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研究生:胡恬
研究生(外文):Tian Hu
論文名稱:探討生薑中成分6-shogaol在脂多醣誘導發炎反應的抗發炎作用
論文名稱(外文):Study on anti-inflammatory effect of Ginger constituent 6-shogaol in lipopolysaccharide-induced inflammation
指導教授:曾志正曾志正引用關係
指導教授(外文):Jason T.C. Tzen.
口試委員:陳文英錢嘉琳
口試日期:2023-07-24
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:中文
論文頁數:37
中文關鍵詞:生薑6-薑酚發炎反應EP4受體前列腺素E2
外文關鍵詞:Ginger6-shogaolEP4InflammationProstaglandin E2
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6-薑酚 (6-shogaol)是生薑 (Zingiber officinale)中特有的活性成分,在許多研究中也被證實具有抗氧化、抗發炎、抗癌等作用。當發炎反應發生時病灶部位會產生大量的前列腺素 (Prostaglandin, PG),其中前列腺素E2 (Prostaglandin E2, PGE2)是人體內表現量最多的PG並對於發炎反應的調控十分重要。研究指出,前列腺素EP4受體 (Prostaglandin EP4 receptor)可能是參與人類發炎相關疾病的關鍵,在細胞平台的文獻中顯示PGE2可通過活化EP4降低LPS誘導的細胞因子TNF- , IL-1及NO的產生達到抑制發炎反應的效果。6-shogaol已被證實能降低發炎反應,因其結構與PGE2相似,故在本研究中建立以LPS誘導小鼠巨噬細胞RAW 264.7的發炎平台以探討生薑中的6-shogaol是否也能透過PGE2的作用路徑去影響發炎反應。首先以分子對接模擬軟體釐清其是否與PGE2相同能與EP4受體活性中心產生鍵結,在模擬結果中顯示兩者具有相同的活性鍵結位置,推測6-shogaol可能會與EP4受體產生交互作用。接著以Western blot進一步探討6-shogaol於EP4受體路徑調控LPS誘導小鼠巨噬細胞發炎的抑制效果,結果顯示,給予1 M EP4 antagonist並不影響6-shogaol調控LPS 誘導之發炎相關蛋白TNF-的表現,因此推測在平台中6-shogaol對於調控炎相關蛋白表達可能不受EP4受體路徑所影響。COX蛋白在許多文獻中被證明是發炎反應重要的一部分,其活性也是決定前列腺素的產量主因,因此在研究中也進而去探討6-shogaol的抗發炎作用是否透過上游COX途徑所影響。在LPS誘導的發炎平台中可以看到,6-shogaol能顯著抑制COX-2而非COX-1蛋白的表現量,由此往COX-2途徑做進一步的討論,根據結果,給予20 μM COX-2 inhibitor並不能完全抑制6-shogaol對於LPS 誘導之iNOS及COX-2的蛋白的調控,推測在平台中6-shogaol對於調控發炎相關蛋白表達可能還會受COX-2外其他路徑所影響。在本研究中以分子模擬和拮抗EP4受體及COX-2蛋白路徑方式初步評估6-shogaol在LPS誘導之發炎反應中的抗發炎作用,在Western blot結果顯示6-shogaol的抗發炎作用可能部分透過COX-2蛋白而不受EP4受體路徑所影響,因此推測在平台中6-shogaol可能還能夠透過其他路徑達到抗發炎的效果。希望未來能對於6-shogaol抗發炎路徑有更深入的研究,進而探討生薑作為天然抗發炎藥物開發之淺力。
The active ingredient 6-shogaol found in ginger (Zingibe) has been confirmed in various studies to possess antioxidant, anti-inflammatory, and anti-cancer effects. Prostaglandin E2 (PGE2) is the most abundant PG in the human body and is crucial in regulating inflammatory responses. Research suggests that the prostaglandin EP4 receptor may play a key role in inflammatory-related diseases in humans. In vitro studies, it has been shown that PGE2 can inhibit the production of inflammatory cytokines TNF-α, IL-1β, and NO induced by LPS by activating EP4. 6-shogaol has been proven to reduce inflammation, and due to its structural similarity to PGE2, this study aims to establish an inflammatory platform using LPS-induced mouse macrophage RAW 264.7 cells to investigate whether 6-shogaol in ginger can also affect inflammatory responses through the PGE2 pathway. Molecular docking simulation software was initially used to clarify whether 6-shogaol can bind to the active center of the EP4 receptor, similar to PGE2. The molecular docking simulation results showed that both have the same active binding position, suggesting that 6-shogaol may interact with the EP4 receptor. Western blot analysis further explored the inhibitory effects of 6-shogaol on LPS-induced inflammation in mouse macrophages through the EP4 receptor pathway. The results showed that EP4 antagonist did not affect the expression of the inflammatory protein TNF-α regulated by 6-shogaol. Therefore, it is speculated that 6-shogaol's regulation of inflammatory protein expression in the platform may not be influenced by the EP4 receptor pathway. COX proteins have been proven to be crucial in the inflammatory response, and their activity is a major determinant of prostaglandin production. Therefore, the study also investigated whether the anti-inflammatory effects of 6-shogaol are influenced by the upstream COX pathway. In the LPS-induced inflammatory platform, 6-shogaol significantly inhibited the expression of COX-2 but not COX-1 protein. Further study on the COX-2 pathway revealed that COX-2 inhibitor did not completely inhibit the regulation of iNOS and COX-2 proteins by 6-shogaol, suggesting that 6-shogaol's regulation of inflammatory protein expression in the platform may also be influenced by pathways other than COX-2. In this study, molecular simulation and antagonism of the EP4 receptor and COX-2 protein pathways were used to assess the anti-inflammatory effects of 6-shogaol in LPS-induced inflammatory responses. The Western blot results indicated that the anti-inflammatory effects of 6-shogaol may partially occur through the COX-2 protein and are not influenced by the EP4 receptor pathway. Therefore, it is speculated that in the platform, 6-shogaol may achieve its anti-inflammatory effects through other pathways.
中文摘要 i
Abstract ii
目次 iv
表目錄 v
圖目錄 vi
附圖目錄 vii
壹、文獻回顧 1
一、生薑 1
二、發炎反應 (Inflammatory Response) 3
三、前列腺素 (Prostaglandin) 4
四、抗發炎藥物的治療及發展 7
貳、實驗材料與方法 8
一、實驗材料 8
二、儀器與設備 11
三、實驗方法 12
參、結果 15
ㄧ、評估 6-shogaol 在 LPS 誘導小鼠巨噬細胞發炎反應中的抑制效果 15
二、6-shogaol與EP4受體分子對接模擬運算 15
三、評估6-shogaol是否通過EP4受體調控LPS誘導小鼠巨噬細胞發炎相關蛋白的表達 16
四、探討6-shogaol是否會透過COX-1及COX-2路徑達到抗發炎的效果 16
肆、討論 18
伍、圖表 21
陸、附件圖表 29
柒、參考文獻 34
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