以急性發炎小鼠模式探討山苦瓜及其乙酸乙酯萃物對血清及肝臟發炎指標的影響

Effect of Momordica charantia L. and it’s ethyl acetate extract on serum and liver inflammatory markers in LPS induced acute inflammation model

蔡侑倫(You-Lun Tsai)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/生化科技學系/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00535

摘要

許多研究以巨噬細胞體外培養或LPS致急性發炎小鼠模式，篩選具影響發炎反應之樣品。研究指出山苦瓜具抗發炎之潛力，本研究主要探討山苦瓜（Momordica charantia Linn. var. abbreviata ser.）對發炎反應的影響。
以LPS刺激之RAW 264.7細胞株分析花蓮三號(H3-)與花蓮四號(H4-)山苦瓜萃物對TNF-alpha與IL-6分泌的影響，結果顯示，三號與四號之酒精萃物(-EtOH)、乙酸乙酯萃物(-EA)與正己烷萃物(-Hex)，皆顯著抑制TNF-alpha及IL-6分泌並呈現劑量效應，但花蓮四號水萃物(-W)卻顯著促進IL-6分泌。以初代腹腔細胞分析山苦瓜萃物對TNF-alpha、IL-1beta及IL-6分泌的影響，H3-W與H4-W皆顯著促進自發性分泌TNF-alpha、IL-1beta及IL-6，但H3-EA與H4-EA對細胞激素分泌皆無顯著影響。LPS刺激下，H3-W亦顯著促進腹腔細胞分泌TNF-alpha及IL-1beta，於25 ug/mL時有促進IL-6分泌的趨勢，但於75 ug/mL時顯著抑制IL-6分泌，H4-W亦顯著促進IL-1beta分泌並有促進TNF-alpha分泌的趨勢。H3-EA顯著促進TNF-alpha分泌，但顯著抑制IL-1beta及IL-6分泌，H4-EA亦顯著抑制IL-1beta及IL-6分泌。
LPS致急性發炎小鼠模式中，餵食山苦瓜樣品一週後以腹腔注射15 mg LPS/kwBW誘發BALB/c小鼠急性發炎，於LPS刺激後不同時間點採血，分析血清細胞激素含量與存活時數之相關性，並分析血清與肝臟發炎指標。結果顯示小鼠腹腔注射LPS 後9小時，血清IL-6含量與存活時數達顯著負相關，且血清IL-6含量能預測小鼠存活情況，顯示小鼠腹腔注射LPS 後9小時血清IL-6含量為發炎嚴重程度的良好指標。餵食H3-EA 30 mg/KgBW/day不影響小鼠存活時數，及腹腔注射LPS 後9小時血清IL-6含量； H4-EA 18.8 mg/KgBW/day雖不影響小鼠存活時數，卻顯著增加9小時血清IL-6含量。餵食含5%三號或四號全果之飼料(-BGP)，皆顯著加速小鼠死亡，並顯著增加9小時血清IL-6含量。分析小鼠腹腔注射LPS後9小時小鼠肝臟mRNA表現，H4-EA顯著增加NOS2表現，並有增加IL-1beta、IL-6與IL-10表現的趨勢，H4-BGP顯著增加肝臟TNF-alpha、IL-1beta、IL-6、IL-10、IFN-gamma 與NOS2表現。
最後分析山苦瓜對正常小鼠血清與肝臟發炎指標的影響。H4-BGP顯著增加血清CXCL10含量，並有增加IL-6含量的趨勢，顯著促進腹腔細胞分泌IL-6，顯著增加肝臟TNF-alpha、IL-1beta、IL-6、IL-10、IFN-gamma、IL-4、IL-12b、NOS2與CXCL10 mRNA表現，和有增加Chi3l3表現的趨勢，但H4-EA萃物不影響血清細胞激素含量、腹腔細胞分泌細胞激素以及肝臟各mRNA表現。
以上結果顯示，H4-BGP促進正常小鼠肝臟表現促炎性細胞激素mRNA，和誘發巨噬細胞分泌IL-6，亦加劇LPS致急性發炎小鼠之發炎反應，而H4-EA雖不影響正常小鼠，但加劇急性發炎小鼠發炎反應，因此若欲開發山苦瓜為保健食品，必須審慎評估山苦瓜對發炎反應的影響。

關鍵字

山苦瓜 ； 細胞激素 ； 巨噬細胞 ； 急性發炎 ； 肝臟

並列摘要

Effect of sample on cytokines secretion of macrophage in vitro and LPS-induced acute inflammatory mice model are often used to screen sample with inflammatory modulation effect. Many researchs has revealed that wild bitter gourd (Momordica charantia Linn. var. abbreviata ser) has anti-inflammatory potential. The main purpose of this study is to analyze the effect of wild bitter gourd (Hualian NO.4 and NO.4, H3- and H4- respectively) on inflammation.
Analyzing the effect of H3- and H4- bitter gourd extracts on LPS-stimulated Raw 264.7 cells, etanol extract(-EtOH), ethyl acetate extract(-EA) and hexane extract(-Hex) significantly inhibit TNF-alpha and IL-6 secretion in a dose-dependent manner in both strain, but water extract(-W) significantly promote IL-6 secretion in both strain.
Analyzing the effect of extracts on secretion by peritoneal exudate cells(PEC) of mice, H3-W and H4-W extracts significantly promote spontaneous secretion of TNF-alpha, IL-6 and IL-1beta, but EA extracts have no effect on secretion of cytokine. For LPS-stimulated PEC, H3-W significantly promotes TNF-alpha and IL-1beta, has a promotive trend and significantly inhibits IL-6 secretion at 25ug/mL and 75ug/mL respectively, H4-W significantly promotes IL-1beta secretion and has a promotive trend in TNF-alpha secretion, H3-EA significantly promote TNF-alpha secretion but significantly inhibit IL-6 and IL-1beta secretion, H4-EA significantly inhibit TNF-alpha and IL-6 secretion.
In LPS-induced acute inflammatory mice model, feeding mice with wild bitter gourd samples for a week and provokes acute inflammation by 15mg LPS injection intraperitonealy. The results shows that serum level of IL-6 and survival time are highly correlated 9hr post LPS challenge, and is able to predict survival status, revealing that Serum level of IL-6 9hr post LPS challenge is a ideal marker for severity of inflammation. Feeding 30mg H3-EA/kgBW/day does no affect survival time and serum IL-6 level 9hr post LPS challenge, feeding 18.8mg H4-EA /kgBW/day does no affect survival time but signigicantly elevating serum IL-6 level 9hr post LPS challenge, feeding diet with 5% H3 or H4 wild bitter gourd powder(BGP) significantly accelerates death and elevating serum IL-6 level 9hr post LPS challenge. Analyzing liver mRNA expression 9hr post LPS challenge, H4-EA significantly promote NOS2 expression, has a higher IL-6, IL-1beta and IL-10 expression trend, H4-BGP significantly promotes TNF-alpha, IL-6, IL-1beta ,IL-10, IFN-gamma and NOS2 expression.
Analyzing wild bitter gourd samples on serum and liver inflammatory markers of normal mice, H4-BGP significantly increases CXCL10 level and has a higher serum IL-6 trend in serum, significantly promotes IL-6 secretion by PEC, signigicantly increases TNF-alpha, IL-6, IL-1b, IL-10, IFN-gamma, IL-4, IL-12b, NOS2 and CXCL10 mRNA expression in liver, and has a higher chi3l3 mRNA expression trend in liver, but H4-EA dose not affect cytokine level of serum, productivity of PEC and mRNA expression pattern in liver.
The results shows that, H4-EA does not affect liver and serum inflammatory markers of normal mice, but exaggerate inflammatory response in acute model. H4-BGP promotes normal mice expresses inflammatory cytokines mRNA in liver, inducing IL-6 secretion by macrophage, and exaggerates inflammatory response in acute model. Therefore, in development of functional food, it must cautiously evaluate the effect of wild bitter gourd on inflammatory response.

並列關鍵字

wild bitter gourd ； cytokine ； acute inflammation ； macrophage ； liver

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