臺灣博碩士論文加值系統

發炎疼痛是一種人類常見的症狀，但疼痛的詳細機制與止痛機轉探討依然有其限制，發炎反應形成之後會導致周邊組織疼痛過敏化，接著引起組織酸化、痛覺接受器活化以及發炎反應的相關物質釋放。針灸常被應用於治療疼痛相關疾病，但其止痛的機制仍未全然了解，第三型酸敏性離子通道、第一與第四型瞬態電位類香草素受器常被認為與機械力、溫度相關性發炎疼痛有關，然而其在電針止痛的機制當中尚未被探討。
我們利用注射鹿角膠或完全佐劑至老鼠腳掌來誘導老鼠產生發炎反應，然後運用數種動物行為實驗，如針刺回縮反應、熱輻射、與冷熱板來探討發炎疼痛如何誘發與電針足三里穴位止痛的效果，更運用西方墨點法、免疫螢光染色與即時定量聚合酶連鎖反應來觀察第三型酸敏性離子通道、第一與第四型瞬態電位類香草素受器表現量之增減，進一步運用全細胞紀錄來探討電針足三里穴位前後，背根神經節生理特性的改變。
我們的結果發現透過鹿角膠或完全佐劑誘導老鼠產生發炎反應，可以經由電針足三里進行治療。在第四天後，發炎反應所誘導的第三型酸敏性離子通道、第一與第四型瞬態電位類香草素受器在背根神經節大量表現的現象，可以經由電針足三里穴位而調降，進一步我們運用全細胞紀錄來觀察背根神經節的生理特性，發現發炎所導致神經過度興奮的情形可以被電針所調控，動作電位的閾值與開啟關閉的時間皆受到電針所調控，另外給予第一與第四型瞬態電位類香草素受器的專一性致效劑，來誘導離子通道開啟的比例亦會被電針所調降。我們的研究進一步發現，電針後會調控背根神經節與脊椎神經的前列腺酸性磷酸酶的表現，進而活化腺苷酸接受器進而降低痛覺接受器的活性來達到止痛效果。同時發現針灸能有效調節發炎反應所導致的神經過度興奮的現象。我們的研究針對針灸止痛的機制進行探討，對於往後臨床運用會有所助益。

Inflammatory pain is a major human affliction, but our understanding of pain mechanisms is limited. Peripheral tissue inflammation initiates hyperalgesia accompanied by tissue acidosis, nociceptor activation, and inflammation mediators releasing. Acupuncture analgesia is widely used and accepted, but the mechanism is still an outstanding question. Acid-sensing ion channel 3 (ASIC3), transient receptor potential vanilloid subtype 1 (TRPV1) and transient receptor potential vanilloid subtype 4 (TRPV4) are crucial mechanical and thermal receptors involved in inflammatory pain, but their roles in electroacupuncture- (EA) mediated analgesia are unclear.
We injected mice with carrageenan or complete Freund’s adjuvant to induce intraplantar inflammatory pain and investigated the analgesic effect of EA using animal behavior tests, immunostaining, Western blotting, real-time PCR, and a whole-cell recording technique. The inflammatory pain model mice developed both mechanical and thermal hyperalgesia.
Notably, EA at the Zusanli ST36 acupoint reversed these phenomena, indicating its curative effect in inflammatory pain. The protein levels of ASIC3, TRPV1 and TRPV4 in dorsal root ganglion (DRG) neurons were increased at day 4 after the initiation of inflammatory pain and were attenuated by EA, as demonstrated by immunostaining and Western blot analysis. We verified DRG electrophysiological properties to confirm that EA ameliorated peripheral nerve hyperexcitation. Our results indicated that the action potential threshold, rise time, and fall time, and the percentage and amplitude of TRPV1 and TRPV4 were altered by EA, indicating that EA has an antinociceptive role in inflammatory pain. Our results demonstrated a novel role for EA in regulating prostatic acid phosphatase protein expression in DRG and spinal cord then to activate the adenosine A1 receptor followed decreasing the nociceptors activity and expression. We also found that EA could mediate nerve hyperexcitation in mouse inflammatory pain models. These results indicate highly valuable data from investigating acupuncture mediated analgesia mechanisms and can be further applied to clinical medicine.

中文摘要 i
Abstract iii
Table of Contents v
List of Figures viii
List of Tables x
Abbreviations xi
Chapter 1 12
Introduction 12
1.1 Inflammatory Pain 14
1.2 Animal Models of Pain 15
2.1 Nociceptors: 17
2.2 Transient Receptor Potential (TRP) families 19
2.2.1 Transient Receptor Potential Vanilloid Subtype 1 (TRPV1) 20
2.2.2 Transient Receptor Potential Vanilloid Subtype 4 (TRPV4) 21
2.3 Acid-Sensing Ion Channel 23
2.3.1 Acid-Sensing Ion Channel 3 (ASIC3) 26
3.1 Acupuncture 27
3.2 Adenosine A1 Receptor 29
3.3 Prostatic Acid Phosphatase (PAP) 30
Chapter 2 32
Materials and Methods 32
2.1 Animals 32
2.2 EA Treatment 32
2.3 Inflammatory Pain Models. 33
2.4 Animal Behavior of Mechanical, Thermal Hyperalgesia 34
2.5 Cold/Hot Plate 35
2.6 Immunofluorescence 36
2.7 Western Blot Analysis and Tissue Sampling 37
2.8 RNA Isolation and Real-Time PCR 38
2.9 DRG Primary Cultures and Whole-Cell Patch-Clamp Recording 40
2.10 Statistic Analysis 41
Chapter 3 42
Results 42
3.1 Carra- or CFA-induced Inflammatory Hyperalgesia was Attenuated Using 2 Hz Low Frequency EA at the ST36 Acupoint 42
3.2 Low-Frequency EA at the ST36 Acupoint Decreased Carra-or CFA-Induced Inflammatory Pain by the von Frey Filament and Hargreaves’ Tests 44
3.3 EA Attenuated Carra-Elicited Inflammatory Pain by the Hot/Cold-Plate Test 46
3.4 EA Attenuated CFA-Elicited Inflammatory Pain by the Hot/Cold-Plate Test 48
3.5 ASIC3 Expression in Both Carra-and CFA-mediated Inflammation by Using Immunofluorescence Staining 49
3.6 Using Real-time PCR to Investigate the Expression of ASIC3 51
3.7 To Quantity of ASIC3 by Using Western Blot 52
3.8 TRPV1 and TRPV4 Expression in DRG Neurons from Carra- and CFA-Induced Hyperalgesia Was Decreased by EA 54
3.9 EA at ST36 Ameliorated Overexpression of TRPV1 and TRPV4 in DRG Neurons by Western Blotting 55
3.10 EA at the ST36 Acupoint Altered Electrophysiological Properties in Inflamed DRG Neurons 56
3.11 Analyzing the Acupoint, DRG and Spinal Cord PAP by Western Blot 58
Chapter 4 60
4.1 Discussion 60
4.2 Conclusion 68
References 70

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