% and 19% in the normal group, but the level of DA, DOPAC, HVA increased gradually and was dose-dependent after the treatment of TSG. The level of DA, DOPAC, HVA level of the mice given to TSG was compared with the normal group. There was no statistical difference (P > 0.05), but after treatment with NAC, the levels of DA, DOPAC and HVA also significantly recovered (Fig.4, P < 0.05).
The results of 4. full band autofluorescence enzyme labeled ROS showed that in the MPTP treatment group, the level of ROS was significantly higher than that in the blank control group (P0.01). After the treatment of TSG (20 and 40mg/kg) or NAC in different doses, the level of ROS decreased significantly (P0.01) compared with the model group, and TSG was dose-dependent. Statistical significance (P > 0.05) indicates that the protective effect of TSG is similar to that of NAC (Fig.5).
The results of 5. Western Blot assay showed that the TSG (20 and 40mg/kg) intervention group could significantly improve the activation of JNK, P38 and mitochondrial pathway, but had no effect on ERK pathway. The mice given NAC therapy were similar to TSG, but also inhibited the activation of JNK, P38, and mitochondrial pathways.
[Conclusion]
1. TSG has neuroprotective effect on MPTP induced PD mice.
2. TSG may play a neuroprotective role in MPTP-induced PD mice by inhibiting ROS-mediated JNK, P38 and mitochondrial pathways.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R742.5
本文編號(hào)：2155852
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a common central nervous system degenerative disease in middle-aged and elderly people with static tremor, muscle stiffness, motion retardation, and postural reflex disorder. The pathological features of the middle cerebral cortex (substantia nigra pars compact, SNpc) dopamine (dopamine, DA) can be deity The current drug treatment does not prevent or delay the process of PD and can only alleviate the symptoms, and long-term treatment will result in diminishing effect and many side effects. Studies have shown that the important factors in the pathogenesis of PD may be oxidative stress or mitochondrial dysfunction. Further studies have shown that the MAPK pathway plays an important role in the pathogenesis of PD. Blocking the activation of JNK and P38 can inhibit MPTP induced neurotoxicity.
1- methyl -4 phenyl -1,2,3,6- four hydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyrid-ine, MPTP) is one of the classic PD modeling drugs. Its metabolite, 1- methyl -4 phenyl pyridine ion (1-methy-4-phenylpyridinium, MPP+), is the main substance to induce neurotoxicity. In DA neurons, MPP+ can inhibit the mitochondrial complex. Destroying the electron respiratory chain, resulting in a large number of ROS, further leading to the deletion of DA neurons in substantia nigra.
The root of Polygonum multiflorum is a traditional Chinese herbal medicine that has anti aging effect. Two 2,3,5,4 '-tetrahydroxystilbene-2-O- beta -D-glucoside (TSG) is one of its main active components. It has many biological characteristics, such as antioxidant, anti-inflammatory, and anti arteriosclerosis. It is found that TSG can not only inhibit ROS protection. Brain tissue, and can block JNK activation to protect the brain from ischemia injury, and TSG can reduce the overexpression of alpha synuclein (alpha -synuclein, alpha -syn) (APP transgenic Alzheimer's disease model). It also inhibits the neurotoxic effect of MPP+ on SH-SY5Y cells.
Recent experiments in our team showed that TSG could inhibit the apoptosis of PC12 cells induced by MPP+ by blocking the JNK pathway of the ROS-NO. In order to further study the neuroprotective effect of TSG in the body, the aim of this experiment is as follows: 1) use the PD mouse model to verify the neuroprotective effect of TSG; 2) determine whether this neuroprotective effect is possible. It is related to ROS mediated JNK, P38, and mitochondrial pathway.
[Objective]
1. the PD mouse model induced by MPTP was established.
2. to observe the effects of TSG on the behavior, morphology and biochemistry of MPTP induced PD mice.
3. to explore whether TSG regulates ROS mediated JNK, P38 and mitochondrial pathways, and plays a neuroprotective role in MPTP induced PD mice.
[method]
1. animal groups and model building: the experimental animals were randomly divided into 7 groups, 10.A groups in each group: normal group, continuous 7d intraperitoneal injection (i.p.) 0.1ml/ saline, equal amount of normal saline (i.g.), /d, 7d, 1 times / D, 7d, B group: MPTP model group, 1 consecutive (physiological saline solution), 1 H was given the same amount of physiological saline (i.g.), 1 times /d, followed by 7d, given physiological saline (i.g.), 1 times /d, C, D group as TSG treatment group, MPTP (i.p.) 30mg/kg. I.p. (i.p.) 0.1ml/ times, 1h after TSG (i.g.) 20mg/kg or 40mg/kg, /d, then 7d only TSG (i.g.) 20mg/kg. Poles and open field experiments to measure behavioral changes.
2. after completing behavioral tests, the mice were perfused, and the brains were quickly taken and frozen sections were made by immunofluorescence staining.
The number of tyrosine hydroxylase (TH) positive neurons was measured.
The level of ROS was detected by 3. full band automatic enzyme labeling instrument.
4. the contents of DA, two hydroxyphenylacetic acid (DOPAC) and high vanillic acid (HVA) in striatum were detected by HPLC.
5. Western Blot detection JNK, p-JNK, P38, p-P38, ERK, p-ERK, Bcl-2, Bax, Cyt C.
[results]
1. behavioral results showed that in the climbing pole experiment, after the mice were given MPTP, the climbing pole time (TLa) was significantly increased (P 0.01) compared with the blank control group. But after the treatment of TSG, the TLa was significantly shorter than the model group, while the 40mg/kgTSG group improved significantly (P 0.01). There was no significant difference between the group of TSG and the blank control group (P 0.01). P > 0.05). After NAC, TLa was also improved significantly compared with the model group (Fig.2a, P 0.01). In the field test, the speed of movement (V) of mice decreased significantly after MPTP (P 0.01) compared with the blank control group (P 0.01), and the central residence time (T) was significantly prolonged (Fig.2b, P 0.01). There was no significant difference between TSG group and blank control group (P > 0.05). After NAC treatment, compared with MPTP group, V increased and T decreased significantly (Fig. 2c, P 0.05).
2. the results of immunofluorescence staining showed that the count of TH positive neurons in the substantia nigra was double blind. The reduction of TH positive neurons in the model group was about 66.9% (P0.01) compared with the blank control group under the fluorescence microscope. However, after TSG treatment, the TH positive neurons were in a dose-dependent manner. The TH positive neurons in the TSG group were only given the TH positive nerve. Similar to the high dose group, TH positive neurons increased significantly after NAC treatment (Fig. 3, P < 0.05).
3. the results of high performance liquid phase showed that compared with the blank control group, the DA level in the striatum decreased by 82%, and the level of DOPAC and HVA decreased to 15% and 19% in the normal group, but the level of DA, DOPAC, HVA increased gradually and was dose-dependent after the treatment of TSG. The level of DA, DOPAC, HVA level of the mice given to TSG was compared with the normal group. There was no statistical difference (P > 0.05), but after treatment with NAC, the levels of DA, DOPAC and HVA also significantly recovered (Fig.4, P < 0.05).
The results of 4. full band autofluorescence enzyme labeled ROS showed that in the MPTP treatment group, the level of ROS was significantly higher than that in the blank control group (P0.01). After the treatment of TSG (20 and 40mg/kg) or NAC in different doses, the level of ROS decreased significantly (P0.01) compared with the model group, and TSG was dose-dependent. Statistical significance (P > 0.05) indicates that the protective effect of TSG is similar to that of NAC (Fig.5).
The results of 5. Western Blot assay showed that the TSG (20 and 40mg/kg) intervention group could significantly improve the activation of JNK, P38 and mitochondrial pathway, but had no effect on ERK pathway. The mice given NAC therapy were similar to TSG, but also inhibited the activation of JNK, P38, and mitochondrial pathways.
[Conclusion]
1. TSG has neuroprotective effect on MPTP induced PD mice.
2. TSG may play a neuroprotective role in MPTP-induced PD mice by inhibiting ROS-mediated JNK, P38 and mitochondrial pathways.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R742.5
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