| 摘要: | 腦中風是最常見的腦血管疾病，是指顱內血管因為急性阻塞或破裂導致該區域的腦細胞缺乏養分而無法維持正常的生理活性。根據世界衛生組織統計，中風是造成全球的第二大死因。腦中風依照其病理機轉可以分為出血性和缺血性中風，其中缺血性中風佔百分之八十七。
缺血性中風中因為血流受阻而造成缺乏養分及氧氣的組織，在回復血液循環時會引起多種細胞毒性物質過度生成，已被推測會進一步惡化腦部損傷的可能，這些過量的毒性物質除了常見的自由基及發炎前驅物質以外，還有就是代謝環境壓力，糖解作用代謝產物甲基乙二醛的累積，而產生的醣化壓力就是常見的代謝壓力，在正常生理情況下細胞會藉由乙二醛酶系統清除甲基乙二醛的毒性，但過量的甲基乙二醛與蛋白質結合後會使蛋白質醣化，使蛋白質失去功能，且最後會形成糖化終產物(AGEs)，有研究指出同樣發生中風的情形下，糖尿病患者的死亡率是非糖尿病患者的4倍，因為糖尿病患者本身的血糖偏高，造成中風後會產生更多的醣化壓力，造成中風損傷的惡化更為嚴重。
因此，本篇研究主要想探討，在正常血糖的情況下，腦中風後，腦內的甲基乙二醛是否會有所變化，研究結果發現在中風再灌流1小時後，患側半腦的甲基乙二醛修飾蛋白有顯著下降的現象，而隨著時間的增加，到了中風再灌流12小時後，患側半腦的甲基乙二醛修飾蛋白與對側半腦相比有顯著上升的現象，而之後我們藉由重組腺相關病毒轉導的方式，使小鼠的GLO1過度表現，增強清除甲基乙二醛的效率，並在缺血性中風前後進行神經缺陷性行為測試以及最後的腦損傷範圍評估。以神經學狀態評估去評判小鼠的運動、感覺、反射及平衡行為是否異常，發現AAV-GLO1組的小鼠中風後的神經損傷分數有下降的現象。以拉力測試讓小鼠抓取測量儀器去評估小鼠的抓握能力，發現AAV-GLO1組的小鼠其抓握能力數值有顯著改善的現象。另外在滾輪測試及自發性運動試驗，分別去觀測小鼠運動功能及移動能力，皆發現AAV-GLO1組的小鼠在兩種測試中都有改善的趨勢。最後，我們以2 %氯化三苯基四氮唑染色去觀察小鼠腦梗塞的區域，可以發現AAV-GLO1組的小鼠腦損傷區域有顯著的下降，表示過度表現GLO1蛋白以後可以有效減少中風後腦損傷的情形發生。並且在細胞實驗也發現過度表現GLO1蛋白的Neuro2a神經細胞，可以降低甲基乙二醛刺激引發的內質網壓力並有效保護神經細胞死亡的情況。
總結來說，我們發現在缺血性中風後，腦內的甲基乙二醛會隨著時間慢慢累積，並且藉由過度表現GLO1後，可以有效改善小鼠神經缺陷性行為表現以及腦梗塞的情形，而這可能是因為GLO1蛋白可以降低內質網壓力來保護神經細胞免於細胞死亡的原因。
The most common cerebrovascular disease is stroke, which is defined by the lack of nutrients due to acute occlusion and rupture in cerebral vessel so that the brain cells are unable to maintain normal physiological activity. According to the World Health Organization, stroke is the second of top ten global deaths. In addition, stroke can be divided into hemorrhagic and ischemic type according to the pathological mechanism. Of all strokes, ischemic stroke, which usually caused by blood flow occlusion due to thrombosis or embolism, accounts for eighty seven percent.
In ischemic stroke, tissue will be lack of nutrients and oxygen due to occlusion of blood flow. At reperfusion, tissue and cells will over produce various cytotoxic substances which have been speculated the possibility of further worsening brain damage after ischemic stroke. Besides free radicals and inflammatory precursors, metabolic stress is another toxic substances. The accumulation of glycolysis metabolite like methylglyoxal resulting dicarbonyls stress is a common metabolic stress. Under normal physiological conditions, the cells will eliminate the toxicity of methylglyoxal by the glyoxalase system. However, the excessive methylglyoxal combining with the proteins will lead to the proteins glycated, causing the proteins to lose its function and eventually becoming advanced glycation end products (AGEs). Epidemiological studies indicated diabetic stroke patients have a 4-fold higher mortality than their non-diabetic counterparts. In addition, the high level of blood sugar will increase the production of methylglyoxal which will lead to more severe dicarbonyls stress after stroke. This is one of the reasons why the stroke injury will be larger and more severe in diabetes patients after stroke.
Therefore, we aimed to investigate whether the level of methylglyoxal will pathologically change following stroke under the normal blood sugar. According to our results, the level of MG-modified proteins significantly decreased in the ipsilateral side compared with the contralateral side following 1 hour reperfusion after stroke. However, as time progressed, the level of MG-modified proteins significantly increased in the ipsilateral side compared with the contralateral side following 12 hour reperfusion after stroke. Furthermore, we also investigated whether overexpressing GLO1 by recombinant adeno-associated virus (rAAV) can improve stroke injury. According to our data, AAV-GLO1 group would significantly improve at neurological severity scores and grip strength test comparing to AAV-GFP group after ischemic stroke. In addition, AAV-GLO1 group had an improvement trend at rota-rod performance test and locomotor activity. Finally, we observed the infarction size by staining with 2 % TTC. It could be found that there were a significant decrease in the area of brain damage in the AAV-GLO1 group, indicating that overexpression of GLO1 protein could effectively reduce brain damage after ischemic stroke. Furthermore, in vitro, it was also found that n2a cell overexpressed the GLO1 protein could effectively decrease methylglyoxal-induced ER stress and protect neuron from death.
In summary, we found that methylglyoxal in the ipsilateral brain slowly accumulated after ischemic stroke, and overexpressing GLO1 could effectively improve neurodegenerative behavior and infarction size in mice by decreasing methylglyoxal-induced ER stress and cell death. |
