鱸鰻魚油抑制RAW264.7巨噬細胞株分泌促發炎介質之研究

Study on Anguilla marmorata Oil Inhibiting the Secretion of Pro-inflammatory Mediators by RAW264.7 Macrophages

黃大恆(Ta-Heng Huang)

指導教授 ： 林翰佑

共同指導教授 ： 韓玉山(Yu-San Han)

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202004170

摘要

自古以來，鰻魚被認為是最有營養的魚類之一，許多古籍記載鰻魚的抗炎症及過敏之效用。本研究旨在探討鱸鰻魚油的抗發炎活性。首先，使用含不同omega 3/6脂肪酸成分的膳食油脂樣品，在細胞培養模式下研究促發炎介質（包括NO、IL-6、TNF-α和PGE2）的抑制作用。將RAW264.7小鼠巨噬細胞株與不同劑量的樣品: 鱸鰻魚油（AM）、深海魚油（DO）、大豆油（SO）以及純EPA培育24小時，再以LPS/IFNγ活化24小時。樣品對於細胞之毒性測試則通過MTT測定法測量。實驗結束後，收集上清液，並且通過酵素結合免疫吸附分析法（ELISA）或Griess試驗測量NO、IL-6、TNF-α和PGE2的產生。結果顯示，在200 μg/mL濃度下不同樣品的細胞毒性實驗中，AM擁有最高的細胞存活率。在100〜200 μg/mL的濃度下，AM顯著抑制IL-6和PGE2兩種發炎介質。純EPA和DO在同樣濃度下則能顯著抑制所有促發炎介質的產生，並呈現劑量反應。此外，為了探討AM更進一步的抑制促發炎介質潛力，在更進一步的實驗中，測定了濃度在100〜500 μg/mL下的AM、相同AM濃度之EPA/DHA含量（ED）及兩種AM fraction（F2和F4）的抑制發炎介質能力比較。結果顯示，小鼠巨噬細胞RAW 264.7於LPS/IFNγ活化下，ED和兩種AM萃取物在100~500 μg/mL之細胞存活率皆大於80%，顯示無細胞毒性。在100 μg/mL的濃度下，AM有相較ED顯著的抑制NO的能力。而在兩種AM fraction（F2和F4）的測試實驗中，F4以劑量反應相關的方式顯著降低了所有促發炎介質NO、IL-6、TNF-α和PGE2，顯示其相較AM和F2更佳的抑制發炎介質能力。綜合上述結果，AM在濃度100〜500 μg/mL下對RAW 264.7巨噬細胞有低的細胞毒性，並且在500 μg/mL濃度下有最佳的抑制NO，IL-6和PGE2產生的能力。F4作為鱸鰻魚油的高極性萃取物，擁有更高的抑制發炎介質能力，值得進一步研究。整體來說，鱸鰻魚油在500 μg/mL下的抗促炎症因子分泌能力最佳，值得作為之後動物實驗之參考。膳食油樣品中不飽和脂肪酸的含量和組成可能是導致抑制發炎介質能力的差異。鱸鰻魚油作為日常食用魚油補充劑，可減少特定促發炎因子NO，IL-6和PGE2的產生，顯示出預防和調節發炎反應的潛力。

關鍵字

抗發炎反應 ； 促發炎介質 ； NO ； IL-6 ； TNF-α ； PGE2 ； RAW264.7 細胞株 ； 鱸鰻魚油

並列摘要

Since ancient times, anguillid eels, such as Anguilla japonica and Anguilla marmorata, have been considered among the most nutritious fish species. Some Chinese medical books suggest that consuming the eel could help combat inflammatory conditions and allergies, making it a good medicine. This study aims to examine the anti-inflammatory activity of A. marmorata oil (AM). First, we compared the extent of inflammatory mediator inhibition offered by AM with that offered by some other dietary oils: deep sea fish oil (DO), soybean oil (SO), and pure eicosapentaenoic acid (EPA). At different concentrations, these were used to pretreat RAW264.7 macrophage cells for 24 h, which were then treated with LPS/IFNγ for another 24 h. The supernatants were then harvested, and the concentrations of TNF-α, IL-6, prostaglandin E2 (PGE2), and NO were measured by enzyme-linked immunosorbent assay (ELISA) and Griess test. Cell viability was measured using the MTT assay. The results showed that among different samples, AM at 200 μg/mL had the highest cell viability in cytotoxicity experiments. At 100–200 μg/mL, AM significantly inhibited two inflammatory mediators, IL-6 and PGE2. Pure EPA and DO could significantly inhibit the production of all pro-inflammatory mediators at the same concentration, and show in the dose response way. To explore the potential of AM in inhibiting pro-inflammatory mediators, EPA/DHA(ED) with the same concentration as EPA/DHA concentration in the AM oil and AM oil fractions (F2 and F4) were tested. The results showed that the cell survival rates with both ED and two AM fractions at 100–500 μg/mL were greater than 80%, with no cytotoxicity. At a concentration of 100 μg/mL, AM could inhibit NO significantly better than ED. In the test experiment with the two AM fractions F2 and F4, F4 significantly reduced all pro-inflammatory mediators, namely NO, IL-6, TNF-α, and PGE2, in a dose-response-related manner, showing that it was more effective than AM and F2. As a highly polar extract fraction of AM, F4 has a higher ability to inhibit inflammatory mediators and is worthy of further study. Taken together, AM at 500 μg/mL had the best inflammatory inhibition ability. The differences in anti-inflammatory performances were possibly influenced by the capacity and composition of PUFAs among dietary oil supplements. The AM oil may contain potent immunomodulatory substances that modulate the inflammatory responses. This oil, as a fish oil supplement for daily consumption, reduces the production of specific inflammatory mediators like NO, IL-6, and PGE2 and shows potential in preventing and down-regulating the inflammatory response.

並列關鍵字

Anti-inflammatory response ； Pro-inflammatory mediators ； NO ； IL-6 ； TNF-α ； PGE2 ； RAW264.7 cells ； Anguilla marmorata oil

參考文獻

Ahn, J. C., Chong, W.-S., Na, J. H., Yun, H. B., Shin, K. J., Lee, K. W. A. (2015). An Evaluation of Major Nutrients of Four Farmed Freshwater Eel Species (Anguilla japonica, A. rostrata, A. bicolor pacifica and A. marmorata). 44-50.

Arai, T., Chino, N. (2018). Opportunistic migration and habitat use of the giant mottled eel Anguilla marmorata (Teleostei: Elopomorpha). Scientific Reports, 8(1), 5666.

Axelrod, L., Camuso, J., Williams, E., Kleinman, K., Briones, E., Schoenfeld, D. (1994). Effects of a small quantity of omega-3 fatty acids on cardiovascular risk factors in NIDDM. A randomized, prospective, double-blind, controlled study. Diabetes Care, 17(1), 37-44. doi:10.2337/diacare.17.1.37

Calder, P. C. (2013). Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology, 75(3), 645-662. doi:10.1111/j.1365-2125.2012.04374.

Heinsbroek, L. (2008). A review of eel culture in Japan and Europe. Aquaculture Research, 22, 57-72. doi:10.1111/j.1365-2109.