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研究生:王定仁
研究生(外文):WANG, TING-JEN
論文名稱:豬結締組織水解產物應用於調節異位性皮膚炎小鼠模式之潛在效果
論文名稱(外文):The Potential Effects of Porcine Tissue Hydrolysate on Modulated Atopic Dermatitis Murine Model
指導教授:謝長奇謝長奇引用關係
指導教授(外文):HSIEH, CHANG-CHI
口試委員:劉登城
口試委員(外文):LIU, DENG-CHENG
口試日期:2024-07-18
學位類別:博士
校院名稱:東海大學
系所名稱:畜產與生物科技學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:英文
論文頁數:123
外文關鍵詞:Atopic DermatitisPorcine byproducts hydrolysatesBioactive peptidesImmunomodulationHair follicles regenerationEpoxide hydrolases
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異位性皮膚炎 (Atopic dermatitis, AD) 是一種常見的過敏型發炎性皮膚病,其特徵是多種病因。本研究探討了皮膚接種卵清蛋白(ovalbumin, OVA)和複合2,4,6-三硝基-1-氯苯(2,4,6,-trinitrochlorobenzene, TNCB)誘導的BALB/c小鼠模式中研究了異位性皮膚炎的發病機制。本動物模式模擬慢性 AD 的特徵,包括皮疹、肥大細胞浸潤、血清抗 OVA 特異性 IgE 和細胞激素的調昇以及毛囊的病理變化。本研究探討了豬肺和豬腎水解產物在 AD 發展中對過敏和免疫反應的調節作用。在 AD 癒後水解物對促進皮膚傷口癒合和毛囊毛髮再生的潛在性效果。結果顯示,水解豬臟器副產物處理可調節 T 細胞激素並顯著降低第二型 T 輔助細胞 (type 2 T helper cell, Th2)極化,最終產生對降低OVA特異性免疫球蛋白 E (immunoglobulin E, IgE)含量的過敏免疫抑制。進一步利用組織免疫染色學分析顯示對髮根再生的調控蛋白LGR5、LGR6和CD34的表達增強。這些結果顯示,本研究的豬組織水解物所含寡肽可能會調節異位性皮膚炎所致傷口癒合過程中的上皮角質化,促進毛囊新生,毛髮再生的過程。本研究透過胜肽測序法來確定豬肺和豬腎水解物中生物活性肽的序列,解構對於抗發炎免疫調節之分子對接,來研究水解液中之機能性寡肽對已知的可溶性環氧化物水解酶(soluble epoxide hydrolase, sEH)與白三烯素A4水解酶(leukotriene A4 hydrolase, LTA4H)抑制活性。我們的發現揭示了豬副產物水解胜肽在AD 免疫調節作用中的用途,並進一步應用以改善伴侶動物皮膚和毛囊再生的傷口癒合。綜上所述,這項研究發展了一種治療和/或管理異位性皮膚炎的新方法。
關鍵字:異位性皮膚炎,水解豬副產物,機能性胜肽,免疫調節,毛囊再生,環氧化物水解酶

Atopic dermatitis (AD) is a prevalent inflammatory skin condition characterized by multifaceted etiologies in allergy effect. This study investigated the pathogenesis of atopic dermatitis in BALB/c murine model, induced with skin inoculated ovalbumin (OVA) with 2,4,6-trinitro-1-chlorobenzene (TNCB). This model simulates features of chronic dermatitis, including skin rashes, mast cell infiltration, upregulation of serum anti-OVA specific IgE and cytokines, and pathological changes in hair follicles. This study investigates the potential effects of hydrolysate derived from porcine lung and kidney on modulating allergic reactions and immune responses in atopic dermatitis (AD). Additionally, it examines the effects on skin wound healing and hair follicle regeneration. Results revealed that treatment with porcine tissue hydrolysate increased T regulatory cells and suppressed cytokine profiles and anti-OVA IgE levels, leading to a significant reduction in Th2 polarization. Immunohistochemical analysis showed enhanced expression of hair regeneration biomarkers including Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6), and Cluster of Differentiation 34 (CD34). These findings collectively suggest that porcine tissue hydrolysate may facilitate epithelialization during wound healing, promote follicle neogenesis, and support hair regeneration in atopic dermatitis conditions. De novo sequencing was used to identify the potential oligopeptides in the porcine lung and kidney hydrolysate. Furthermore, molecular docking determined the antagonistic activity of these oligopeptides against epoxide hydrolases such as soluble epoxide hydrolase (sEH) and leukotriene A4 hydrolase (LTA4H). Our finding expounds the usage of porcine byproducts in the immunomodulatory effect of AD and improved wound healing of skin and hair follicles regeneration in companion animals. Overall, this study highlights a novel approach to the treatment and/or management of atopic dermatitis.
Keywords: Atopic Dermatitis; Porcine byproducts hydrolysates; Bioactive peptides; Immunomodulation; Hair follicles regeneration; Epoxide hydrolases
TABLE OF CONTENTS
LIST OF FIGURES ................................................................................................................... v
LIST OF TABLES .................................................................................................................. viii
LIST OF ABBREVIATIONS.................................................................................................... ix
ABSTRACT............................................................................................................................. xii
CHAPTER Ⅰ.............................................................................................................................. 1
INTRODUCTION.................................................................................................................... 1
1.1. Background ................................................................................................................. 1
1.2. Objectives of study ...................................................................................................... 2
CHAPTER II ............................................................................................................................ 3
LITERATURES REVIEW...................................................................................................... 3
2.1. Skin anatomy and physiology ....................................................................................... 3
2.2. Atopic dermatitis (AD) ................................................................................................. 5
2.3. Chronic inflammation and inflammatory cytokines in AD........................................... 7
2.4. Allergic responses in AD............................................................................................. 12
2.5. Wound healing and skin regeneration in AD .............................................................. 16
2.6. Hair follicles regeneration in AD ................................................................................ 18
2.7. Atopic dermatitis in pets ............................................................................................. 19
2.8. Porcine tissue hydrolysate and bioactive peptides (BAP) .......................................... 29
CHAPTER Ⅲ ........................................................................................................................ 32
MATERIALS & METHODS ................................................................................................ 32
3.1. Materials...................................................................................................................... 32
3.2. Preparation of porcine tissue hydrolysate ................................................................... 32
3.3. Animals and experimental design ............................................................................... 33
3.4. Determination of serum and tissue biochemical parameters ...................................... 36
3.5. Immunohistochemistry examination (IHC) ................................................................ 39
3.6. Porcine lung and kidney hydrolysate sequencing ....................................................... 41
3.7. Molecular docking analysis ........................................................................................ 42
3.8. Statistical analysis ....................................................................................................... 46
CHAPTER Ⅳ......................................................................................................................... 47
RESULTS ................................................................................................................................ 47
4.1. Effect of porcine tissue hydrolysate on body, thymus and spleen weight of the mice 47
4.2. Effect of porcine tissue hydrolysate on spleen immune cell proliferation in
OVA+TNCB-induced AD mice ......................................................................................... 47
4.3. Effect of porcine tissue hydrolysate on serum total antibody levels in OVA+TNCBinduced
AD mice................................................................................................................ 48
4.4. Effect of porcine tissue hydrolysate on serum anti-OVA antibody levels in
OVA+TNCB-induced AD mice ......................................................................................... 48
4.5. Effect of porcine tissue hydrolysate on splenocyte cytokines IFN-γ, IL-4, IL-5, and IL-
6 levels in OVA+TNCB-induced AD mice ........................................................................ 48
4.6. Effect of porcine tissue hydrolysate on immunohistochemical analysis .................... 49
4.7. Effect of porcine tissue hydrolysate on LC-MS Spectrum analysis ........................... 50
4.8. Effect of porcine tissue hydrolysate on Peptide De novo Sequencing ....................... 50
4.9. Effect of porcine tissue hydrolysate on molecular docking ........................................ 50
CHAPTER Ⅴ .......................................................................................................................... 83
DISCUSSION ......................................................................................................................... 83
CHAPTER Ⅵ......................................................................................................................... 86
CONCLUSION ...................................................................................................................... 86
REFERENCES....................................................................................................................... 89
Appendix A ........................................................................................................................... 106
Appendix B ........................................................................................................................... 107
Appendix C ........................................................................................................................... 108
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