本文選題：腦卒中 + SAA�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：在世界范圍內(nèi),腦卒中是一種伴有較高發(fā)病率和致死率的常見健康問題,它在發(fā)展中國家的發(fā)生率呈快速上升趨勢。世界衛(wèi)生組織預(yù)估,每年有1500萬人新發(fā)腦卒中,70%伴有殘疾甚至死亡。在老齡人口中,腦卒中導(dǎo)致的死亡率高達(dá)三分之二,是第二大致死性疾病。近年來,在中國等發(fā)展中國家中由于生活方式的改變,腦卒中的發(fā)病率呈現(xiàn)上升趨勢,我們亟需了解腦卒中的潛在發(fā)病機(jī)制并發(fā)展新的治療方法。在腦血管病的發(fā)展進(jìn)程中,活性氮及硝化應(yīng)激(Nitrosative stress)參與介導(dǎo)細(xì)胞損傷。缺血性腦損傷引起腦部血液供應(yīng)中斷,導(dǎo)致缺氧和低糖,以致神經(jīng)元無法維持細(xì)胞穩(wěn)態(tài)和基本功能所需的離子濃度梯度。研究表明,在缺血的腦組織中,谷氨酸含量在缺血發(fā)生后快速上升。過量激活NMDA谷氨酸受體并導(dǎo)致鈣離子(Ca2+)濃度上升,Ca2+的釋放會使各種信號通路激活最終損傷神經(jīng)元功能甚至引起細(xì)胞死亡。Ca2+介導(dǎo)鈣調(diào)蛋白(CaM)和一氧化氮合酶(NOS)的偶聯(lián),改變內(nèi)皮型一氧化氮合酶(eNOS)的功能。阻斷eNOS的功能會因?yàn)镹ADPH氧化酶的激活而引起超氧根離子(O2-·)濃度上升。一氧化氮(NO)和O2-·濃度上升,最終反應(yīng)生成過氧亞硝基陰離子(ONOO-)。以過氧亞硝基陰離子為代表的活性氮形成的硝化應(yīng)激,會影響包括血管舒張,神經(jīng)傳遞,免疫防御和細(xì)胞運(yùn)動性調(diào)控等多種細(xì)胞生理過程。硝化應(yīng)激與多種疾病發(fā)生發(fā)展進(jìn)程相關(guān),它對細(xì)胞結(jié)構(gòu)的損傷包括脂類,細(xì)胞膜,蛋白質(zhì)和DNA。大腦脈管系統(tǒng)是危險(xiǎn)因素下硝化應(yīng)激介導(dǎo)的缺血性腦損傷病理過程中的重要靶組織。因此,靶向硝化應(yīng)激的神經(jīng)保護(hù)策略可能對腦卒中的治療具有積極的作用。在本研究中,我們分別使用了兩種天然強(qiáng)效抗氧劑——丹酚酸A(SAA)和β-胡蘿卜素(BC)。利用小鼠短暫性中腦動脈閉塞(tMCAO)模型,考察它們保護(hù)缺血性腦損傷的藥理學(xué)作用及潛在分子機(jī)制。在中藥相關(guān)文獻(xiàn)中,SAA被稱為丹參。SAA是一種多靶點(diǎn)藥物,抑制多種缺血損傷因子所激活的胞內(nèi)信號通路。SAA是從中藥丹參中提取而得,已有文獻(xiàn)報(bào)道,它對心腦血管疾病有治療作用。丹參根和莖的切片經(jīng)薄層色譜分析,柱色譜提純,高效液相色譜制備粗產(chǎn)。我們考察了 SAA抗神經(jīng)血管損傷的藥理學(xué)效應(yīng),此外也探索了其藥理作用與激活鈣蛋白酶和eNOS脫偶聯(lián)之間的關(guān)系。C57BL/6雄性小鼠在中腦動脈梗阻60min模型建立前連續(xù)七天給予腹腔注射SAA(1 mg/kg和5mg/kg)或生理鹽水作為對照。高分辨率正電子發(fā)射斷層(PET)掃描儀可用于小型動物成像以檢測18F-FDG葡萄糖在腦內(nèi)的代謝,用于再灌注24小時(shí)后藥物改善腦代謝功能的檢測;再灌注23h后,轉(zhuǎn)棒實(shí)驗(yàn)及神經(jīng)功能學(xué)評分實(shí)驗(yàn)用于評估小鼠功能性行為;腦梗阻程度采用Nissl染色進(jìn)行考察。鈣蛋白水解酶的活性,eNOS脫偶聯(lián)以及其它細(xì)胞凋亡級聯(lián)反應(yīng)關(guān)聯(lián)蛋白也作為檢測SAA抗神經(jīng)血管損傷的重要指標(biāo)。相較于模型組,SAA(1 mg/kg和5 mg/kg)預(yù)處理的組可有效減輕神經(jīng)功能障礙。在旋轉(zhuǎn)實(shí)驗(yàn)中,SAA處理組相較于模型組跌倒傾向明顯降低。Nissl染色腦片顯示模型組存在大面積腦梗塞,而SAA(1 mg/kg和5 mg/kg)處理組腦梗塞面積明顯有所減少。PET結(jié)果提示,SAA預(yù)處理可以有效提高模型組葡萄糖的代謝率。相較于假手術(shù)組,模型組小鼠腦內(nèi)缺血側(cè)相較于對照側(cè)區(qū)域的攝取值的比值明顯減少,而SAA預(yù)處理可有些改善兩側(cè)的差異。此外,SAA可以抑制鈣蛋白酶的水解能力。血影蛋白Spectrin的裂解量間接反映了鈣蛋白酶的活性。蛋白質(zhì)免疫印跡分析顯示,相較于假手術(shù)組,缺血側(cè)腦組織的血影蛋白崩解片段明顯增多。用SAA(1 mg/kg和5 mg/kg)處理可以有效減輕保護(hù)血影蛋白崩解片段的產(chǎn)生。腦卒中也會加強(qiáng)鈣蛋白酶介導(dǎo)的鈣調(diào)磷酸酶(CaN)的激活,SAA(1 mg/kg和5 mg/kg)處理同樣也會抑制鈣蛋白酶介導(dǎo)的CaN降解。此外,eNOS二聚體的形成可以產(chǎn)生生理效應(yīng)的NO,然而單體的形成則作為eNOS脫偶聯(lián)的標(biāo)志物,可促進(jìn)OO-生成。給予SAA(1 mg/kg和5 mg/kg)后,可以顯著減少tMCAO后eNOS二聚體和單體的比率,具有一定量效關(guān)系。在免疫印跡分析中,我們觀察到模型組缺血腦區(qū)AKT,ERK和FKHR的去磷酸化現(xiàn)象。我們推測磷酸化水平的下降可能是由eNOS脫偶聯(lián)引起的O2-·上升及硝化應(yīng)激導(dǎo)致的。SAA預(yù)處理(1 mg/kg和5 mg/kg)能顯著提高AKT,ERK和FKHR的磷酸化水平。我們的實(shí)驗(yàn)結(jié)果顯示SAA具有較好的抗缺血性神經(jīng)損傷作用。SAA給藥提高缺血小鼠腦葡萄糖的代謝及抗神經(jīng)功能障礙與其抑制鈣蛋白酶的激活、抑制eNOS脫偶聯(lián)及硝化應(yīng)激損傷相關(guān),從而保護(hù)大腦免遭缺血損傷。β-胡蘿卜素(BC)是一種具有抗氧化功能的重要類胡蘿卜素。β-胡蘿l卜素因具有抗氧化功能而降低多種心臟和腦部疾病的風(fēng)險(xiǎn),有報(bào)道稱低劑量的β-胡蘿卜素可降低心肌缺血的發(fā)病風(fēng)險(xiǎn)。血腦屏障可以阻擋大量的中樞神經(jīng)系統(tǒng)激動劑。過去幾十年,納米粒子在中樞神經(jīng)系統(tǒng)給藥領(lǐng)域占據(jù)重要地位,納米給藥系統(tǒng)有加速藥物穿過BBB的功能。腦損傷靶標(biāo)分子的修飾可幫助納米給藥系統(tǒng)靶向到特定損傷區(qū)域,本實(shí)驗(yàn)考察利用硝基酪氨酸抗體修飾給藥系統(tǒng)的靶向性。實(shí)驗(yàn)中,我們采用了三種β-胡蘿卜素制劑,包括提高β-胡蘿卜素溶解度的乳劑(BC-EM),BC偶聯(lián)PEG修飾的脂質(zhì)納米粒(BC-PLNs),抗3-硝基酪氨酸抗體修飾的BC偶聯(lián)PEG修飾的脂質(zhì)納米粒(BC-NT/PLN)。實(shí)驗(yàn)分為七個(gè)組,包括假手術(shù)組,模型組,BC-EM(10 mg/kg)組,BC-PLNs(2.5 mg/kg)組,BC-NT/PLNs(0.625 mg/kg,1.25mg/kg 或 2.5 mg/kg)組。MCAO模型建立后 60 min,根據(jù)小鼠體重按比例靜脈注射藥物或藥物載體。在再灌注23小時(shí)末估算神經(jīng)功能損傷程度,并取出大腦樣本進(jìn)行梗阻損傷區(qū)域的Nissl染色。通過腦片與熒光探針NP3孵育的結(jié)果評價(jià)ONOO-的產(chǎn)生量。免疫印跡技術(shù)可見鈣蛋白酶的激活,緊密連接蛋白,phospho-AKT(Ser 473),phospho-FKHR(Ser256)和 phospho-ERK(Thr202/Tyr204)的磷酸化。BBB 的完整性由伊文氏藍(lán)對組織溢出物染色來鑒定。我們發(fā)現(xiàn)相較于空白組,BC-EM對神經(jīng)損傷有明顯的修復(fù)作用。而相較于BC-EM,BC-PLNs在極低濃度時(shí)就能顯示神經(jīng)保護(hù)功能。2.5 mg/kg劑量的BC-PLNs在減少神經(jīng)損傷和縮小梗阻區(qū)域方面顯示出特有的優(yōu)勢。但是,為了達(dá)到治療腦卒中的目的,我們要將附有3-硝基酪氨酸抗體的BC靶向到缺血區(qū)域。在中腦動脈梗阻和再灌注時(shí)相中有大量ONOO-產(chǎn)生。相較于空白組,低劑量(0.625mg/kg或1.25 mg/kg)被NT/PLNs偶聯(lián)的BC即可對功能損傷有所改善,而2.5 mg/kg的劑量對損傷沒有顯著改變。同時(shí),靜注低劑量(0.625mg/kg或1.25 mg/kg)被3-NT/PLNs修飾的BC有效減少M(fèi)CAO模型中的梗阻區(qū)域。NT/BC/PLNs在低劑量時(shí)具有神經(jīng)保護(hù)作用并且能對抗腦缺血引起的損傷。我們的結(jié)果顯示,BC-EM(10 mg/kg),PLNs 包載 2.5 mg/kg BC 和 0.625mg/kg 3NT/PLNS能有效激活鈣蛋白酶并抑制血影蛋白和CaN的降解。我們發(fā)現(xiàn),給予BC-EM(10 mg/kg)可有效抑制 phospho-P38(Thr180/Tyr182)和 phospho-JNK(Thr183/Tyr185)的上調(diào)。缺血會引起許多蛋白 phospho-AKT(Ser 473),phospho-FKHR(Ser256)和 phospho-ERK(Thr202/Tyr204)磷酸化水平的下降。NT/BC/PLNs(0.625mg/kg)有效抑制上述蛋白質(zhì)磷酸化水平的下調(diào)。過氧亞硝基陰離子(ONOO-)是一種對組織有損傷作用的強(qiáng)氧化劑和強(qiáng)硝化劑,硝化應(yīng)激在缺血過程中能導(dǎo)致腦微血管損傷及破壞神經(jīng)血管單元。NP3作為一種對ONOOC敏感的熒光探針,孵育使用后,相比于對照組,模型組大腦受損側(cè)局部微血管處顯示極強(qiáng)的NP3熒光強(qiáng)度,提示ONOO-濃度出現(xiàn)顯著上升。NT/PEG納米結(jié)合物(0.625mg/kg)有效抑制硝化應(yīng)激并保護(hù)微血管受損。利用受損的血管中滲漏出的伊文氏藍(lán)可以判斷微血管損傷程度。在空白組中,MCAO 24小時(shí)后,伊文氏藍(lán)溶液從血管滲入腦實(shí)質(zhì)的量大大提高。相比之下,NT/PEG-納米結(jié)合物(0.625mg/kg)組則有效減少伊文氏藍(lán)的滲漏并且抑制了緊密連接蛋白ZO-1和閉合蛋白Occludin的退化。相較于常規(guī)β-胡蘿卜素乳劑,較低劑量的BC腦靶向遞藥系統(tǒng)NT/BC/PLNs有效地改善了腦損傷。利用抗體偶聯(lián)策略,NT/BC/PLNs遞藥系統(tǒng)將β-胡蘿卜素靶向輸送到ONOO-生成的腦缺血損傷區(qū)域。此外,該系統(tǒng)減少ONOO-的生成,并且抑制伊文氏藍(lán)滲漏,降低微血管的破損。同時(shí)通過抑制鈣蛋白酶的激活,減少缺血損傷中血影蛋白和CaN的降解,最終起到保護(hù)神經(jīng)血管單元作用。綜上所述,硝化應(yīng)激參與缺血后腦損傷病理過程。丹酚酸A和β-胡蘿卜素具有一定的抗缺血性腦損傷作用,其分子機(jī)制與減輕eNOS脫偶聯(lián)和硝化應(yīng)激損傷相關(guān)聯(lián)。此外,我們發(fā)現(xiàn)偶聯(lián)Nitrotyrosine抗體的腦靶向納米給藥系統(tǒng)不僅可以定向輸送藥物到缺血腦區(qū),同時(shí)也可以減少負(fù)載藥物劑量起到同等治療作用。
[Abstract]:In the world, stroke is a common health problem with high morbidity and mortality, and its incidence in developing countries is rising rapidly. The WHO estimates that 15 million people have new stroke, 70% with disability or even death every year. The mortality rate of stroke is up to 2/3 in the elderly population. In recent years, the incidence of stroke is rising in China and other developing countries, in China and other developing countries. We need to understand the potential pathogenesis of stroke and develop new treatment methods. In the development of cerebrovascular disease, active nitrogen and nitrification stress (Nitrosative stress) participate in the development of cerebrovascular disease. Mediating cell damage. Ischemic brain damage causes disruption of the blood supply of the brain, resulting in hypoxia and low sugar, so that neurons are unable to maintain the ionic concentration gradient required for cell homeostasis and basic function. The release of ions (Ca2+) increases, and the release of Ca2+ activates the function of the signaling pathway to eventually damage the neuron function and even causes the cell death.Ca2+ to mediate the coupling of calmodulin (CaM) and nitric oxide synthase (NOS), and changes the function of the endothelial nitric oxide synthase (eNOS). The inhibition of the function of eNOS causes SUPEROXYGEN due to the activation of NADPH oxidase. The concentration of the root ion (O2-) rises. The concentration of nitric oxide (NO) and O2- increases, and the final reaction produces peroxy nitroso anion (ONOO-). Nitrification stress formed by the active nitrogen, represented by the peroxy nitroso anions, will affect a variety of cell physiological processes, including vasodilatation, neurotransmission, immune defense and cell motility regulation. Stress is associated with the development of a variety of diseases. Its damage to cell structures, including lipids, cell membranes, protein and DNA. cerebral vascular system, is an important target tissue in the pathological process of ischemic brain damage mediated by nitrification stress. Therefore, the neuroprotective strategy targeted to nitrification stress may be used to treat stroke. In this study, two natural strong antioxidants, salvianolic acid A (SAA) and beta carotene (BC), were used respectively. The pharmacological action and potential molecular mechanism of the mouse transient middle cerebral artery occlusion (tMCAO) were used to investigate the protection of ischemic brain damage. In the related literature of Chinese medicine, SAA was called Salvia miltiorrhiza. .SAA is a multi target drug. The intracellular signaling pathway,.SAA, which is activated by a variety of ischemic injury factors, is extracted from the traditional Chinese medicine Salvia miltiorrhiza. It has been reported in the literature. It has a therapeutic effect on cardiovascular and cerebrovascular diseases. The slices of root and stem of Salvia miltiorrhiza are analyzed by thin layer chromatography, purified by column chromatography and high performance liquid chromatography to produce crude production. We have investigated SAA The pharmacological effects of anti neurovascular injury, and the relationship between its pharmacological effects and the activation of calsin and eNOS deactivation were also explored..C57BL/6 male mice were given SAA (1 mg/kg and 5mg/kg) or physiological salt water for seven days before the establishment of 60min model of middle cerebral artery obstruction. PET) the scanners can be used for small animal imaging to detect the metabolism of 18F-FDG glucose in the brain and to improve the detection of brain metabolism after 24 hours of reperfusion. After reperfusion of 23h, the rotation rod experiment and neurologic score test are used to evaluate the functional behavior of mice; the degree of cerebral infarction is examined by Nissl staining. Enzyme activity, eNOS decoupling and other cell apoptosis cascade reaction proteins are also important indicators for detecting SAA's neurovascular damage. Compared to model groups, SAA (1 mg/kg and 5 mg/kg) pretreated groups can effectively reduce neural dysfunction. In the rotation experiment, the group of SAA is prone to lower.Nissl compared to the model group falling down. Dyed brain slices showed that there was a large area of cerebral infarction in the model group, while the area of cerebral infarction in SAA (1 mg/kg and 5 mg/kg) treatment group was significantly reduced by.PET, and SAA preconditioning could effectively improve the metabolic rate of glucose in the model group. Compared to the sham group, the ratio of the blood deficiency in the model group to the control side of the control group was obvious. SAA pretreatment could significantly reduce the difference between the two sides. In addition, SAA could inhibit the hydrolysis of calpain. The fragmentation of HP Spectrin reflects the activity of calpain indirectly. Protein immunoblotting analysis showed that the disintegration fragment of the ischemic side group was significantly increased compared with the sham group. SAA (1 m) G/kg and 5 mg/kg) treatment can effectively reduce the production of the protection of the disintegration fragments. Stroke also enhances calprotease mediated calcineurin (CaN) activation. SAA (1 mg/kg and 5 mg/kg) can also inhibit calprotease mediated CaN degradation. In addition, the formation of eNOS two polymers can produce physiological effects of NO, but monomers The formation of eNOS, as a marker for eNOS decoupling, can promote OO- formation. After giving SAA (1 mg/kg and 5 mg/kg), the ratio of eNOS two polymers and monomers can be significantly reduced after tMCAO. In Western blot analysis, we observed the dephosphorylation of AKT, ERK and FKHR in the ischemic brain area of the model group. We speculate phosphorylated water. The decrease of the level may be the.SAA preconditioning (1 mg/kg and 5 mg/kg) induced by eNOS dehydration and nitrification stress (1 mg/kg and 5 mg/kg). Our experimental results show that SAA has a better anti ischemic nerve damage effect and.SAA gives the metabolism and anti nerve of glucose in the brain of high ischemic mice. Dysfunction is related to the inhibition of calsin activation, inhibition of eNOS decoupling and nitrification stress damage, which protects the brain from ischemic injury. Beta carotene (BC) is an important carotenoid with antioxidant function. Beta carotene L dopin has the risk of reducing various heart and brain diseases because of its antioxidant function. The low dose of beta carotene can reduce the risk of myocardial ischemia. The blood brain barrier can block a large number of central nervous system agonists. Nanoparticles have played an important role in the central nervous system for the past few decades. The nano drug delivery system has the function of accelerating the drug passing through the BBB. The modification of the target molecules of the brain damage can help. In this experiment, we used three beta carotene preparations, including BC-EM, BC coupled with PEG modified lipid nanoparticles (BC-PLNs), and the anti 3- nitrotyrosine antibody repair. BC coupled PEG modified lipid nanoparticles (BC-NT/PLN). The experiment was divided into seven groups, including sham operation group, model group, BC-EM (10 mg/kg) group, BC-PLNs (2.5 mg/kg) group, BC-NT/PLNs (0.625 mg/kg, 1.25mg/kg, or 2.5 mg/kg) group.MCAO model after establishment 60. The root was injected intravenously or drug carrier according to the weight of mice. In the reperfusion 23 The degree of nerve function damage was estimated at the end of the hour, and the brain samples were taken out for Nissl staining in the area of the obstruction. The production of ONOO- was evaluated by the incubation of brain slices with the fluorescent probe NP3. The immunoblotting technique showed the activation of calsin, the close connexin, phospho-AKT (Ser 473), phospho-FKHR (Ser256) and phospho-ERK (Thr202/T). The integrity of phosphorylated.BBB in yr204 was identified by the staining of eviner blue against tissue spillovers. We found that BC-EM had a significant repair effect on nerve damage compared to the blank group. Compared to BC-EM, BC-PLNs at very low concentrations could show the BC-PLNs of the neuroprotective function of.2.5 mg/kg in reducing nerve damage and narrowing the area of obstruction. The domain shows a unique advantage. However, in order to achieve the purpose of treating stroke, we should target the BC with 3- nitrotyrosine antibody to the ischemic region. There are a large number of ONOO- in the middle cerebral artery obstruction and reperfusion. Compared to the blank group, the low dose (0.625mg/ kg or 1.25 mg/kg) is NT/PLNs coupled BC to the function. The damage was improved, and the dose of 2.5 mg/kg did not change significantly. At the same time, intravenous injection of low dose (0.625mg/kg or 1.25 mg/kg) by 3-NT/PLNs modified BC effectively reduced the.NT/BC/PLNs in the MCAO model, which had a neuroprotective effect at low doses and could antagonize the damage caused by cerebral ischemia. Our results showed that BC-EM (10) Mg/kg), PLNs encapsulation of 2.5 mg/kg BC and 0.625mg/kg 3NT/PLNS effectively activates calsin and inhibits the degradation of spectrin and CaN. We have found that BC-EM (10 mg/kg) can effectively inhibit the up regulation of phospho-P38 (Thr180/Tyr182) and phospho-JNK (473). R256) and the decrease of phosphorylation level of phospho-ERK (Thr202/Tyr204).NT/BC/PLNs (0.625mg/kg) effectively inhibit the downregulation of the protein phosphorylation level. Peroxiso nitroso anion (ONOO-) is a strong oxidant and strong nitrifying agent that has damage to tissue. Nitrification should induce cerebral microvascular damage and destruction of the brain during ischemia. The vascular unit.NP3 was used as a ONOOC sensitive fluorescent probe. After incubation, compared to the control group, the local microvessels of the brain damaged side of the model group showed a strong NP3 fluorescence intensity, suggesting a significant increase in the concentration of ONOO- in the.NT/PEG nanoscale (0.625mg/kg) to inhibit nitrification stress and protect the microvascular damage. In the blank group, the amount of even's blue solution infiltrated from the blood vessels to the parenchyma significantly increased in the blank group. In contrast, the NT/PEG- nanocombination (0.625mg/kg) group reduced the leaking of even blue and suppressed the close connexin ZO-1 and closed eggs in the 0.625mg/kg group. The degradation of white Occludin. Compared to the conventional beta carotene emulsion, the lower dose of the BC brain targeting delivery system NT/BC/PLNs effectively improves brain damage. The NT/BC/PLNs delivery system uses the antibody coupling strategy to deliver beta carotene targeting to ONOO- generated ischemic injury areas. In addition, the system reduces the formation of ONOO- and inhibits the formation of the beta carotene. Evans blue leaks and reduces the damage of microvessels. At the same time, by inhibiting the activation of calsin, reducing the degradation of blood shadow protein and CaN in ischemic injury, and ultimately protecting the neurovascular unit. To sum up, nitrification stress participates in the pathological process of cerebral injury after ischemia. The salvianolic acid A and beta carotene have certain anti ischemic brain damage. The molecular mechanism of injury is associated with the reduction of eNOS decoupling and nitrification stress damage. In addition, we found that the brain targeted nano drug delivery system coupled with Nitrotyrosine antibodies can not only deliver drugs to the ischemic brain area, but also reduce the dose of the loaded drug to the same therapeutic effect.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285

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本文編號：2068275