本文選題：燈盞花乙素 + 腦缺血再灌注��； 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：腦卒中目前在全球范圍內(nèi),已成為被人們廣泛關(guān)注的健康難題,包括缺血性卒中和出血性卒中。缺血性卒中是腦卒中的主要臨床類型,約占60%-80%。氧化應(yīng)激反應(yīng)引起的過氧化損傷是缺血性腦損傷發(fā)生的重要機(jī)制,在腦組織缺血再灌注損傷的病理過程中,氧化應(yīng)激產(chǎn)生大量活性氧,可導(dǎo)致DNA、蛋白質(zhì)和脂質(zhì)等的損傷,并可直接作用于生物膜上的不飽和脂肪酸,損傷生物膜。過量的活性氧會破壞細(xì)胞的正常功能,降低多種酶的活性及DNA和RNA的穩(wěn)定性,誘發(fā)細(xì)胞分子(如蛋白質(zhì)、DNA及脂質(zhì))過氧化損傷,促進(jìn)細(xì)胞的凋亡。在腦缺血再灌注損傷的過程中,活性氧存在多種來源途徑,其中,NADPH(還原型煙酰胺腺嘌呤二核苷酸磷酸)氧化酶(NOX)被認(rèn)為是腦缺血再灌注后活性氧水平異常升高的一個(gè)關(guān)鍵因子。目前研究證明在NADPH氧化酶中,NOX2在缺血性卒中中扮演重要角色,與缺血性腦卒中等疾病密切相關(guān)。大量研究表明,上調(diào)缺血腦組織中NADPH氧化酶的表達(dá)會加重腦缺血再灌注損傷,增加神經(jīng)元死亡,而抑制NOX2的活性可以減輕腦缺血再灌注損傷。"脈為血府,氣為血帥",益氣能夠生血,祛瘀能夠生新,活血能夠生肌。生血、新、肌就包括生新脈和新絡(luò)的意義。故脈絡(luò)的新生無法離開氣血,所以益氣補(bǔ)血藥可以發(fā)揮生成血管的功能。所以益氣可以生脈。對于中風(fēng),古今醫(yī)家均以促進(jìn)腦絡(luò)通暢為最終目的。燈盞生脈膠囊是臨床上在治療腦梗塞等缺血性腦損傷疾病中具有顯著療效的"益氣生脈"中成藥,我們承擔(dān)了國際上首個(gè)探討國產(chǎn)品牌中成藥對缺血性卒中二級預(yù)防作用的大型臨床研究-"燈盞生脈膠囊干預(yù)缺血中風(fēng)二級預(yù)防的多中心、隨機(jī)對照、雙盲臨床試驗(yàn)",結(jié)果顯示:燈盞生脈膠囊可降低首發(fā)癥狀性腦梗死患者非致死性卒中再發(fā)率27.8%,其可有效改善缺血卒中患者殘障程度,且不增加腦出血及其它出血性風(fēng)險(xiǎn),在臨床上已廣泛用于缺血性心腦血管疾病的防治,燈盞生脈膠囊以燈盞細(xì)辛為主藥,而燈盞花乙素(Scutellarin)是從燈盞細(xì)辛中提取的主要黃酮類活性成分之一,化學(xué)名為4',5,6三羥基黃酮-葡萄糖醛酸苷。近年來基礎(chǔ)研究表明燈盞花乙素對腦血管的損傷具有保護(hù)作用。那么燈盞花乙素保護(hù)腦缺血再灌注損傷的作用機(jī)制是什么?本研究利用星形膠質(zhì)細(xì)胞缺糖缺氧模型和大鼠大腦中動脈栓塞模型,探討燈盞花乙素保護(hù)腦缺血再灌注損傷的可能作用機(jī)制。目的:通過細(xì)胞模型(星型膠質(zhì)細(xì)胞缺氧/再復(fù)氧模型)及整體動物(大鼠大腦中動脈栓塞模型,tMCAO)研究燈盞花乙素處理對NOX2及過氧化損傷的改變,明確燈盞花乙素保護(hù)腦缺血再灌注后神經(jīng)損傷的信號機(jī)制。方法:1.體外培養(yǎng)原代星形膠質(zhì)細(xì)胞,制備缺糖缺氧/復(fù)糖復(fù)氧損傷的細(xì)胞模型。將星形膠質(zhì)細(xì)胞分為正常對照組、缺糖缺氧/復(fù)糖復(fù)氧組、Scu低濃度(2 μM)+缺糖缺氧/復(fù)糖復(fù)氧組、Scu中濃度(10μM/L)+缺糖缺氧/復(fù)糖復(fù)氧組、Scu高濃度(50 μ M/L)+缺糖缺氧/復(fù)糖復(fù)氧組。分子對接技術(shù)研究燈盞花乙素和NOX2蛋白對接情況;Western Blot檢測NOX2蛋白表達(dá)情況;活性氧試劑盒檢測星形膠質(zhì)細(xì)胞內(nèi)活性氧含量。2.制備大鼠MCAO動物模型,采用簡單隨機(jī)分組法將SD大鼠隨機(jī)分為以下幾組:假手術(shù)組(sham)組、手術(shù)組、燈盞花乙素低濃度(50mg/kg)+手術(shù)組、燈盞花乙素高濃度(l00mg/kg)+手術(shù)組、不同濃度夾竹桃麻素+手術(shù)組。Bederson 5分法對大鼠進(jìn)行神經(jīng)行為學(xué)評分;TTC染色法檢測大鼠腦梗死面積的變化;Western Blot法檢測NOX2蛋白表達(dá)的變化情況;ELISA法檢測氧化應(yīng)激相關(guān)指標(biāo)的變化;免疫組化檢測大鼠腦組織中的細(xì)胞凋亡情況。結(jié)果:1.分子對接研究結(jié)果顯示燈盞花乙素和NOX2蛋白的活性口袋形狀相互吻合,二者的3D分子結(jié)構(gòu)對接良好。原代培養(yǎng)的星形膠質(zhì)細(xì)胞純度98%。缺糖缺氧2 h復(fù)糖復(fù)氧24 h后,燈盞花乙素(10,50 μM)可以改善細(xì)胞存活率下降(P0.05)。Western Blot顯示缺糖缺氧2 h后復(fù)糖復(fù)氧24 h的星形膠質(zhì)細(xì)胞NOX2表達(dá)水平高于正常對照組,燈盞花乙素(2,10,50 μM)預(yù)處理的缺糖缺氧2 h后復(fù)糖復(fù)氧24 h的星形膠質(zhì)細(xì)胞中NOX2表達(dá)水平與同時(shí)間點(diǎn)溶劑對照組相比下降(P0.05)。缺糖缺氧導(dǎo)致細(xì)胞內(nèi)活性氧水平升高,而燈盞花乙素(10,50 μM)能降低細(xì)胞內(nèi)活性氧的水平(P0.05)。2.大鼠神經(jīng)行為學(xué)評分、腦梗死面積百分率的檢測結(jié)果顯示,與sham組相比,I/R組大鼠神經(jīng)行為學(xué)評分,腦梗死面積百分率增高(P0.05),與I/R組相比,Scu(低、高濃度)+I/R組大鼠神經(jīng)行為學(xué)評分,腦梗死面積百分率降低(P0.05)。3.大鼠神腦組織細(xì)胞凋亡及氧化應(yīng)激相關(guān)指標(biāo)(8-羥基脫氧鳥苷,8-OHdG;4-羥基壬烯醛,4-HNE;3-硝基酪氨酸,3-NT)的檢測結(jié)果顯示與sham組相比,I/R組大鼠免疫組化陽性細(xì)胞率和氧化應(yīng)激相關(guān)指標(biāo)增高(P0.05)。假手術(shù)組、I/R3d組的Caspase-3陽性細(xì)胞百分率分別為12.20±1.62%、55.45±4.99%。假手術(shù)組、I/R 3d 組的 8-OHdG 分別為 100%、177.2%±31.2%;4-HNE 分別為 100%、163.6%± 28.7%;3-NT 分別為 100%、159.3%±32.1%。與 I/R 組相比,Scu(低、高濃度)+I/R組大鼠免疫組化陽性細(xì)胞率和氧化應(yīng)激相關(guān)指標(biāo)降低(P0.05)。Scu低濃度組、Scu高濃度組的Caspase-3陽性細(xì)胞百分率分別為22.36±3.37%、11.70± 1.79%。Scu 低濃度組、Scu 高濃度組的 8-OHdG 分別為 113.1%±33.1%、99.9%± 11.3%;4-HNE 分別為 112.0%± 32.1%、95.2%± 18.9%;3-NT 分別為 85.6%±31.3%、90.0%±20.5%。4.Western Blot檢測NOX2蛋白的結(jié)果顯示,與sham組相比,I/R組大鼠3d的NOX2蛋白表達(dá)水平增高(P0.05)。與I/R組相比,Scu(低、高濃度)+I/R組大鼠3 d的NOX2蛋白表達(dá)水平下降(P0.05)。夾竹桃麻素+I/R組大鼠3 d的NOX2蛋白的表達(dá)水平也下降(P0.05)。結(jié)論:1.燈盞花乙素對缺糖缺氧/復(fù)糖復(fù)氧誘導(dǎo)的星形膠質(zhì)細(xì)胞損傷具有保護(hù)作用。燈盞花乙素能夠抑制缺糖缺氧星形膠質(zhì)細(xì)胞NOX2蛋白的表達(dá),降低缺糖缺氧星形膠質(zhì)細(xì)胞內(nèi)活性氧水平。2.燈盞花乙素能夠保護(hù)腦缺血再灌注損傷,發(fā)揮NOX2蛋白抑制劑的作用。3.燈盞花乙素保護(hù)腦缺血再灌注損傷的作用機(jī)制與靶向調(diào)控NOX2引起的過氧化損傷有關(guān)。
[Abstract]:Cerebral apoplexy has become a worldwide concern, including ischemic stroke and hemorrhagic stroke. Ischemic stroke is the main clinical type of stroke. The oxidative damage caused by 60%-80%. oxidative stress is an important mechanism for ischemic brain injury, and it is in cerebral ischemia reperfusion injury. During the pathological process of injury, oxidative stress produces a large amount of active oxygen, which can cause damage to DNA, protein and lipid, and can directly affect the unsaturated fatty acids on the biofilm and damage the biofilm. Excessive active oxygen can destroy the normal function of the cells, reduce the viability of various enzymes and the stability of DNA and RNA, and induce cell molecules (such as protein). In the process of cerebral ischemia reperfusion injury, there are many sources of reactive oxygen species in the process of cerebral ischemia-reperfusion injury, in which NADPH (NOX) is considered to be a key factor in the abnormal increase of active oxygen level after cerebral ischemia reperfusion. In NADPH oxidase, NOX2 plays an important role in ischemic stroke and is closely related to ischemic stroke secondary disease. A large number of studies have shown that the up-regulated expression of NADPH oxidase in ischemic brain can aggravate cerebral ischemia reperfusion injury and increase neuron death, while inhibiting the activity of NOX2 can reduce cerebral ischemia reperfusion injury. "Pulse Blood can produce blood, Qi can produce blood, blood stasis can be born new, blood circulation can produce muscle. Blood, new, new, the meaning of new veins and new collateral. Therefore, the newborn can not leave Qi and blood, so Qi enriching blood medicine can play the function of generating blood vessels. So Qi Qi can produce pulse. For stroke, ancient and modern doctors all promote brain collaterals patency. End objective. Breviscapine Shengmai capsule is a clinical medicine which is clinically effective in the treatment of cerebral infarction and other ischemic brain injury diseases. We undertake the first international study of the first large-scale clinical research on the preventive effect of Chinese traditional Chinese patent medicine on the two stage of ischemic stroke - "breviscapine Shengmai capsule" in the intervention of two levels of ischemic stroke. A multi center, randomized controlled, double blind clinical trial, "the results showed: breviscapine Shengmai capsule can reduce the recurrence rate of non fatal stroke in patients with first onset symptomatic cerebral infarction by 27.8%. It can effectively improve the degree of disability in patients with ischemic stroke, without increasing cerebral hemorrhage and other hemorrhagic risk, and has been widely used in ischemic cardio cerebral vascular disease." The prevention and control of the disease, breviscapine Shengmai capsule with Breviscapine as the main drug, and breviscapine (Scutellarin) is one of the main flavonoids extracted from Erigeron breviscapus, named 4', 5,6 three hydroxy flavonoid glucuronide. In recent years, the basic research shows that breviscapine has protective effect on the damage of cerebral blood vessels. What is the mechanism of the protection of cerebral ischemia reperfusion injury by acetic acid? This study uses the model of astrocyte glucose deficiency anoxia and rat middle cerebral artery embolization to explore the possible mechanism of the protective effect of erigerin on the protection of cerebral ischemia reperfusion injury. Objective: to use the cell model (astrocyte anoxia / reoxygenation model) and the whole Body animals (rat model of middle cerebral artery embolism, tMCAO) study the changes in NOX2 and peroxidation damage by breviscapine treatment, and make clear the signal mechanism of breviscapine to protect the nerve injury after cerebral ischemia and reperfusion. Method: 1. the primary astrocytes were cultured in vitro, and the cell model of oxygen deficiency anoxia / reoxygenation injury was prepared. The quality cells were divided into normal control group, hypoxic hypoxia / reoxygenation group, low concentration of Scu (2 mu M) + oxygen deficiency anoxia / reoxygenation group, concentration of Scu (10 M/L) + oxygen deficiency anoxia / reoxygenation group, high concentration of Scu (50 mu M/L) + oxygen deficiency anoxia / reoxygenation group. The docking of breviscapine and NOX2 protein by molecular docking technique; Western Blot detection NOX The expression of 2 protein; the MCAO animal model of rats was prepared by active oxygen kit to detect the active oxygen content in astrocytes. The SD rats were randomly divided into groups of the following groups: the sham operation group (sham) group, the operation group, the low concentration of breviscapine (50mg/kg) + operation group, the high concentration of breviscapine (l00mg/kg) + operation group, and the operation group. The neurobehavioral scores of rats were scored with the same concentration of.Bederson 5. The changes in the area of cerebral infarction in rats were detected by TTC staining; the changes of the expression of NOX2 protein were detected by Western Blot method; the changes of the related indexes of oxidative stress were detected by ELISA; the apoptosis in the brain tissue of rats was detected by immunohistochemistry. Results: the results of 1. molecular docking show that the shape of the active pocket of breviscapine and NOX2 protein is consistent with each other, and the 3D molecular structure of the two is well butted. The purity of the primary cultured astrocytes is 98%. deficient, 2 h h complex and 24 h, and breviscapine (10,50 M) can improve the decrease of cell survival rate (P0.05).Western Blot display The expression level of NOX2 in astrocytes was higher than that in the normal control group. The level of NOX2 expression in astrocytes after glucose deficiency and oxygen deficiency 2 h pretreated by breviscapine (2,10,50 mu M) was lower than that of the control group at the same time at the same time (P0.05). The level of intracellular reactive oxygen species caused by glucose deprivation (2,10,50 mu M) pretreated by breviscapine ethyl (2,10,50 mu M) was higher than that of the control group (P0.05). Increase, and breviscapine (10,50 mu M) can reduce the level of intracellular reactive oxygen level (P0.05).2. rats' neurobehavioral score, and the percentage of cerebral infarction area showed that compared with the sham group, the score of neurobehavioral score in the group I/R and the percentage of cerebral infarction area increased (P0.05), and compared with the I/R group, the +I/R group of Scu (low, high concentration) was the rat nerve. Behavioral score, the percentage of cerebral infarction area decreased (P0.05).3. rats' apoptosis and oxidative stress related indexes (8- hydroxy deoxy guanosine, 8-OHdG; 4- hydroxy nonylaldehyde, 4-HNE; 3- nitrotyrosine, 3-NT) detection results showed that compared with the sham group, the rate of immuno histochemical positive cells in the I/R group was higher than that of oxidative stress. (P0.05) in sham operation group, the percentage of Caspase-3 positive cells in group I/R3d was 12.20 + 1.62%, 55.45 + 4.99%. in sham operation group, 8-OHdG in group I/R 3D was 100%, 177.2% + 31.2%, 4-HNE was 100%, 163.6% + 28.7%, 3-NT was 100%, 159.3% + 32.1%. was compared with I/R group, Scu (low, high concentration) immunohistochemical positive rats The index of cell rate and oxidative stress decreased (P0.05).Scu low concentration group, the percentage of Caspase-3 positive cells in Scu high concentration group was 22.36 + 3.37%, 11.70 + 1.79%.Scu low concentration group, 8-OHdG in Scu high concentration group was 113.1% + 33.1%, 99.9% + 11.3%, 4-HNE was 112% + 32.1%, 95.2% +, respectively, 3-NT was 85.6%, respectively. 31.3%, 90% + 20.5%.4.Western Blot detection of NOX2 protein showed that the NOX2 protein expression level of 3D in the I/R group was higher than that in the sham group (P0.05). The expression level of 3 D in +I/R group was lower than that in the I/R group. Conclusion: 1. breviscapine B has protective effect on astrocyte damage induced by oxygen deficiency and hypoxia / reoxygenation. Breviscapine can inhibit the expression of NOX2 protein in hypoxia starlike astrocytes and reduce the level of reactive oxygen species (.2.) in starlike astrocytes, which can protect cerebral ischemia reperfusion injury and play NO. The role of X2 protein inhibitor.3. breviscapine in protecting cerebral ischemia-reperfusion injury is related to targeted regulation of NOX2 induced oxidative damage.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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