|摘要: ||同半胱胺酸(Homocysteine; Hcy)為甲硫胺酸(Methionine )代謝的產物，主要是由腺?化的化合物：S-腺核?甲硫胺酸(S-adenosylmethionine; SAM)和S-腺核?同半胱胺酸(S-adenosylhomocysteine; SAH)代謝而成。以往許多證據顯示Hcy是心血管疾病和神經損傷相關疾病的危險因子之一。然而，近年卻有學者指出高濃度Hcy與心血管疾病以及中風之間較無直接關係，且在這些疾病中也往往會伴隨著SAH(Hcy生合成的前驅物)濃度的增加，反而SAH比較被關注。證據顯示SAH在心血管疾病和神經損傷疾病中，血漿及腦中SAH有明顯增加的趨勢。研究指出SAH亦在阿茲海默症、心血管疾病和腦部神經損傷疾病中扮演重要角色。有學者也指出SAH和Hcy之間可能存在某種互補或協同的作用。因此，無論是心血管或是神經損傷相關疾病病理作用影響，SAH和Hcy似乎皆有可能參與其中的作用機制。但現今的研究，卻鮮少有文獻報告將兩者一併在同一試驗模式下觀察討論。而熱中風好發於高溫下工作者或處於熱環境者，會造成高溫、腦缺血、神經異常和多發性器官失調。而心血管方面疾病的高危險因子者，也較易罹患熱中風。目前已知熱中風所造成的傷害，主要病理因素就是心臟血管功能喪失或損傷、細胞介質素(特別是TNF-α與IL-1β被探討最多)媒介之發炎反應所致多重器官衰竭和腦部缺氧缺血所致神經細胞損傷或死亡所引起。另外許多文獻報告，在誘發腦部缺氧缺血模式的動物實驗中，血中高濃度的Hcy或SAH會促進神經損傷或死亡的情形。根據以上探討，我們推測SAH或Hcy也極可能參與了熱中風損傷的病理發展。然而，截至目前為止卻沒有任何的研究文獻指出SAH或Hcy與熱中風所致損傷的相關性，於是我們創先提出研究計畫探討。在未來三年的研究計畫中，我們將進行以下研究。探討熱中風大鼠體內SAH和Hcy含量的變化，及外加給予高量SAH或Hcy對大鼠熱中風損傷參數之影響--- 第一年：1)我們將分別檢測於誘發動物體生成熱中風前後，血漿和腦中SAH和Hcy濃度變化情形。(2)為了確定SAH和Hcy於熱中風中的影響，我們將外加給予高濃度SAH或Hcy，觀察對動物體熱中風損傷參數的影響(如存活時間、動脈血壓和細胞介質素：TNF-α和IL-1β的表現等)。(3)同時也將觀測腦部紋狀體區神經細胞缺氧缺血指標(麩胺酸含量和乳酸/丙酮酸比值)與神經損傷指標(甘油含量)，以評估神經損傷程度。探討針對體內高SAH與Hcy表現之大鼠(由餵食高含量甲硫胺酸飲食誘發)，給予高含量葉酸與維生素B12飲食處理後，對熱中風病理生成之影響--- 第二年：我們將計劃利用飲食預防醫學的處理方式，來評估是否具有預防熱中風損傷的作用。(1)因此，?予動物體餵食高含量甲硫胺酸飲食(可促使血中高SAH及Hcy表現)或合併高含量葉酸及維生素B12飲食，來觀察動物體內血漿和腦中SAH和Hcy濃度變化情形，評估高含量葉酸及維生素B12飲食對SAH與Hcy濃度變化程度。(2)觀測對動物體熱中風損傷參數的影響(如存活時間、動脈血壓和細胞介質素：TNF-α和IL-1β的表現等)，而達到改善或預防熱中風所造成的傷害。(3)進一步評估腦部缺血缺氧與神經損傷程度，也可更確定SAH及Hcy對熱中風損傷的影響。利用小鼠微神經膠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，探討SAH或Hcy對神經膠細胞於缺氧狀況下的影響及其可能作用機制--- 第三年：由於熱中風所致的神經傷害，引發的主因可能是腦部神經細胞缺氧缺血及細胞介質素所媒介之發炎反應所造成。因此，我們將做一體外微神經膠細胞試驗(BV-2 cells)培養於低氧槽中，來模擬活體熱中風缺氧缺血狀況，再分別給予外加SAH或Hcy處理細胞，觀察(1)細胞內外SAH與Hcy濃度變化。(2)細胞DNA損傷的影響。(3)觀測細胞介質素(TNF-α與IL-1β)的釋放程度，以探討神經細胞缺氧時SAH或Hcy所致的細胞損傷，是否與細胞介質素所媒介之發炎反應有關。對於熱中風所造成的傷害，目前並無一個可完全治療或預防的有效策略，而其傷害的完整病理作用機制也未完全被釐清。在本三年研究試驗中，我們率先提出觀察探討SAH或Hcy與熱中風所致損傷的相關性，也將試圖利用飲食預防醫學的處理方式，來評估是否具有預防熱中風損傷的作用，再以細胞作用機制探討SAH或Hcy，於熱中風中可能參與的病理角色。我們預實驗初步結果發現，動物體於熱中風生成後，腦中SAH含量確實明顯增加。此一研究計畫值得更深入探討。|
Homocysteine (Hcy), which was produced during methionine metabolic cycle, mainly formed from metabolism of adenosyl compound: S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). It has been proposed that Hcy was one of the high risk factors for cardiovascular diseases and neurodegenerative diseases. However, some recent studies have found no significant associations between plasma elevated Hcy and cardiovascular diseases or neurodegenerative diseases. These diseases frequently followed by SAH accumulation (the immediate precursor for Hcy biosynthesis). Therefore, SAH has also been concentrated. Many evidences showed that the plasma and cerebral levels of SAH in cardiovascular and neurodegenerative diseases were significantly increased. Studies have been attributed to SAH may play an important role in Alzheimer』s disease, cardiovascular, and neurodegenerative diseases. Some scientists also suggested that it might have some possible interaction of complement or coordination between SAH and Hcy in the pathological development of these diseases. So far, little attention has been given to observe and discuss the effects of both SAH and Hcy on the same experimental model. Heatstroke developed hyperthermia, cerebral ischemia, neurological abnormalities, and multiple organ disorders with physical work in warm environments, or at rest in a hot environment. Patients with the high risk factors of cerebrovascualr disorders were also easy to get heatstroke. The cardiovascular dysfunctions or injuries, the inflammatory response with cytokines (especially TNF-α and IL-1β)-mediated multiple organs failure, and the cerebral hypoxia and ischemia-evoked neuronal injuries or death were thought the main causes to develop the pathological formation of heatstroke-induced damage. Literatures reported that the plasma or cerebral high levels of Hcy or SAH cloud enhance the situations of neuronal injuries or death in various animal models of cerebral ischemia and hypoxia. According to above observation, we speculate that the SAH and Hcy might be involved in the pathological development of heatstroke-induced damage. Up to now, no studies have ever tried to investigate the correlation in SAH and Hcy, and the heatstroke-induced damage. Therefore, we are the first to address hypotheses in this 3-year project and plan to conduct the following studies. Investigation of the changes of the SAH and Hcy levels in rats during heatstroke, and the effects of exogenous treatment with high levels of SAH or Hcy on parameters of heatstroke-induced damage--- 1st year: (1) As the first step, we will assess the changes of the plasma and cerebral SAH and Hcy levels before or after heatstroke induction. (2) In order to ascertaining the effects of SAH and Hcy on heatstroke, we will treat exogenous high levels of SAH or Hcy, and monitor the effects on parameters of heatstroke-induced damage, such as the survival time, mean arterial pressure and cytokines levels of TNF-α and IL-1β. (3) Accordingly, the degree of neuronal damage will be also evaluated by measuring the cerebral cellular ischemia (e.g. glutamate and lactate/pyruvate ratio) and injury (e.g. glycerol) markers in corpus striatum of rats. Investigation of the effects of rats with high levels of SAH and Hcy (induced by feeding diet of high content methionine) on pathological formation of heatstroke after treatment with diet of high content folate and vitamin B12 --- 2nd year: We will plan to use the method of diet treatment as a prophylaxis to estimate whether it has influences on prevention for the heatstroke-induced damage. First, (1) we will measure the changes of the plasma and cerebral levels of SAH and Hcy in rats after giving treatment with diet of high content methionine (enhancing the high levels of SAH and Hcy in plasma) or the combined treatment with diet of high content both folate and vitamin B12. (2) We will attempt to use the diet treatment as a prophylaxis, and examine the effects on parameters of heatstroke-induced damage, and to evaluate the efficacy on improvement or prevention. (3)Furthermore, we also assess the degree of cerebral ischemia, hypoxia, and neuronal injuries after giving diet treatment in every rat of each group. Investigation of the effects of the hypoxia condition on mouse microglia cell line (BV-2 cells) with incubation in modular incubator chamber (mimicking the ischemic and hypoxic conditions in rats during heatstroke induction), and trying to find possible working mechanism--- 3rd year: The cerebral ischemia and hypoxia, and inflammatory response with cytokines might be main causes to participate in pathological formation of heatstroke-induced neuronal damage. Consequently, we will use the mouse microglia cell line (BV-2 cells) with incubation in modular incubator chamber to mimic the ischemic and hypoxic conditions in vivo experiment in heatstroke, and observe (1) the changes of intracellular and extracellular concentrations of SAH and Hcy, (2) the degree of DNA damage, and (3) the releasing levels of cytokines (TNF-α and IL-1β) before or after exogenous treatment of SAH or Hcy. We attempt to study the SAH or Hcy-induced cellular injury under hypoxic condition whether was associated with cytokines-evoked inflammatory response. Until now, the intact mechanisms of heatstroke-induced damage remained unclear, and no one therapeutic strategy could be effective to completely improve or prevent. In this 3-year project, we are the first to investigate the correlation between SAH or Hcy and heatstroke-induced damage in animal model, and will also try to use the manner of diet treatment as a prophylaxis to estimate whether it has influences on prevention for the heatstroke-induced damage. Next, we will also explain the role of SAH or Hcy in pathological development of heatstroke-induced damage by an in vitro cell line experiment. Our preliminary results found that the levels of SAH in rats』 brain were apparently increased after heatstroke induction. This study will be worthy to investigate further more closely.