腦中風(stroke)是指因血流阻斷所造成的腦部傷害。除了名列全球第二大死因外，腦中風也造成患者長期失能。根據血流阻斷的方式，又可將腦中風分為出血性腦中風(hemorrhagic stroke)和缺血性腦中風 (ischemic stroke)兩種。其中又以缺血性腦中風佔大宗，每年約造成六百萬人死亡。胞漿素原活化劑(recombinant tissue plasminogen activator, rtPA)為目前唯一被美國食品藥品管理局(FDA)核准，應用於治療缺血性腦中風的藥物。然而rt-PA仍存在許多待克服的問題，如：嚴格的施用標準和潛在的出血風險，故缺血性腦中風仍需新的醫療策略或治療方法介入。淫羊藿甙(icariin)和淫羊藿素(icaritin)為淫羊藿的主要活性類黃酮萃取物。目前，已知淫羊藿甙和淫羊藿素可在動物模式中作為神經保護劑治療阿茲海默症。此外，研究亦指出淫羊藿甙可治療腦部缺血再灌流 (ischemia-reperfusion)傷害，然而其對於缺血性腦中風的預防效果及其作用機制仍待探究。因此，本篇研究的目的為探討淫羊藿甙和淫羊藿素對於缺血性腦中風所致傷害的預防效果。為了建立急性缺血性腦中風之動物模式，本篇研究將小鼠中腦動脈(middle cerebral artery)阻塞50分鐘後再恢復血流24小時以模擬缺血再灌流傷害。結果顯示，中腦動脈阻塞前1小時預處理淫羊藿甙和淫羊藿素能預防缺血再灌流所致體重流失、神經受損、腦部梗塞以及腦組織型態學改變。此外，預處理淫羊藿甙和淫羊藿素可降低凋亡細胞數量和反轉凋亡相關蛋白的表現，神經發炎相關蛋白iNOS 和eNOS表現量亦下降，內皮-間質轉換(endothelial-mesenchymal transition)相關蛋白fibronectin和vimentin的表現量也顯著降低，而細胞衰老(cellular senescence)相關蛋白p53, p21表現亦減少。組織免疫染色(Immunohistochemical stain)結果也發現預處理淫羊藿甙和淫羊藿素可減少細胞衰老指標蛋白p21和beta-半乳糖苷酶 (beta-galactosidase)的表現。另一方面，預處理淫羊藿甙和淫羊藿素可透過提升海馬迴中神經保護相關蛋白smad2/3來對抗缺血再灌流傷害。在體外實驗的部分，本研究將小鼠微血管內皮細胞(bEnd.3)和小鼠微膠細胞(BV-2)置於缺氧箱(O2 < 1%)中2或8小時再使其恢復常氧狀態24-72小時以模擬缺氧再灌流(Hypoxia-reperfusion)傷害。而實驗結果顯示預處理淫羊藿甙和淫羊藿素可回復小鼠內皮細胞和微膠細胞的細胞存活率。此外，在預處理淫羊藿甙和淫羊藿素的小鼠內皮細胞中亦發現老化相關半乳糖酶(senescence-associated beta-galactosidase)的表現量減少。綜上所述，本研究發現預處理淫羊藿甙和淫羊藿素能有效降低缺血再灌流傷害。然而，淫羊藿甙和淫羊藿素是否在對抗缺血再灌流中扮演抗氧化的角色以及其詳細分子機轉仍待後續研究探討。

Stroke is a cerebral damage caused by the interruption of the blood supply to the brain which is the second leading cause of death in the world and an important cause of long-term disability. Based on the pathophysiological mechanism, stroke can be divided into hemorrhagic stroke and ischemic stroke. Among them, ischemic stroke is the major type which caused about 6 million deaths every year. Currently, recombinant tissue plasminogen activator (rtPA) is the only drug approved by FDA for ischemic stroke. However, the strict prescription rule and potential bleeding risk are the limitations to overcome. Therefore, new medical strategies or therapies are urgently required for ischemic stroke. Icariin (ICA) and Icaritin (ICT) are two major active flavonoid components extracted from Epimedium Herba which have been regarded as a neuroprotective agent in Alzheimer’s disease animal model. Studies also demonstrated that icariin treatment could decrease ischemia-reperfusion (I/R) injury-induced brain injury, but the preventive effects of ICA still unknown. Hence, the purpose of this study is to investigate the preventive effects of ICA and ICT on ischemic stroke. In order to establish a mouse model of ischemic stroke, mice were operated with middle cerebral artery occlusion (MCAO) for 50 min and followed by 24 h reperfusion in vivo. Our results showed that both ICA and ICT (60 mg/kg) pretreatment could decrease mice body weight loss, neurological injury, infarct volumes and tissue morphology changes induced by MCAO in acute ischemic stroke animal model. Additionally, both ICA and ICT pretreatment could also reduce the apoptotic cell numbers and reverse the apoptosis associated protein expressions. Moreover, the results of endothelial-mesenchymal transition and neuron inflammation were shown by upregulating the protein expressions of fibronectin, vimentin, iNOS and eNOS and downregulating the protein expression of CD31 after MCAO surgery, which could be significantly reversed by both ICA and ICT pretreatment. In addition, both ICA and ICT pretreatment could reverse the cellular senescence through diminishing the expressions of p53, p21 and beta-galactosidase activities in both protein expressions and IHC staining. Furthermore, both ICA and ICT pretreatment could provide neuroprotective effects by abundantly raising the smad2/3 expression to against the ischemia-reperfusion injury in hippocampus. To mimic the hypoxia-reperfusion injury, mouse microvascular endothelial cells (bEnd.3) and mouse microglia cells (BV-2) were cultured in hypoxia tank (O2 <1%) for 2 or 8 h and followed by 24 to 72 h reperfusion in vitro. The results showed that both ICA and ICT pretreatment significantly reversed the reduction of cell viability caused by hypoxia-reperfusion injury both in the bEnd.3 and BV-2 cells. Moreover, both ICA (40 μM) and ICT (2.5 μM) pretreatment could attenuate ischemia-reperfusion injury-induced cellular senescence by decreasing the beta-galactosidase activity in bEnd.3. Taken together, these results suggested that both ICA and ICT pretreatment effectively alleviate the brain injury by acute cerebral ischemic stroke. However, whether the antioxidative function is involved in the protection of both ICA and ICT against acute cerebral ischemic stroke remains further investigation in the future.

致謝 i
中文摘要 iii
Abstract v
Abbreviations vii
Contents ix
Chapter I Introduction 1
1.1 Stroke 1
1.1.1 Burden of stroke 1
1.1.2 Etiologic classification of stroke 2
1.1.3 Brain and ischemic stroke 3
1.2 Pathophysiological mechanism of ischemic stroke 4
1.2.1 Energy failure, malfunction of ion gradient and excitotoxicity 5
1.2.2 Peri-infarct depolarization 7
1.2.3 Oxidative stress 8
1.2.4 Inflammation 9
1.2.5 Programmed cell death 11
1.3 Therapy for ischemic stroke 13
1.3.1 Thrombolytic therapy 13
1.3.2 Neuroprotective agents 14
1.4 Icariin and icaritin 15
Chapter II Aims 16
Chapter III Materials and Methods 17
3.1 Chemicals 17
3.2 Acute cerebral ischemia-reperfusion (I/R) injury model 17
3.3 Neurological score assessment 18
3.4 Determination of infarct volume 20
3.5 Tissue preparation 20
3.6 Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay 20
3.7 Histopathological and immunohistochemical analysis 21
3.8 Double stain immunohistochemical analysis 22
3.9 Western blotting analysis 22
3.10 Cell culture 23
3.11 Hypoxia-reperfusion (H / R) treatment 24
3.12 Measurement of cell viability 24
3.13 Senescence-associated beta-galactosidase activity analysis 24
3.14 Primary antibodies 25
3.15 Statistical analysis 26
Chapter IV Results 27
4.1 The therapeutic effects of icaritin (ICT) on body weight loss, neurological severity, and infarct volume in mice after MCAO 27
4.2 The preventive effects of icariin (ICA) and icaritin (ICT) on body weight loss, neurological severity, and infarct volume in mice after MCAO 27
4.3 ICA and ICT reduced the number of apoptotic cells induced by MCAO in the cerebral hippocampus and cortex of mice 28
4.4 Both ICA and ICT alleviated the histological changes induced by MCAO in the cerebral hippocampus and cortex of mice 29
4.5 Both ICA and ICT diminished the expressions of senescence markers induced by MCAO in the cerebral hippocampus and cortex of mice 29
4.6 Both ICA and ICT altered the β-galactosidase and CD31 contents in cerebral ischemia mouse mice brain tissue 30
4.7 Both ICA and ICT reversed the expressions of apoptosis markers in mouse brain tissue caused by ischemia-reperfusion 31
4.8 Both ICA and ICT decreased inflammation and endothelial-mesenchymal transition in mouse brain tissue after ischemia-reperfusion 32
4.9 Both ICA and ICT diminished the cell senescence in mouse brain tissue induced by ischemia-reperfusion 33
4.10 Both ICA and ICT inverted the depletion of Smad2/3 in cerebral ischemia mouse brain tissue 33
4.11 Both ICA and ICT reversed the cell viability reduction induced by OGD treatment 34
4.12 Both ICA and ICT attenuated cell senescence induced by OGD treatment in bEnd.3 cells 34
Chapter V Discussion 35
Chapter VI Conclusion 40
Chapter VII References 41
Chapter VIII Figures and figure legends 49
Figure 1. The therapeutic effects of icaritin (ICT) on body weight loss, neurological severity, and infarct volume in mice after MCAO. 49
Figure 2. The preventive effectss of icariin (ICA) and icatitin (ICT) on body weight loss, neurological severity, and infarct volume in mice after MCAO. 51
Figure 3. ICA and ICT reduced the number of apoptotic cells induced by MCAO in the cerebral hippocampus and cortex of mice. 54
Figure 4. Both ICA and ICT alleviated the histological changes induced by MCAO in the cerebral hippocampus and cortex of mice. 55
Figure 5. Both ICA and ICT diminished the expressions of senescence markers induced by MCAO in the cerebral hippocampus and cortex of mice. 56
Figure 6. Both ICA and ICT altered the β-galactosidase and CD31 contents in cerebral ischemia mouse mice brain tissue. 57
Figure 7. Both ICA and ICT reversed the expressions of apoptosis markers in mouse brain tissue caused by ischemia-reperfusion. 58
Figure 8. Both ICA and ICT decreased inflammation and endothelial-mesenchymal transition in mouse brain tissue after ischemia-reperfusion. 59
Figure 9. Both ICA and ICT diminished the cell senescence in mouse brain tissue induced by ischemia-reperfusion. 61
Figure 10. Both ICA and ICT inverted the depletion of Smad2/3 in cerebral ischemia mouse brain tissue. 62
Figure 11. Both ICA and ICT reversed the cell viability reduction induced by OGD treatment. 63
Figure 12. Both ICA and ICT attenuated cell senescence induced by OGD treatment in bEnd.3 cells. 64

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