敗血症(sepsis)是一種嚴重的發炎反應，也是臨床上常發生於重症病人的併發症， 當細菌外膜上的 lipopolysaccharide (LPS)侵入人體後，會藉由 Toll like receptor-4 (TLR-4) 路徑活化免疫細胞，並引發體内的發炎反應。在敗血症初期中性多形核白血球 (polymorphonuclear leukocyte, PMN)如受到LPS刺激後亦會藉由TLR-4路徑引發免疫反 應，促使PMN上黏著分子表現並migration至發炎組織，TLR-4路徑的活化亦會使PMN 發生去顆粒作用，產生自由基及蛋白質分解酵素毒殺外來物質，但分泌過多時會傷害 正常組織，這可能是敗血症引發多重器官衰竭的主要原因之一。microRNA(miRNA)是 一種長度很短的non-codingRNA，可調節細胞的分化、增殖和凋亡，其作用類似干擾 RNA的運作並抑制基因的表現，目前對其如何調控PMN所引發之非特異性免疫能力 的研究極少機制也仍不明確。Arginine (Arg)是一種非必需胺基酸，但過去許多研究顯 示它具有免疫調節及抑制發炎反應的作用，在壓力狀態下被視為一條件性必需胺基 酸。另外Arg為nitric oxide (NO)的前趨物質，故在敗■症時若體内有足夠的Arg，貝1J 可經由Arg-NO pathway產生足量NO，來毒殺外來物並促進免疫力。TLR-4路徑是發 炎過程中非常重要的免疫機制，而miRNA又扮演基因調控的角色，目前並無Arg添加 對白血球受到刺激後TLR-4 pathway及miRNA影響之相關文獻。因此本實驗將以三年 的時間，分別以體内及體外實驗之模式來探討Arg添加對敗血症時體内PMNTLR-4 pathway及miRNA之影響。第一、二年為細胞實驗，先探討給予PMN不同濃度Arg 培養，再予以LPS刺激後利用miRNA microarry及PCR array觀察TLR-4 pathway的 變化，並利用inducibleNOS抑制劑觀察NO的影響。第二年將特定miRNA利用 locked-nucleic acid-modified oligonucleotide (LNA) knock-down 後探討對其對應蛋白質 的影響，第三年為動物實驗，給予小鼠餵食含或不含Arg及NO抑制劑之配方後引致 敗血症，觀察Arg或NO對miRNA及TLR-4路徑之影響。本結果可作為營養基因學 在臨床上應用之參考。 Sepsis is one of the major causes of death in critically ill patients. When bacterial toxin lipopolysaccharide (LPS) insults the body, immune cells especially polymorphonuclear leukocyte (PMN) are activated at the early stage of sepsis via the Toll like receptor-4 (TLR-4) pathway. This procedure may result in the generation of excessive free radicals and proteolytic enzymes which may contribute to tissue injury and multiple organ failure in sepsis. MicroRNAs are small nucleotide sequences of non-coding RNA and have been demonstrated to play important roles in regulating gene expressions of various systems including immune-related proteins. Arginine (Arg) is a semi-essential amino acid with numerous useful physiologic properties. It is the specific precursor of nitric oxide (NO). Our previous reports have revealed that supplemental dietary Arg reduces inflammatory reaction and modulates immune function in septic rats. So far, there is no study investigating the effect of Arg on TLR-4 pathway and miRNA expressions in PMN stimulated by LPS. Therefore, we plan to carry out this study in 3 consecutive years. In the first year, we plan to administer different Arg levels and inducible NO inhibitor to PMN to investigate the effect of Arg on the alteration of TLR-4 pathway and miRNA expressions stimulated by LPS. In the second year, a locked-nucleic acid-modified oligonucleotid (LNA) will be used to knockdown the specific miRNA responsible for the changes of TLR-4 proteins found in the first year to reconfirm the effect of Arg . In the third year, animal study with or without Arg/NO inhibitor will be performed in septic mice to observe the influence of Arg or NO on TLR-4 pathway and miRNA expressions in septic condition. This study will be the first nutrigenomic study to investigate the effects of Arg on inflammatory-related gene and corresponding protein expressions in sepsis, and may provide useful information for clinical application in patients with sepsis.
