本文選題：雙環(huán)醇 + 炎癥反應(yīng)��； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：腦血管病被認(rèn)為是危害人類健康的主要疾病之一，以其較高的發(fā)病率、致殘率和病死率給人類生命造成嚴(yán)重威脅，隨著人們生活方式的改變，其發(fā)病率有逐年增高的趨勢(shì)，給社會(huì)和家庭帶來(lái)沉重的負(fù)擔(dān)。腦血管病中以缺血性腦卒中最常見(jiàn)，約占所有腦血管病的60%～80%。腦缺血后繼發(fā)性腦損傷是病情加重及影響預(yù)后的重要原因，繼發(fā)性腦損傷機(jī)制復(fù)雜，如氧化應(yīng)激、炎癥反應(yīng)、鈣超載、興奮氨基酸毒性、細(xì)胞凋亡及線粒體功能異常等因素均參與了繼發(fā)性腦損傷的病理過(guò)程。其中過(guò)度的炎癥反應(yīng)在腦梗死的病理生理過(guò)程中發(fā)揮著關(guān)鍵性的作用。如何減輕炎癥損傷成為治療急性期腦梗死的一種重要途徑。 Toll樣受體（Toll like receptors，TLRs）是近年來(lái)發(fā)現(xiàn)的一類天然免疫受體，分布十分廣泛，屬于模式識(shí)別受體，可識(shí)別并結(jié)合病原體相關(guān)分子模式，通過(guò)激活一系列信號(hào)轉(zhuǎn)導(dǎo)，引起炎性介質(zhì)的釋放，最終激活天然免疫防御及獲得性免疫系統(tǒng)。 目前確定的TLRs共有11種。研究表明，TLR2，TLR4和TLR9在缺血后被激活。我們已經(jīng)證實(shí)了TLR2，TLR4所介導(dǎo)的核因子-κB（nuclearfactor-kappaB，NF-κB）的激活參與了缺血后繼發(fā)性腦損傷。而TLR9作為一種炎癥介質(zhì)同樣參與了缺血后繼發(fā)性損傷。腫瘤壞死因子受體相關(guān)因子6（Tumour-necrosis factor receptor-associated factor6，TRAF6）是Toll樣/白細(xì)胞介素-1受體（Toll/IL-1receptor，TIR）超家族重要的接頭分子，可以直接和白細(xì)胞介素受體相關(guān)激酶（interleukin-1receptor associated kinase，IRAK）結(jié)合、從而激活轉(zhuǎn)錄因子NF-κB，引起一系列炎癥細(xì)胞的激活以及炎癥因子的釋放加重缺血后腦損傷。 雙環(huán)醇（Bicyclol）是我國(guó)第一個(gè)擁有自主知識(shí)產(chǎn)權(quán)的國(guó)家一類新藥，從中藥五味子丙素中提取。雙環(huán)醇具有減少氧自由基、保護(hù)抗氧化酶系統(tǒng)、抗炎、抗凋亡、抗腫瘤增殖的作用。廣泛應(yīng)用于肝炎、肝纖維化、肝癌的治療，研究表明其對(duì)缺血再灌注后的肝臟，腎臟均有保護(hù)作用，雙環(huán)醇能否下調(diào)缺血后腦組織TLR4，TLR9，TRAF6，NF-κB和MMP-9；上調(diào)Claudin-5表達(dá)產(chǎn)生腦保護(hù)作用有待進(jìn)一步研究。 本實(shí)驗(yàn)在大鼠永久性大腦中動(dòng)脈栓塞（Middle Cerebral ArteryOcclusion，MCAO）所致腦梗死模型上觀察TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5在腦組織的表達(dá)及局部腦缺血后雙環(huán)醇的腦保護(hù)作用及其可能的機(jī)制。 方法：采用成年健康雄性Sprague-Dawley大鼠，應(yīng)用改良Longa線栓法建立大鼠右側(cè)MCAO模型。實(shí)驗(yàn)動(dòng)物隨機(jī)分為假手術(shù)組（Sham），pMCAO組，Vehicle組（pMCAO+0.5%羧甲基纖維素鈉），雙環(huán)醇小劑量組（Vehicle+bicyclol50mg/kg，By-L），雙環(huán)醇大劑量組（Vehicle+bicyclol100mg/kg，By-H）。溶劑對(duì)照組和藥物干預(yù)組動(dòng)物分別預(yù)給藥（灌胃）3天每天一次，最后一次給藥后1小時(shí)行右側(cè)大腦中動(dòng)脈栓塞術(shù)。術(shù)后24h對(duì)大鼠進(jìn)行神經(jīng)功能評(píng)分，評(píng)分完畢后將動(dòng)物斷頭處死，用2％2,3,5-三苯基四唑氮紅（triphenyltetrazolium chloride，TTC）染色法測(cè)定腦梗死體積，用干濕重法測(cè)定腦組織含水量，用免疫組化，免疫印記（Western blot），實(shí)時(shí)熒光定量PCR（RT-qPCR）測(cè)定TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5的表達(dá)。 結(jié)果： 1神經(jīng)功能評(píng)分：Sham組無(wú)明顯神經(jīng)功能缺損，而pMCAO組、Vehicle組、雙環(huán)醇小劑量組和大劑量組大鼠均出現(xiàn)不同程度的左側(cè)肢體偏癱。雙環(huán)醇大劑量組與pMCAO組和Vehicle組相比，神經(jīng)功能評(píng)分有所改善，差異有統(tǒng)計(jì)學(xué)意義（P 0.05）。但雙環(huán)醇小劑量組與pMCAO組和Vehicle組相比，神經(jīng)功能評(píng)分有所改善，差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05）。 2腦組織含水量測(cè)定：雙環(huán)醇大劑量組病變側(cè)腦組織含水量在24h時(shí)低于pMCAO組和Vehicle組，差異有統(tǒng)計(jì)學(xué)意義（By-H vs. pMCAO andVehicle:83.38%±0.70%vs.85.73%±1.17%and85.90%±1.01%，P 0.05）。雙環(huán)醇小劑量組病變側(cè)腦組織含水量低于Vehicle組，但差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.5）。然而，雙環(huán)醇大小劑量組之間比較無(wú)統(tǒng)計(jì)學(xué)意義（P0.5）。 3腦梗死體積（%HLV）的測(cè)定：與pMCAO組和Vehicle組相比，雙環(huán)醇大劑量組梗塞體積明顯減小，差異有統(tǒng)計(jì)學(xué)意義（By-H vs. pMCAO andVehicle:31.89%±6.16%vs.45.16%±3.94%and46.10%±4.36%，P 0.05）。雙環(huán)醇小劑量組與pMCAO組和Vehicle組相比，梗塞體積有所減小，但差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 4雙環(huán)醇對(duì)TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5表達(dá)的影響：在腦梗死24h后，TLR4，TLR9，TRAF6，NF-κB和MMP-9蛋白和基因表達(dá)及陽(yáng)性細(xì)胞數(shù)明顯增多。Claudin-5蛋白和基因表達(dá)明顯降低。雙環(huán)醇干預(yù)后，大劑量組TLR4，TLR9，TRAF6，NF-κB和MMP-9蛋白和基因表達(dá)及陽(yáng)性細(xì)胞數(shù)明顯下降，與pMCAO組和Vehicle組相比結(jié)果有統(tǒng)計(jì)學(xué)意義（P 0.05）；而Claudin-5蛋白和基因表達(dá)明顯升高，與pMCAO組和Vehicle組相比結(jié)果有統(tǒng)計(jì)學(xué)意義（P 0.05）。而小劑量組各項(xiàng)指標(biāo)的變化與pMCAO組和Vehicle組相比結(jié)果無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論：在腦缺血的損傷過(guò)程中TLR4，，TLR9，TRAF6，NF-κB和MMP-9表達(dá)上調(diào)，Claudin-5表達(dá)下調(diào)，給予雙環(huán)醇干預(yù)后可以有效減輕腦損傷。其作用可能與下調(diào)TLR4，TLR9，TRAF6，NF-κB和MMP-9，減輕炎癥損傷；上調(diào)Claudin-5，保護(hù)血腦屏障有關(guān)。
[Abstract]:Objective: cerebrovascular disease is considered to be one of the main diseases which are harmful to human health. With its high incidence, the rate of disability and mortality are serious threat to human life. With the change of people's life style, the incidence of the disease has a trend of increasing year by year and a heavy burden to the society and family. Secondary brain injury, which is most common in 60% ~ 80%. of cerebrovascular disease, is an important cause of aggravation and prognosis, and secondary brain injury is complicated, such as oxidative stress, inflammatory reaction, calcium overload, excitatory amino acid toxicity, apoptosis and abnormal function of grain body and other factors involved in secondary brain injury. The excessive inflammatory response plays a key role in the pathophysiological process of cerebral infarction. How to reduce the damage of inflammation is an important way to treat acute cerebral infarction.
Toll like receptor (Toll like receptors, TLRs) is a kind of natural immune receptor found in recent years. It is widely distributed and belongs to the pattern recognition receptor. It can identify and combine the model of the pathogen associated molecules, activate a series of signal transduction, cause the release of inflammatory mediators, and eventually activate the natural immune defense and acquired immune system.
Currently, there are 11 identified TLRs. Studies have shown that TLR2, TLR4 and TLR9 are activated after ischemia. We have confirmed that TLR2, the activation of nuclear factor kappa B (nuclearfactor-kappaB, NF- kappa B) mediated by TLR4 is involved in secondary cerebral injury after ischemia. TLR9, as an inflammatory medium, is also involved in secondary ischemic injury. The cause of the tumor's bad death. The subreceptor related factor 6 (Tumour-necrosis factor receptor-associated factor6, TRAF6) is an important junction molecule of the Toll like / interleukin -1 receptor (Toll/IL-1receptor, TIR) superfamily, which can bind directly with the interleukin receptor related kinase (interleukin-1receptor associated kinase) and activate the transcription factor. The activation of a series of inflammatory cells and release of inflammatory factors aggravate brain damage after ischemia.
Bicyclic alcohol (Bicyclol) is the first kind of new drug in China with independent intellectual property rights. It is extracted from Chinese medicine Schisandra propane. Bicyclic alcohol has the effect of reducing oxygen free radicals, protecting antioxidant enzyme system, anti-inflammatory, anti apoptosis, and anti-tumor proliferation. It has been widely used in hepatitis, liver fibrosis, and liver cancer treatment. After the injection, the liver and kidney have protective effects. Can the TLR4, TLR9, TRAF6, NF- - kappa B and MMP-9 be down - regulated by double ring alcohols, and the protective effect of the expression of Claudin-5 to produce brain remains to be further studied.
In this experiment, TLR4, TLR9, TRAF6, NF- kappa B and MMP-9 were observed on the cerebral infarction model of Middle Cerebral ArteryOcclusion (MCAO) in rats. The expression of Claudin-5 in the brain tissue and the protective effect of bicyclic alcohol on the brain after local cerebral ischemia and its possible mechanism.
Methods: the adult healthy male Sprague-Dawley rats were used to establish the right MCAO model of rats by modified Longa thread suppository. The experimental animals were randomly divided into sham operation group (Sham), pMCAO group, Vehicle group (pMCAO+0.5% carboxymethyl cellulose sodium), small dose of bicyclic alcohol group (Vehicle+ bicyclol50mg/kg, By-L), and large dose of bicyclic alcohol group (Vehicle+bicyclol100m) G/kg, By-H). In the solvent control group and the drug intervention group, the animals were pretreated each day (gavage) for 3 days, and the right middle cerebral artery embolization was performed 1 hours after the last administration. After the operation, 24h was used to score the nerve function of the rats. After the score was finished, the animals were killed with 2% 2,3,5- three phenyl four azolium nitrogen red (triphenyltetrazolium chloride, TT). C) the volume of cerebral infarction was measured by the staining method. The water content of the brain tissue was measured by dry wet weight method. The expression of TLR4, TLR9, TRAF6, NF- kappa B and MMP-9, and Claudin-5 were measured by immunohistochemistry, Western blot and real-time fluorescence quantitative PCR (RT-qPCR).
Result:
1 nerve function score: there was no obvious nerve function defect in group Sham, but in group pMCAO, group Vehicle, small dose group of double ring alcohol and large dose group, there were different degrees of left limb hemiplegia. Compared with group pMCAO and Vehicle group, the score of neurologic function was improved, the difference was statistically significant (P 0.05). Compared with group pMCAO and group Vehicle, the score of neurological function was improved, and the difference was not statistically significant (P0.05).
2 the measurement of water content in the brain tissue: the water content of the lateral brain tissue in the large dose group of the double ring alcohol group was lower than that of the pMCAO group and the Vehicle group at 24h. The difference was statistically significant (By-H vs. pMCAO andVehicle:83.38% + 0.70%vs.85.73% + 1.17%and85.90% + 1.01%, P 0.05). The water content of the diseased brain tissue in the small dose group of double ring alcohol was lower than that in the Vehicle group, but the difference was no unification. Significance (P0.5). However, there was no significant difference between the two groups in size and dose (P0.5).
3 measurement of volume of cerebral infarction (%HLV): compared with group pMCAO and group Vehicle, the infarct volume decreased significantly in the large dose group of double cyclic alcohol (By-H vs. pMCAO andVehicle:31.89% + 6.16%vs.45.16% + 3.94%and46.10% + 4.36%, P 0.05). The infarct volume decreased, but the infarct volume decreased, but the difference between the small dose group and the Vehicle group decreased, but the difference was less than that of the Vehicle group. There was no statistical significance (P0.05).
4 the effects of bicyclic alcohol on TLR4, TLR9, TRAF6, NF- kappa B and MMP-9, Claudin-5 expression: TLR4, TLR9, TRAF6, NF- kappa, protein and gene expression and the number of positive cells were significantly increased after cerebral infarction. The number of positive cells decreased significantly, compared with the pMCAO group and Vehicle group (P 0.05), while the Claudin-5 protein and gene expression increased significantly, compared with the pMCAO group and the Vehicle group (P 0.05), but the changes in the small dose group were not statistically significant compared with those in the pMCAO group and the Vehicle group (P0., P0.). 05).
Conclusion: the expression of TLR4, TLR9, TRAF6, NF- kappa B and MMP-9 is up regulation during the injury of cerebral ischemia, and the expression of Claudin-5 is down regulated. The prognosis of Claudin-5 can effectively reduce the brain damage. The effect may be related to the reduction of TLR4, TLR9, TRAF6, NF- kappa B and reducing inflammation damage, the upper modulation and the protection of the blood brain barrier.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R743.33

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