山苦瓜與苦瓜萃出物對脂多醣體RAW264.7巨噬細胞發炎反應之探討

Effects of the wild bitter gourd and white bitter gourd extracts on the inflammatory responses of LPS-induced murine Raw 264.7 macrophages

雲文姿(Wen-Tzu Yun)

指導教授 ： 李宗貴

中山醫學大學/健康管理學院/營養學研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00002

摘要

臨床研究顯示，慢性發炎與心臟病、中風或粥狀動脈硬化症等心血管疾病的發生有密切關係，顯示減少慢性發炎反應，將有助於預防心血管疾病的發生。本實驗預計比較山苦瓜與苦瓜不同溶劑萃取物在脂多醣體(lipopolysaccharide, LPS)活化巨噬細胞 RAW264.7狀況下，對NO、PGE2、IL-1β等促發炎因子生成之效應，並探討可能機轉。本實驗採用連續萃取方式製備各溶劑萃出物，細胞毒性(MTT)分析結果指出，山苦瓜熱水與冷水萃出物的IC50分別為5.01 mg/ml與0.51 mg/ml，而苦瓜熱水與冷水萃出物的IC50則為12.84 mg/ml與2.04 mg/ml；山苦瓜熱水萃出物(0.5-2mg/ml)及95%乙醇萃出物(25μg/ml-50μg/ml)皆可有效抑制LPS誘發RAW264.7巨噬細胞之NO生成(p<0.05)，且以乙醇萃出物抑制效果最強，在50μg/ml處理下，對NO生合成有36%的抑制能力；除NO外，50 μg/ml山苦瓜乙醇萃出物也可抑制37% PGE2分泌量。比較山苦瓜萃出物與苦瓜萃出物抑制NO與PGE2生合成效應，也以山苦瓜優於苦瓜。進一步分析iNOS、COX2與pro-IL-1β蛋白質表現，結果顯示：山苦瓜乙醇萃出物可有效抑制胞內iNOS與pro-IL-1β蛋白質含量，然而COX2蛋白表現的抑制現象則僅出現於乙醇萃出物處理組；EMSA也證實山苦瓜乙醇及熱水萃出物均可抑制NFκB活化。總酚類化合物含量分析結果，也是山苦瓜的酚類含量高於苦瓜，而山苦瓜中又以乙醇萃出物的酚類含量最高(31.82±4.91mg/g)，其次是乙酸乙酯萃出物(17.55±3.58mg/g)、熱水萃萃出物(11.15±1.16mg/g)，最低為冷水萃出物(8.53±0.15mg/g)。綜合上述結果，山苦瓜乙醇萃出物可有效抑制LPS誘發iNOS、pro-IL-1β蛋白質表現及NO與PGE2生合成，其可能機轉與其抑制NF-κB的活化有關。

關鍵字

山苦瓜 ； 苦瓜 ； RAW264.7 ； iNOS ； PGE2 ； IL-1β ； NF-κB

並列摘要

Recent clinic research shows the etiology of cardiovascular diseases is associated with the chronic inflammation. This evidence implicates that decrease of chronic inflammation may help to maintain normal cardiovascular function. This study was designed to investigate the biological activity of wild bitter gourd and white bitter gourd extracts on NO, PGE2, and IL-1β production in LPS-stimulated RAW 264.7 macrophages. Result indicates the hot water (0.5-2mg/ml) and 95% ethanol (25-50μg/ml) extracts of the wild bitter gourd are effectively inhibit NO production (p<0.05). At 50μg/ml, a 36% decrease of NO production was noted on ethanol extract-treated cells as compared to the cells treated with LPS alone. In addition, a 37% suppression of PGE2 secretion was also noted by 50μg/ml ethanol extract. As compared to the effect of the wild bitter gourd, extracts from the white bitter gourd showed a much less inhibition on NO and PGE2. Western blot analysis revealed that iNOS and pro-IL-1β levels were dose-dependently decreased in the hot water- and ethanol-extracts of the wild bitter gourd. However, the suppression of COX2 expression was noted only in cells treated with the ethanol extract. Electrophoresis mobility shift assay further showed both wild bitter gourd hot water- and ethanol extracts inhibit NF-κB activation. The phenolic compound contents of each wild bitter gourd extract was in an order of ethanol (31.82±4.91mg/g)>ethyl acetate (17.55±3.58mg/g)>hot water (11.15±1.16mg/g)>cold water (8.53±0.15mg/g). These results suggest the ethanol-extract of wild bitter gourd is effectiveness on the inhibition of LPS-induced iNOS and pro-IL-1β protein expression and NO and PGE2 production. The possible mechanism is probably related to its inhibition on the
NF-κB activation.

並列關鍵字

Bitter gourd ； Raw 264.7 macrophages ； iNOS ； PGE2 ； IL-1β ； NF-κB

參考文獻

Abu el Asrar, A. M., Maimone, D., Morse, P. H., Gregory, S., and Reder, A. T. (1992). Cytokines in the vitreous of patients with proliferative diabetic retinopathy. Am J Ophthalmol 114, 731-736.

Ahmed, I., Adeghate, E.,Sharma, A. K., Pallot, D. J., Singh, J. (1998).Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-direct rat. Diabetes Res Clin Pract 40,145-151.

Ahmed, I., L., Lakhani, M. S., Gillett, M., John, A., Raza, H. ( 2001). Hypotriglyceridemic and hypocholesterolemic effects of anti-diabetic Momordica charantia (karela) fruit extract in streptozotocin-induced diabetic rats. Diabetes Res Clin Pract 51, 155-161.

Alexander, C., and Rietschel, E. T. (2001). Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 7, 167-202.

Aley, K. O., McCarter, G., and Levine, J. D. (1998). Nitric oxide signaling in pain and nociceptor sensitization in the rat. J Neurosci 18, 7008-7014.