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研究生:謝治宇
研究生(外文):Chih-Yu Hsieh
論文名稱:探討台灣綠蜂膠及聚乙烯吡咯衍生物 4-HAB 對尿酸結晶引起 NLRP3 發炎體活化之影響
論文名稱(外文):Effect of Taiwanese Green Propolis and Polyenylpyrrole Derivative 4-HAB on MSU-induced NLRP3 Inflammasome Activation
指導教授:花國鋒花國鋒引用關係
指導教授(外文):HUA, KUO-FENG
口試委員:吳世雄朱俐潔羅禮強陳裕文
口試委員(外文):WU, SHIH-HSIUNGCHU, LICHIEH JULIELO, LEE-CHIANGCHEN, YUE-WEN
口試日期:2018-07-30
學位類別:博士
校院名稱:國立宜蘭大學
系所名稱:生物技術與動物科學系動物科學碩士班
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:2018
畢業學年度:107
語文別:英文
論文頁數:116
中文關鍵詞:NLRP3 發炎體痛風台灣綠蜂膠聚乙烯吡咯
外文關鍵詞:NLRP3 inflammasomeGoutTaiwanese Green PropolisPolyenylpyrrole
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發炎體是由多數種蛋白組成,其包括 NOD 受體 (NOD-like receptors, NLR)、凋亡相關斑點樣蛋白 (Apoptosis-associated speck-like protein containing a CARD, ASC) 和胱天蛋白酶-1 (Caspase-1),發炎體活化後會將介白素 1β 前驅物 (proIL-1β) 和 IL-18 前驅物 (proIL-1β) 切割為具有活性的 IL-1β 和 IL-18。環境中許多物質都會使發炎體活化,如病原體分子、代謝物、環境刺激物 (石棉、二氧化矽或納米顆粒) 和自體產生的危險因子 (膽固醇結晶、ATP 或 DNA) 。發炎體活化所釋放的 IL-1β 在對抗病原的宿主防禦機制中佔有重要地位,但在許多慢性發炎疾病中卻發現 IL-1β 的過度表現。在研究中發現發炎體的調控與許多疾病已被證實具有高度的相關性,其中 NLRP3 則最廣為人知。但現今針對 NLRP3 的藥物仍較少,因此本研究探討台灣綠蜂膠 (Taiwanese Green Propolis, TGP) 及聚乙烯吡咯(polyenylpyrrole) 衍生物 4-hydroxy auxarconjugatin B (4-HAB) 與 NLRP3 發炎體間的抗發炎機轉並評估其開發之潛力。首先, TGP 的研究中,發現其能藉由降低 NF-κB 的活化及減少活性氧自由基 (Reactive oxygen species, ROS) 的生成,達到降低 proIL-1β 的表現。並且藉由保護粒線體膜的完整性、降低 ROS 的產生、減少 Cathpsin B 自溶酶體破裂流出以及降低 JNK1/2 的磷酸化及 ASC 的寡聚合,達到抑制 NLRP3 發炎體活化的效果。更進一步發現其抑制發炎體的效果會透過提高細胞自噬作用來達成。在 MSU 誘導腹膜炎動物模式中,觀察到 TGP 能減少細胞浸潤至腹腔中,並降低腹腔沖洗液中 IL-1β 、 Caspase-1 、 IL-6 和 MCP-1 的表現。在 TGP 的純化物 Propolin G 初步的結果也具有抑制 MSU 或 ATP 所誘導的 IL-1β 釋放。第二部分,進行 4-HAB 的探討,前人的研究中已證實其會抑制 LPS/ATP 所誘導的 NLRP3 發炎體活化,在本研究中針對 MSU 所誘導的發炎反應進行更深入的研究。結果中觀察到 4-HAB 具有劑量依賴性的降低 MSU 誘導的 IL-1β 、 IL-18 、 Caspase-1 與 ASC 的釋放。進一步發現其會透過降低 ROS 累積、減少 Cathpsin B 自溶酶體破裂流出及 ASC 的寡聚合達到降低 NLRP3 發炎體活化。此外,4-HAB 會經由 Sirt-1 路徑提高細胞自噬作用,並藉由此現象達到抑制發炎體的活化。在 MSU 腹膜炎動物模式中,4-HAB 能減少細胞浸潤至腹腔中,並降低腹腔沖洗液中 IL-1β 、 Caspase-1 、 IL-6 和 MCP-1 的表現。以上結果顯示, TGP 與 4-HAB 皆在抑制 NLRP3 發炎體的活化具有一定的功效,未來或許能發展為預防 NLRP3 相關發炎的標靶藥物或開發成為保健食品。
Dysregulation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1β precursor (proIL-1β), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported two materials which had the anti-inflammasome effects in vitro and in vivo, the alcoholic extract of Taiwanese Green Propolis and polyenylpyrrole derivative, Compound 1H (4-hydroxy auxarconjugatin B, 4-HAB). First, TGP inhibited proIL-1β expression by reducing nuclear factor kappa B (NF-κB) activation and reactive oxy gen species (ROS) production in LPS-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-jun N-terminal kinase 1/2 (JNK1/2) phosphorylation and apoptosis-associated speck-like protein (ASC) oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo, the monosodium urate crystals (MSU)-induced peritonitis model, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1β, active caspase-1, IL-6 and monocyte chemoattractant protein-1 (MCP-1) in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Second, 4-HAB has dose-dependently reduced the MSU-induced release of IL-1β, IL-18, active caspase-1 and ASC secretion in macrophages. In a mechanic study, 4-HAB inhibited the accumulation of ROS, lysosomal cathepsin B release, ASC oligomerization and speck formation in MSU-activated macrophages. Furthermore, 4-HAB increased Sirt1 expression and induced autophagy in macrophages. Importantly, inhibition or knockdown of Sirt1 and autophagic protein microtubule-associated protein light chain 3 (LC3) reversed the inhibitory effect of 4-HAB on the activation of NLRP3 inflammasome. Our results also showed that Sirt1 was the upstream of autophagy in 4-HAB-treated macrophages. On the other hand, the anti-inflammasome properties of 4-HAB further confirmed in a mice model of MSU-induced peritonitis by the reduced IL-1β, active caspase-1, IL-6 and MCP-1 secretion in peritoneal lavage fluids. In conclusion, Propolis or 4-HAB attenuated gouty inflammation, in part, by attenuated activation of NLRP3 inflammasome. In particularly 4-HAB also through enhanced Sirt1/autophagy induction pathway, but not propolis. Overall, TGP and 4-HAB both showed the potential for anti-MSU-induced inflammation which could be a novelty drug of the NLRP3 inflammasome.
Catalog
全文摘要 I
Abstract II
Acknowledgements III
Catalog IV
List of Figures VII
Abbreviations IX
Chapter I Introduction 1
Section 1 Taiwanese Green Propolis 2
Section 2 Polyenylpyrrole Derivative 4-HAB 3
Section 3 Inflammation and Inflammasome 5
Section 4 NLRP3 Inflammasome Signaling Transduction 6
Item 1: The channel model- potassium efflux. 6
Item 2: ROS model. 7
Item 3: Lysosome rupture model. 7
Section 5 Regulation of the NLRP3 Inflammasome. 9
Item 1: Negative regulators. 9
Item 2: Autophagy. 9
Item 3: Other mechanisms. 9
Section 6 Autophagy 11
Item 1: Signaling transduction 11
Item 2: SIRT1 12
Section 7 Aim 12
Chapter II Materials and Methods 13
Section 1 Materials 14
Item 1: Cell culture 14
Item 2: TGP, Propolin G and 4-HAB 14
Item 4: Antibodies 19
Item 5: Kits 21
Item 6: Instrument 22
Item 7: Experimental consumables 23
Section 2 Methods 24
Chapter III Results 29
Section 1 Taiwanese Green Propolis study 30
Item 1: TGP reduced MSU-induced NLRP3 inflammasome activation and cytokine secretion 30
Item 2: TGP reduced inflammasome-mediated IL-1β secretion 30
Item 3: TGP reduced MSU-induced lysosomal rupture and JNK1/2 phosphorylation 31
Item 4: TGP reduced MSU-induced mitochondrial damage and ASC oligomerization 31
Item 5: TGP suppressed the LPS-mediated priming signal of NLRP3 32
Item 6: TGP attenuated the NLRP3 inflammasome via increased autophagy 32
Item 7: TGP reduced inflammation in a mouse model of MSU-induced peritonitis 33
Section 2 4-HAB study 34
Item 1: 4-HAB reduced MSU-induced NLRP3 inflammasome activation in LPS-primed macrophages 34
Item 2: 4-HAB prevented mitochondrial damage and increased its biogenesis 34
Item 3: 4-HAB reduced lysosome rupture and ASC oligomerization 35
Item 4: 4-HAB attenuated enhanced NLRP3 inflammasome activity through autophagy induction 36
Item 5: Sirt1 was upstream of autophagy in 4-HAB attenuated enhanced NLRP3 activity 36
Item 6: 4-HAB inhibited NLRP3 inflammasome activation in a mouse model of gouty inflammation 37
Chapter IV Discussion 38
Section 1 Taiwanese Green Propolis 39
Section 2 4-HAB 41
Chapter V Conclusion 45
Chapter VI Figures & Figure Legends 48
Section 1 Taiwanese Green Propolis 49
Section 2 4-HAB 75
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