格蘭氏陰性細菌上的脂多糖，又稱作內毒素是引起敗血症的主要物質，而嚴重的敗血症會造成很高的死亡率。因此，進一步的發展出同時擁有抑制細菌生長和中和內毒素能力的抗菌胜肽非常具有臨床運用的價值。在我們之前的研究中發展出S1系列抗菌胜肽，發現其中幾個胜肽可能有中和內毒素的能力，所以在這次的研究中，我們進一步設計出極短鏈的抗菌胜肽，發現其中KWWK-Nal2具有很好的抗菌及中和內毒素的能力，此外，我們證實了之前所假設的模型，所有的S1和極短鏈KWWK系列胜肽皆可以促使脂多糖聚集，但只有少數具有兩個以上體積較大β-naphthylalanine (Nal)胺基酸的胜肽(S1-Nal2, S1-Nal3, Nal2-S1, kwwk-Nal2)才具有中和內毒素的能力。更重要的是我們發現S1-Nal2和kwwk-Nal2可以有效的抑制老鼠巨噬細胞由內毒素所引起NO和TNF-α的釋放，另外，在動物實驗中我們也發現這兩條胜肽可以明顯的減少被內毒素感染的老鼠血清中TNF-α的含量和降低肺臟的損害，所以我們的研究結果指出S1-Nal2和KWWK-Nal2這兩條胜肽具有潛力發展成抗內毒素的藥物來治療由內毒素所引起的敗血症。

Lipopolysaccharide (LPS), known as endotoxin, plays an important role in triggering of sepsis due to gram-negative bacterial infection, which is accompanied with high mortality in patients. Therefore, developing dual antimicrobial and LPS-neutralizing peptide as therapeutic agents could be useful in clinical application. Previously, we developed S1 series antimicrobial peptide and revealed that some of them might have endotoxin neutralization activity. In this study, we further developed ultra-short series peptide and found that KWWK-Nal2 peptide also showed efficient antimicrobial and endotoxin neutralization activity. Furthermore, we demonstrated a proposed model that all of S1 series and ultra-short KWWK series peptide could promote LPS aggregation, but only few peptides (S1-Nal2, S1-Nal3, Nal2-S1, KWWK-Nal2) containing more than two bulky amino acid β-naphthylalanine (Nal) residues could actually neutralize the LPS toxicity in LAL assay. More importantly, we showed that S1-Nal2 and KWWK-Nal2 significantly inhibited nitric oxide(NO) and tumor-necrosis factor alpha (TNF-α) release from LPS-induced murine macrophage; furthermore, in vivo tests also indicated that those two peptides significantly reduced serum TNF-α levels and alleviated the lung tissue damage in LPS-stimulated mice. Overall, our results indicate that S1-Nal2 and KWWK-Nal2 are potential endotoxin neutralization agents in treatment of LPS-induced sepsis.

Contents
中文摘要 I
Abstract II
Acknowledgement III
Contents IV
Chapter 1. Introduction 1
1.1 Overview of sepsis 1
1.2 The therapy in sepsis 2
1.3 Development of antimicrobial peptide 4
1.4 Endotoxin neutralizing activity of antimicrobial peptide 5
1.5 The aim of this study 6
Chapter 2. Materials and methods 8
2.1 Materials 8
2.2 Quantization of the peptides 8
2.3 Minimum inhibitory concentration assay 9
2.4 Limulus amebocyte lysate (LAL) assay 10
2.5 Dynamic light scattering (DLS) 11
2.6 Cell culture and treatments 13
2.7 Cytotoxicity assay 13
2.8 Quantification of nitric oxide(NO) production in murine macrophage J774A.1 14
2.9 Quantification of pro-inflammatory cytokine production in murine macrophage J774A.1 15
2.10 LPS animal model 16
2.11 Quantification of pro-inflammatory cytokine production in mice 17
2.12 Histological analysis 18
Chapter 3. Results 20
3.1 Antimicrobial activities of the peptides 20
3.2 Dynamic light scattering of lipopolysaccharide aggregation in the presence of peptides 20
3.3 Endotoxin-neutralizing activity of peptides 21
3.4 Cytotoxicity of peptides on murine macrophages J774A.1 22
3.5 Inhibition of nitric oxide produces in LPS-stimulated murine macrophage J774A.1 22
3.6 Inhibition of pro-inflammatory cytokine produces in LPS-stimulated murine macrophage J774A.1 23
3.7 Suppression of pro-inflammatory cytokine produces in LPS-stimulated mice model 24
3.8 Reduction of lung injury in LPS-stimulated mice model 24
Chapter 4. Conclusion and Discussion 26
Figures 30
Figure 1-1 A schematic diagram of structure in the Gram-negative bacteria cell wall and structure of lipopolysaccharide (LPS) [38, 39]. 30
Figure 1-2 The mechanism for inflammatory effects in LPS-induced macrophages. Modified from [40]. 30
Figure 1-3 Multiple targeting pathways attenuate the body’s inflammatory response to endotoxin [8]. 31
Figure 1-4 The structure of non-nature amino acid – Naphthylalanine. 31
Figure 2 MIC values displayed on a color scale for KWWK-Nal, KWWK-FNal, KWWK-Nal2 with different sodium concentrations. 32
Figure 3 Size distributions of lipopolysaccharide aggregate in the presence of peptides. 33
Figure 4 Endotoxin-neutralizing activity in the presence of peptides. 34
Figure 5 Cytotoxicity of peptides on murine macrophages J774A.1. 35
Figure 6 Inhibition of nitric oxide produces in LPS-stimulated murine macrophage J774A.1. 36
Figure 7 Inhibition of pro-inflammatory cytokine (TNF-α) produces in LPS-stimulated murine macrophage J774A.1. 37
Figure 8 Suppression of pro-inflammatory cytokine (TNF-α) produces in LPS-stimulated mice model. 38
Figure 9 Reduction of lung injury in LPS-stimulated mice model. 39
Table 1 Antimicrobial peptide sequence 40
References 41

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