Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions. / In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones. / Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models. / PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1. / In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions. / In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease. / 缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。 / 當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。 / 蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。 / PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。 / 不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。 / 總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。 / Zhen, Xia. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 194-206). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1291499 |
| Date | January 2014 |
| Contributors | Zhen, Xia (author.), Lam, Fu Yuen (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (xviii, 206 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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