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研究生:傅耀賢
研究生(外文):Yaw-syan Fu
論文名稱:以熱水浴方式引起高體溫現象對於大鼠呼吸道神經性發炎反應的抑制作用與機轉之研究
論文名稱(外文):Inhibitory Effect of Warm Water Immersion-induced Hyperthermia on Neurogenic Inflammation in Rat Airways and the Possible Mechanisms
指導教授:劉昭成黃宏圖黃宏圖引用關係
指導教授(外文):Jau-Cheng LiouHung-Tu Hyang
學位類別:博士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:148
中文關鍵詞:熱休克反應呼吸道神經性發炎
外文關鍵詞:heat shock responseneurogenic inflammationairway
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神經性發炎反應是由位於哺乳動物周邊之小型無髓鞘感覺神經纖維受局部刺激或興奮後,除向中樞回傳感覺訊息外,並於周邊釋出神經胜肽。而這些神經胜肽包括物質 P (substance P, SP)、neurokinin A、CGRP 等,其中主要引起之神經性發炎反應為 SP,能透過 NK1 受器作用造成血管壁內平滑肌擴張及血管內皮細胞間隙之形成,導致局部組織血漿滲漏與發炎現象。
許多研究指出短暫熱休克前處理能活化動物熱休克蛋白質之表現,此類反應能協助動物對抗與降低後續其他類型壓力之傷害與程度。本研究將大鼠以恆溫水浴方式,使其核心體溫達到 42.0±0.5 °C,並持續 15 分鐘引發熱休克反應,控制處理則以 37.0 °C 水浴處理相同時間。24 小時後再以靜脈內注射辣椒素 (90μg/kg)、SP (3μg/kg) 或用電刺激迷走神經方式,以誘發呼吸道局部性神經性發炎反應,實驗期間同時記錄動物血壓變化,並利用預先注入循環中之 India ink 來標定出現滲漏之血管密度、或 Evans blue 滲漏量,來評估呼吸道組織血漿滲漏情形。實驗結果顯示,熱休克前處理能有效抑制由辣椒素、SP 及電刺激迷走神經所引起之呼吸道神經發炎反應,並降低神經性發炎期間對於血壓影響的程度。如在事先給予 aminoguanidine 抑制並阻斷誘導型一氧化氮合成酵素 (iNOS) 之活性時,對於 SP 所引起之神經性發炎反應並無明顯影響;但卻能消除熱休克前處理抑制神經性發炎的效果。而在給予 diphenhydramine 阻斷組織胺的 H1 受器活性下,能降低給予 SP 所引起之神經性發炎反應。本部份結果顯示在神經性發炎反應中,iNOS 活性與對於神經性發炎結果之影響不大,但在熱休克反應的啟動上扮演決定性角色;而 SP 引起之發炎反應中組織胺透過 H1 受器路徑來加成其結果,熱休克反應則對兩者所引起之發炎均有抑制效果。
在新生幼鼠時期給予高劑量辣椒素處理,成長後成為缺乏 C 型神經纖維之成鼠給予靜脈注射 SP,能引起較正常 SD 大鼠更為嚴重之神經性發炎反應。熱休克前處理對於後天缺乏 C 型神經纖維之動物同樣具有降低 SP 引起神經性發炎之效果。顯示初生時期以辣椒素處理引起後天缺乏 C 型神經纖維支配之動物,成長後依然保有對於 SP 誘發神經性發炎之生理反應,並對其出現敏感化現象。
綜合本研究之結論,熱休克前處理對不同方式誘導產生之呼吸道神經性發炎反應均具有顯著的抑制效果,且 HSP72 表現有關。在 SP 引起神經性發炎的過程中,於局部組織間能透過組織胺的 H1 受器的作用來加成其所誘發之神經性發炎反應與血漿滲漏之程度;但在此過程中組織內生性 iNOS 的活性則對 SP 引起之神經性發炎的急性反應上無明顯影響。
In mammals, the neurogenic inflammatory response can be induced by stimulation or activation on the peripheral sensory C-fibers to release neuropeptides from the peripheral terminals, at the same time their afferent functions are enhanced. There are several neuropeptides stored and released from peripheral terminals of the afferent fibers, such as substance P (SP), neurokinin A, and calcitonin gene related peptide (CGRP). SP is one of the major inflammatory mediators of neurogenic inflammation that can act on neurokinin-1 receptors on smooth muscles and endothelial cells of blood vessels, causing vasodilatation, endothelial gap formation, and local plasma leakage.
There are many studies and reports indicate that animals pretreated with a short period non-lethal hyperthermia can induce heat shock response and activate the expression of a group of inducible proteins called heat shock proteins (HSPs), and this stress response reduces the injury by same or other following stresses. In this study, the hyperthermia treatment (HT) was implemented by 42℃ hot water bath and the core body temperature of anesthetized rat was elevated and maintained around 42.0±0.5℃ for 15 min, and the normothermia control treatment (NT) was implemented by 37℃ warm water bath with the same period. 24 hours after NT or HT, the neurogenic plasma leakage was induced by intravascular injection with capsaicin (90 μg/kg), SP (3 μg/kg), or electrical stimulation on the right thoracic vagus nerve. The blood pressures of each animal were continually recorded during the neurogenic inflammation induction or sham operation. The amount of neurogenic inflammation of airway was evaluated by the area density leaky blood vessels. The leaking vessels were labeled with India ink and quantitative analysis by morphometric method. Plasma leakage was also measured by interstitial Evans blue concentration. The results indicated that HT could reduce plasma leakage and hypotension of the neurogenic inflammation that induced by capsaicin, SP or electrical stimulation on vagus nerve.
Animals pretreated with aminoguanidine (a selective inhibitor of iNOS) had no significant effect on the neurogenic inflammation by following systemic SP infusion, but that could eliminate the anti-neurogenic inflammatory effect of HT. Animal applied with diphenhydramine (an antagonist of histamine H1 receptor) could attenuate the neurogenic inflammation by following systemic SP infusion, and HT could attenuate the neurogenic inflammation that with or without H1 receptor antagonist. This result indicates that NO synthesis and the activity of iNOS have few effects on neurogenic inflammation of airway, but it plays a critical factor on the initiation of heat shock response. The neurogenic inflammation induced by SP not only direct act on blood vessels but have other indirect effect by the histamine H1 receptor to enhance inflammation.
Neonatal rats received high dose capsaicin treatment would induce irreversible sensory C-fiber denervation. The adult rats that were neonatally treated with capsaicin showed a more serious inflammatory response to systemic SP infusion as compared with animals neonatally treated with vehicle. HT still had the anti-inflammatory effects on the neurogenic inflammation that induced by SP. The results indicated that animals with sensory C-fiber denervation might conserve their neurogenic inflammatory responses and were hypersensitive to SP.
In conclusion, the HT could attenuate the neurogenic inflammation that induced by different drugs or methods, and the anti-inflammatory effects were correlated with the increase in HSP72 expression. In the neurogenic inflammation induced by SP, the activation of histamine H1 receptors may enhance inflammation, but the activity of endogenous iNOS was less effective.
致謝……………………………………………………………………………i
摘要……………………………………………………………………………ii
Abstract…………………………………………………………………………v
Table of contents………………………………………………………………ix
List of Figures…………………………………………………………………xii
Abbreviations………………………………………………………………xiv
Chapter 1. Introduction and Literature Review…………………………………1
1-1 Sensory Nerves and Airway Neurogenic Inflammation………………1
1-2 Heat Shock Response…………………………………………………5
1-3 NO, NOS and Inflammation……………………………………………8
1-4 Histamine and Neurogenic Inflammation……………………………11
1-5 Neonatal Capsaicin Treatment and Neurogenic Inflammation……… 13
Chapter 2. Motive and Research Objective……………………………………15
2-1 Motive…………………………………………………………………15
2-2 Research Objective……………………………………………………15
Chapter 3. Materials and Methods……………………………………………17
3-1 Animals………………………………………………………………17
3-2 Hyperthermia Treatment (Heat Shock Pretreatment) ………………17
3-3 Measurement of Blood Pressure………………………………………18
3-4 Induction of neurogenic inflammation………………………………19
3-5 Measurement of the Degree of Inflammation…………………………21
3-6 The effects of aminoguanidine on inflammation induced by SP…24
3-7. The effects of diphenyhydramine on inflammation induced by SP…25
3-8 The effects of sensory C-fiber denervation on inflammation induced by SP…………………………………………………………………………26
3-9. Histological study……………………………………………………29
3-10 Western Blotting of HSP72 and β-actin……………………………30
3-11 Statistical Analysis…………………………………………………31
Chapter 4. Result………………………………………………………………32
4-1 The effects of heat shock response on neurogenic inflammation that induced by capsaicin and SP………………………………………………32
4-2 The effects of heat shock response on inflammation that induced by electrical stimulation on right vagus trunk………………………………36
4-3 The effects of aminoguanidine and diphenhydrmine on inflammation that induced by SP………………………………………………………38
4-4 The effects of sensory C-fiber denervation on inflammation induced by SP…………………………………………………………………………40
Chapter 5. Discussion…………………………………………………………45
5-1 The effects of heat shock response on neurogenic inflammation that induced by capsaicin and SP………………………………………………45
5-2 The effects of heat shock response on inflammation that induced by electrical stimulation on right vagus trunk………………………………51
5-3 The effects of aminoguanidine and diphenhydrmine on inflammation that induced by SP………………………………………………………55
5-4 The effects of sensory C-fiber denervation on inflammation induced by SP…………………………………………………………………………58
Chapter 6. Conclusion…………………………………………………………63
References……………………………………………………………………64
Figures…………………………………………………………………………82
Table…………………………………………………………………………124
Appendixes…………………………………………………………………125
I. Flowchart of protocol for neurogenic inflammation induction………125
II. Flowchart of protocol for aminoguanidine treatment…………………126
III. Flowchart of protocol for diphenhydramine treatment………………127
IV. Flowchart of protocol for neonatal capsaicin treatment……………128
Publications and Presentations………………………………………………129
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