双环醇对实验性脑梗死大鼠抗炎症损伤机制的研究

发布时间：2018-07-05 10:24

本文选题：双环醇 + 炎症反应　；参考：《河北医科大学》2013年硕士论文

【摘要】：目的：脑血管病被认为是危害人类健康的主要疾病之一，以其较高的发病率、致残率和病死率给人类生命造成严重威胁，随着人们生活方式的改变，其发病率有逐年增高的趋势，给社会和家庭带来沉重的负担。脑血管病中以缺血性脑卒中最常见，约占所有脑血管病的60%～80%。脑缺血后继发性脑损伤是病情加重及影响预后的重要原因，继发性脑损伤机制复杂，如氧化应激、炎症反应、钙超载、兴奋氨基酸毒性、细胞凋亡及线粒体功能异常等因素均参与了继发性脑损伤的病理过程。其中过度的炎症反应在脑梗死的病理生理过程中发挥着关键性的作用。如何减轻炎症损伤成为治疗急性期脑梗死的一种重要途径。 Toll样受体（Toll like receptors，TLRs）是近年来发现的一类天然免疫受体，分布十分广泛，属于模式识别受体，可识别并结合病原体相关分子模式，通过激活一系列信号转导，引起炎性介质的释放，最终激活天然免疫防御及获得性免疫系统。目前确定的TLRs共有11种。研究表明，TLR2，TLR4和TLR9在缺血后被激活。我们已经证实了TLR2，TLR4所介导的核因子-κB（nuclearfactor-kappaB，NF-κB）的激活参与了缺血后继发性脑损伤。而TLR9作为一种炎症介质同样参与了缺血后继发性损伤。肿瘤坏死因子受体相关因子6（Tumour-necrosis factor receptor-associated factor6，TRAF6）是Toll样/白细胞介素-1受体（Toll/IL-1receptor，TIR）超家族重要的接头分子，可以直接和白细胞介素受体相关激酶（interleukin-1receptor associated kinase，IRAK）结合、从而激活转录因子NF-κB，引起一系列炎症细胞的激活以及炎症因子的释放加重缺血后脑损伤。双环醇（Bicyclol）是我国第一个拥有自主知识产权的国家一类新药，从中药五味子丙素中提取。双环醇具有减少氧自由基、保护抗氧化酶系统、抗炎、抗凋亡、抗肿瘤增殖的作用。广泛应用于肝炎、肝纤维化、肝癌的治疗，研究表明其对缺血再灌注后的肝脏，肾脏均有保护作用，双环醇能否下调缺血后脑组织TLR4，TLR9，TRAF6，NF-κB和MMP-9；上调Claudin-5表达产生脑保护作用有待进一步研究。本实验在大鼠永久性大脑中动脉栓塞（Middle Cerebral ArteryOcclusion，MCAO）所致脑梗死模型上观察TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5在脑组织的表达及局部脑缺血后双环醇的脑保护作用及其可能的机制。方法：采用成年健康雄性Sprague-Dawley大鼠，应用改良Longa线栓法建立大鼠右侧MCAO模型。实验动物随机分为假手术组（Sham），pMCAO组，Vehicle组（pMCAO+0.5%羧甲基纤维素钠），双环醇小剂量组（Vehicle+bicyclol50mg/kg，By-L），双环醇大剂量组（Vehicle+bicyclol100mg/kg，By-H）。溶剂对照组和药物干预组动物分别预给药（灌胃）3天每天一次，最后一次给药后1小时行右侧大脑中动脉栓塞术。术后24h对大鼠进行神经功能评分，评分完毕后将动物断头处死，用2％2,3,5-三苯基四唑氮红（triphenyltetrazolium chloride，TTC）染色法测定脑梗死体积，用干湿重法测定脑组织含水量，用免疫组化，免疫印记（Western blot），实时荧光定量PCR（RT-qPCR）测定TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5的表达。结果： 1神经功能评分：Sham组无明显神经功能缺损，而pMCAO组、Vehicle组、双环醇小剂量组和大剂量组大鼠均出现不同程度的左侧肢体偏瘫。双环醇大剂量组与pMCAO组和Vehicle组相比，神经功能评分有所改善，差异有统计学意义（P 0.05）。但双环醇小剂量组与pMCAO组和Vehicle组相比，神经功能评分有所改善，差异没有统计学意义（P0.05）。 2脑组织含水量测定：双环醇大剂量组病变侧脑组织含水量在24h时低于pMCAO组和Vehicle组，差异有统计学意义（By-H vs. pMCAO andVehicle:83.38%±0.70%vs.85.73%±1.17%and85.90%±1.01%，P 0.05）。双环醇小剂量组病变侧脑组织含水量低于Vehicle组，但差异无统计学意义（P0.5）。然而，双环醇大小剂量组之间比较无统计学意义（P0.5）。 3脑梗死体积（%HLV）的测定：与pMCAO组和Vehicle组相比，双环醇大剂量组梗塞体积明显减小，差异有统计学意义（By-H vs. pMCAO andVehicle:31.89%±6.16%vs.45.16%±3.94%and46.10%±4.36%，P 0.05）。双环醇小剂量组与pMCAO组和Vehicle组相比，梗塞体积有所减小，但差异无统计学意义（P0.05）。 4双环醇对TLR4，TLR9，TRAF6，NF-κB和MMP-9，Claudin-5表达的影响：在脑梗死24h后，TLR4，TLR9，TRAF6，NF-κB和MMP-9蛋白和基因表达及阳性细胞数明显增多。Claudin-5蛋白和基因表达明显降低。双环醇干预后，大剂量组TLR4，TLR9，TRAF6，NF-κB和MMP-9蛋白和基因表达及阳性细胞数明显下降，与pMCAO组和Vehicle组相比结果有统计学意义（P 0.05）；而Claudin-5蛋白和基因表达明显升高，与pMCAO组和Vehicle组相比结果有统计学意义（P 0.05）。而小剂量组各项指标的变化与pMCAO组和Vehicle组相比结果无统计学意义（P0.05）。结论：在脑缺血的损伤过程中TLR4，，TLR9，TRAF6，NF-κB和MMP-9表达上调，Claudin-5表达下调，给予双环醇干预后可以有效减轻脑损伤。其作用可能与下调TLR4，TLR9，TRAF6，NF-κB和MMP-9，减轻炎症损伤；上调Claudin-5，保护血脑屏障有关。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R743.33

【参考文献】