視網酸和β-胡蘿蔔素單獨或合併使用維生素E對發炎反應的影響

The effects of retinoic acid and β-carotene alone or in combination with α-tocopherol on inflammation

鄭妃吟(Fei-Yin Jheng)

指導教授 ： 葉姝蘭

中山醫學大學/健康管理學院/營養學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00026

摘要

慢性發炎在許多疾病的發展扮演重要角色。視網酸（RA）、β-胡蘿蔔素（BC）及α-tocopherol (E) 均被報導可能抑制發炎反應，但機制及相關性並不清楚。因此我們比較BC、RA及E單獨或合併作用對發炎反應的影響。RAW264.7細胞，來自BALB/c小鼠的巨噬細胞株，以單獨或合併含有2 μM的BC、RA或10 μM E的培養基培養，並以lipopolysaccharide（LPS）活化24小時，之後分別收集細胞培養液，進一步與C3H10T1/2細胞培養（30%,v/v），以檢測發炎細胞對周邊細胞DNA的傷害情形，並測試這些培養液中發炎調節介質NO和細胞激素TNF-α、IL-1β、IL-6和IL-10含量，另外亦收集RAW264.7細胞測試ROS的生成量及細胞內BC及RA含量。結果顯示，RA＋LPS-條件培養液對C3H10T1/2細胞DNA傷害顯著較LPS-條件培養液低，而BC＋LPS則與LPS沒有差異，而進一步合併α-tocopherol，C3H10T1/2細胞DNA傷害顯著較只有RA或BC更低。RA單獨存在培養基中對LPS誘發的促發炎物質中，除顯著降低TNF-α及ROS產生外，對NO、IL-6沒有影響，並增加IL-1β的分泌; 但合併α-tocopherol後，除IL-1β，其餘促發炎物質均顯著降低，另外亦顯著增加發炎抑制激素IL-10的生成。BC單獨或合併α-tocopherol對LPS所誘發的發炎調節物質表現的影響與RA有相似的趨勢或略差，而對IL-10生成的影響未達顯著。α-tocopherol的存在有降低BC或RA損耗的趨勢。α-tocopherol共同存在下，能增加二者的效果。
為了解BC對發炎作用的影響，與其轉變為RA是否有關，我們比較BC合併使用2,6-di-tert-butyl-4-methylphenol (BHT)及BC單獨對LPS誘發的發炎反應的影響。結果顯示加入BHT後，細胞內BC含量增加，而轉變成RA的量減少。10 μM BHT+LPS條件培養液造成的C3H10T1/2細胞DNA傷害，顯著比單獨LPS條件培養液低，但合併BC後卻無此效果。另外BHT亦降低BC促進IL-10分泌的趨勢。
綜合以上，本研究結果顯示，不論是否合併α-tocopherol ，2 μM RA的抗發炎效果較同濃度BC好，α-tocopherol的存在，可能增加BC或RA的穩定性，而增強其調控發炎調節因子的能力，而BC的調節效果應至少有部分與轉變成RA有關。

關鍵字

β-胡蘿蔔素 ； 視網酸 ； α-tocopherol ； 發炎反應

並列摘要

Chronic inflammation is an important risk factor of the development of various diseases. It has been suggested that retinoic acid (RA), β-carotene (BC) and α-tocopherol (E) may suppress inflammatory reaction. However, the efficiency and the mechanisms underling such effects are unclear. Thus, in the present study, we compared the individual or combined effects of RA, BC and E on inflammation. RAW264.7 cells, BALB/c mouse macrophage-like cell line, were activated by lipopolysaccharide (LPS) for 24 h with the presence of 2 μM BC or RA or 10 μM E alone or combined in the medium. Then, 30 % (v/v) of various conditioned media were incubated with C3H10T1/2 cells to investigate the effect of inflammatory reaction on DNA damage of neighboring cells. In addition, the levels of inflammatory modulators including NO, TNF-α,IL-1β, IL-6 and IL-10 in the conditioned medium as well as the intracellular levels of reactive oxygen species (ROS), BC and RA in RAW264.7 cells were determined. The results showed that the DNA damage in C3H10T1/2 cells induced by RA+LPS-conditioned medium was lower than that induced by LPS-conditioned medium, while BC+LPS-conditioned medium had similar effect to LPS-conditioned medium. Furthermore, the DNA damages induced by RA or BC+E+LPS-conditioned medium, were lower than those induced by the RA or BC+LPS-conditioned medium, respectively. RA alone significantly decreased the secretion of TNF-α and the intracellular ROS level in RAW264.7 cells induced by LPS, but had no effect on the secretion of NO, IL-6 and IL-10. Combination of RA with α-tocopherol significantly decreased the secretion of those proinflammatory mediators induced by LPS except for IL-1β, and significantly increased the secretion of anti-inflammatory cytokine IL-10. The effects of BC alone or in combination with α-tocopherol on the secretion of inflammatory modulators induced by LPS were similar to or lower than that of RA alone or in combination with α-tocopherol, respectively. The existence of α-tocopherol in RAW264.7 cells tended to decrease the depletion of BC or RA induced by LPS.
In order to investigate whether the effect of BC on inflammation is associate with the conversion of BC into RA, we compared the effects of BC+2,6-di-tert-butyl-4-methylphenol (BHT) with that of BC alone on inflammation induced LPS. The results showed that the addition of BHT increased the level of BC while decreased the level of RA in RAW264.7 cells. The DNA damage in C3H10T1/2 induced by 10 μM BHT+LPS-conditioned medium was significantly lower than that induced by LPS-conditioned medium. However, combination of BC with BHT had no such effect. In addition, BHT tended to reduce the secretion of IL-10 induced by BC in LPS-activated RAW 264.7 cells.
In conclusion, our results suggested that the overall effects of 2 μM RA alone or in combination with E on anti-inflammation were stronger than that of 2 μM BC alone or in combination with E, respectively. Combination of RA or BC with E may increase their stability in cells and the efficiency of BC or RA on inflammatory modulation. The effects of BC, at least partly, are association with the conversion of BC into RA.

並列關鍵字

β-carotene ； retinoic acid ； α-tocopherol ； inflammation

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