|摘要: ||人體能將飲食中甲硫胺酸(Methionine )透過S-腺核苷甲硫胺酸(S-adenosylmethionine; SAM)和S-腺核苷同半胱胺酸(S-adenosylhomocysteine; SAH)中間代謝產物代謝成同半胱胺酸(Homocysteine; Hcy)。許多證據顯示Hcy和SAH都可能是心血管疾病和神經損傷相關疾病的危險因子。最近學者們指出SAH和Hcy之間可能存在某種互補或協同的作用。但現今的研究，卻鮮少有文獻報告將兩者合併在同一試驗模式下觀察討論。在大鼠熱中風模式中許多證據指出循環性休克、缺氧缺血損傷是熱中風發展的主因。目前已知熱中風呈現血管內皮受損會造成循環休克、多發性器官功能喪失、腦部缺氧缺血和損傷，而最終導致減短熱中風存活時間。近期有證據顯示出熱中風所生成的反應與熱中風動物體體內增加自由基生成(特別是氫氧自由基和超氧自由基)、增加脂質過氧化和降低酵素性抗氧化防禦系統有關。而心血管方面疾病的高危險因子者，也較易罹患熱中風。根據以上探討，我們推測SAH和Hcy也極可能同時參與了熱中風損傷的病理發展。而我們先前研究證實SAH合併Hcy確實能藉由增加腦部缺氧缺血、發炎反應進而加重熱中風損傷。目前研究對SAH和Hcy造成細胞或動物的傷害機制著重於氧化壓力上升。然而，截至目前為止卻沒有任何的研究文獻指出SAH合併Hcy與熱中風損傷引發的氧化傷害之間的相關性。再者，榖胱甘肽(Glutathione，GSH) 是一種小分子三胜肽，是細胞內濃度最高的的抗氧化劑，存在於身體細胞中，其中以肝臟內含量最多。它具有強抗氧化力、為一有效解毒劑。許多研究指出補充GSH在缺血缺氧模式中能抑制氧化傷害、抑制活性氧生成，也可增加抗氧化防禦作用。因此，GSH可能具有預防或治療SAH和Hcy促進熱中風的損傷反應。目前並無文獻探討GSH對熱中風所致損傷之影響。於是在未來三年的研究計畫中，我們將進行以下研究。 第一年：探討給予SAH合併Hcy對大鼠熱中風氧化損傷可能作用機制---我們將外加給予SAH合併Hcy，監測熱中風動物體體內抗氧化防禦系統變化。本實驗主要的目的在於觀察，是否給予SAH合併Hcy處理會透過降低抗氧化防禦系統，而加重惡化熱中風所造成病理生理參 數，而導致減短動物體存活時間。並觀察腦與肝損傷程度、自由基生成、GSH相關分子變化、抗氧化酵素活性與表現的情形，以探討可能的作用機制。 第二年：探討口服GSH後給予SAH合併Hcy促進大鼠熱中風病理生成之影響---我們將計劃利用口服方式處理GSH後，探討GSH是否具預防因SAH合併Hcy降低抗氧化防禦系統加重惡化熱中風所造成的病理作用。在此一試驗中，我們不僅可以再次確認SAH和Hcy參與在熱中風所致損傷病理生理發展的角色，而且也點出和Hcy相關的疾病可能可視為是熱中風發生率的危險因子。預計给予動物體餵食GSH之大鼠觀測因SAH合併Hcy所致熱中風氧化損傷參數的影響，而達到預防因SAH合併Hcy促進熱中風所造成的傷害。 第三年：探討缺氧下GSH對SAH合併Hcy誘導神經膠細胞與肝細胞氧化傷害之保護作用及可能作用機制--利用小鼠微神經膠細胞與肝細胞培養處理於低氧槽下的細胞試驗，來模擬活體熱中風缺氧缺血狀態。我們將先評估在培養於缺氧槽的小鼠神經微膠細胞株(BV-2 cells)與人類肝細胞(Chang’s liver cells)中，外加給予SAH和Hcy處理，對GSH相關抗氧化分子、自由基表現、抗氧化酵素與細胞DNA損傷程度的影響。實驗目的強調在，外加給予SAH和Hcy處理在缺氧狀況下的BV-2 cells、Chang’s liver cells，是否會透過促進自由基表現、降低抗氧化防禦系統，而加重細胞和DNA損傷情形。而且，GSH是否會經由降低細胞內自由基，增加抗氧化防禦，進而達到保護效果。 我們預實驗初步結果發現，動物體於SAH合併Hcy加重熱中風後之自由基生成；GSH可增加因SAH合併Hcy處理後熱中風的存活時間。故值得繼續進行研究探討。|
Homocysteine (HCY) is produced as a natural consequence of dietary methionine metabolism in human. Many evidences revealed that Hcy and SAH were one of the high risk factors for cardiovascular diseases and neurodegenerative diseases. The latter studies indicated that SAH and Hcy might have some possible interaction of complement or coordination in the pathological development of these diseases. So far, little attention has been given to observe and discuss the effects of both SAH with Hcy on the same experimental model. Circulatory shock, cerebral hypoxia and ischemia were thought to the main causes to form heatstroke in the heatstroke model of rats. Evidences from clinical and experimental animals with heatstroke have accumulated to indicate that vascular endothelial injury may contribute to circulatory shock, the multiple organs dysfunction, cerebral hypoxia, ischemia and injury, and eventually lead to shorten the survival. Additionally, there are recent evidences to suggest that heatstroke-induced reactions are associated with an increased production of free radicals (specifically hydroxyl radicals and superoxide), increased lipid peroxidation and decreased enzymatic antioxidant defenses in heatstroke rats. Meanwhile, high-risk populations in cardiovascular diseases are also easy to get heatstroke. According to above discussion, we speculate that both of SAH and Hcy may simultaneously participate in pathological formation in heatstroke. Indeed, our previous findings suggest that SAH combined with Hcy could promote the cerebral hypoxia/ischemia situation and inflammation response, and enhanced to aggravate the heatstroke damage. However, no attention has been given to study and discuss the correlation in effects of SAH together with Hcy on the heatstroke-induced oxidative damage. Glutathione (GSH) is a low weight tri-peptide molecule. GSH is the most abundant free radical scavenger synthesized endogenously in liver in humans. GSH is an important antioxidant and plays a major role in the detoxification. The previous study indicate that dietary GSH supplementation can increase myocardial GSH content and antioxidant defense capacity, thereby protecting the intact heart against oxidative damage and functional retardation caused by ischemia-reperfusion. Hence, GSH may prevent or improve the aggravated damage of heatstroke from influence of SAH together with Hcy. Additionally, so far, there is no investigation to study on the correlation between the GSH and heatstroke-induced damage. Therefore, we are the first to address hypotheses in this 3-year project and plan to conduct the following studies.1st year: Investigation of effects of treatment SAH together with Hcy on the heatstroke-induced oxidative damage and probable underlying mechanism. We will monitor changes of the animals’ anti-oxidative defense system parameters during heatstroke after exogenous treatment SAH together with Hcy. The purpose of this study is mainly to observe whether administration of SAH together with Hcy can aggravate the pathological parameters of heatstroke-induced damage via diminishing the anti-oxidative defense system, and results in shrinkage of survival time. Meanwhile, we will try to investigate probable underlying mechanism by acquiring the situations of brain and liver damage, products of radicals and GSH in heatstroke rats, after treatment SAH with Hcy.2nd year: Investigation of effects of SAH together with Hcy on pathological formation during heatstroke, after oral feeding with GSH. We will investigate whether feeding with GSH can prevent or therapy the aggravated heatstroke-induced damage, which is evoked by exogenous treatment SAH together with Hcy. In this experiment, we can not only newly confirm the roles of SAH and Hcy on aggravating heatstroke-induced damage, but also point out the Hcy-related diseases can view as the high risk factors to easily induce heatstroke formation. 3rd year: Investigation of the protective effects of GSH on aggravating microglia and hepatic cells injury under hypoxia condition by giving SAH together with Hcy, and trying to find probable working mechanism. We will firstly evaluate that effects of exogenous SAH with Hcy treatment on expression of radicals and enzymatic or non-enzymatic antioxidants, and the extent of cellular and DNA damage in the mouse microglia cell line (BV-2 cells) and human hepatic cell line (Chang’s liver cells) with incubation in hypoxic incubator chamber (mimicking the heatstroke hypoxic and ischemic condition in vivo).The purpose of this study mainly emphasis on whether exogenous SAH with Hcy treatment under hypoxic conditions in BV-2 cells and Chang’s liver cells can enhance increment of radicals and decrement of anti-oxidative defense system, and lead to cell and DNA damage; however, whether these increment of radicals and decrement of antioxidants can be diminished by GSH, and lead to protect the cell and DNA from injury. According our preliminary results in advance experiment, treatment of SAH together with Hcy can evoke and aggravate the heatstroke-induced high radical products, and apparently revealed the shrinkage in the survival time of heatstroke; however, administration with GSH can reverse these conditions. This study will be worthy to investigate further more closely.