台灣土肉桂葉精油與活性成分枷羅木醇及肉桂醛對於LPS誘發系統性發炎反應小鼠之影響

The effect of the leaf essential oil of Cinnamomum osmophloeum Kanehira and its active ingredients linalool and cinnamaldehyde on LPS-induced systemic inflammatory response in mice

王詩芸(Shih-Yun Wang)

指導教授 ： 劉承慈

中山醫學大學/健康管理學院/營養學研究所/碩士(2015年)

https://doi.org/10.6834/CSMU.2015.00135

摘要

已知台灣土肉桂葉精油（TC）對於LPS誘發之C57BL/6小鼠具有改善腸道絨毛損傷及減緩系統性發炎反應之效果。本研究目的為探討灌食TC中所含的活性成分枷羅木醇（linalool, Lin）和肉桂醛（cinnamaldehyde, Cin）改善LPS誘發腸道黏膜損傷及促進障壁功能之作用及相關機制。實驗以雄性C57BL/6小鼠隨機分為六組，分別灌食：載劑玉米油（4 ml/kg BW）、土肉桂葉精油（13 mg/kg BW）、低劑量肉桂醛（0.4 mg/kg BW）、高劑量肉桂醛（0.9 mg/kg BW）、低劑量枷羅木醇（2.6 mg/kg BW）和高劑量枷羅木醇（5.2 mg/kg BW），每隔一日灌食一次（共八次），於最後一次灌食後隔日將灌食TC、Cin和Lin組別之小鼠注射LPS（10 mg/ml/kg BW），而灌食載劑組分為兩組，分別經由腹腔注射無菌生理食鹽水或LPS，並於禁食12小時後犧牲收集血液及組織/臟器。將製備好之血液及臟器/組織用以分析nitrate/nitrite（即NO）或促發炎激素之含量、NF-κB活化、caspase-1及xanthine oxidase酵素活性，並以western blotting分析各臟器/組織中TLR4訊號路徑和NLRP3發炎體相關分子之表現。此外，亦對於腸黏膜組織分析抗微生物胜肽含量及組織形態學和免疫化學之觀察。結果顯示，在LPS誘發發炎前灌食TC及不同劑量Cin和Lin，均能減緩LPS造成之體重流失及脾臟和腸繫淋巴結之腫大。不論血漿、脾臟、肝臟、腸黏膜組織及腸繫淋巴結，其NO和TNF-α、IFN-γ之含量均獲得改善，這可歸因於土肉桂葉精油成分有效抑制脾臟、肝臟、腸黏膜組織及腸繫淋巴結中TLR4、MD2、MyD88表現量，導致NF-κB活化程度降低。同時土肉桂葉精油成分對於MyD88的表現量之抑制作用，也造成迴腸黏膜抗微生物胜肽REG3-β、REG3-γ、RETNLβ及CRP-ductin的含量下降。誘發前灌食TC及不同劑量Cin、Lin，也抑制脾臟、肝臟、腸黏膜組織及腸繫淋巴結中NLRP3、ASC、caspase-1表現量，而降低臟器/組織中caspase-1之活性。且TNF-α、IFN-γ或IL-1β等發炎介質濃度降低，減少對免疫細胞的刺激，進一步降低周邊血中HMG-1濃度。此外，TC、Cin和 Lin，均可正常化LPS誘發之絨毛形態學，並且降低迴腸組織中嗜中性白血球浸潤及逆轉tight junction結構蛋白claudin-5和ZO-1之流失。總言之，本研究結果顯示在小鼠，肉桂醛及枷羅木醇均會透過降低TLR4訊號路徑分子和NLRP3發炎體相關分子之表現及活性而預防LPS誘發之全身性發炎及迴腸黏膜損傷，二者均是TC抗發炎作用的重要活性成分。而TC及其活性成分在LPS誘發迴腸黏膜之抗發炎作用，伴隨tight junction蛋白上升，顯示其可促進腸道障壁功能，具有應用於抗發炎及促進腸道健康之保健產品的潛力。

關鍵字

LPS ； 迴腸黏膜組織 ； 土肉桂葉精油 ； 枷羅木醇 ； 肉桂醛 ； TLR4 ； NLRP3

並列摘要

We previously reported the protective effect of the leaf essential oil of Cinnamomum osmophloeum Kanehira (TC) on LPS-induced systemic inflammatory response syndrome (SIRS) and the intestinal damage in mice. The aim of the present study is to investigate such effects of linalool (Lin) and cinnamaldehyde (Cin), two of the bioactive components in TC and the effect of TC and these two compounds on intestinal barrier function in LPS-injected mice and the underlying mechanisms. Male C57BL/6 mice were randomly divided into six groups, and gavaged every other day with corn oil (4 ml/kg BW),TC (13 mg/kg BW), Cin (0.4 or 0.9 mg/kg BW) or Lin (2.6 or 5.2 mg/kg BW) for eight times. At the day after the last treatment, corn oil group was randomly divided into control and LPS groups and injected with sterile saline (4 ml/kg BW, i.p.) or LPS (10 mg/ml/kg BW, i.p.), respectively. Animals received TC, Cin, and Lin were all injected with LPS. All mice were sacrificed with CO2 at 12h after the injection followed by the collection of blood and tissues/organs for analysis. It was found that the pretreatment with TC, Cin, and Lin prevented the mice from LPS-induced body weight loss and the enlargement of spleen andmesenteric lymph nodes. In addition, the pretreatment with TC, Cin, and Lin improved LPS-induced elevation of NO, IL-1β, TNF-α, and IFN-γ in peripheral blood, spleen, liver, intestinal mucosa and mesenteric lymph nodes. This is attributable partly to the inhibitory activity of these compositions on the expression of TLR4, MD2, and MyD88, thus resulting in reduced activation of NF-κB in these mice as detected in these organs/tissus. The inhibitory effect of these compositions on MyD88 expression also reflected by the decreased mucosal levels of antimicrobial peptides REG3-β, REG3-γ, RETNLβ and CRP-ductin. Pretreatment with TC, Cin, and Lin before LPS-injection also inhibited the expression of NLRP3, ASC, caspase-1 in spleen, liver, intestinal mucosa and mesenteric lymph nodes and reduced caspase-1 activity in thee organs/tissues. Lowered HMG-1 level in peripheral blood of LPS-injected mice by TC, Cin, and Lin provided another evidence of overall anti-inflammatory activity of these TC components. The results of the histological study showed that TC, Cin and Lin normalized the mucosal morphology, reduced the neutrophil infiltration, and reversed tight junction protein claudin-5 and ZO-1 levels in the ileum mucosa of LPS-injected mice. In conclusion, the result of this study suggested that Cin and Lin to be the active components of TC which prevented mice from LPS-induced SIRS and intestinal damage via suppressing the TLR4 and the NLRP3 pathways and that TC, Cin, and Lin possess the potential to develop their use as anti-inflammatory agents and as intestinal health supplements.

並列關鍵字

LPS ； ileum mucosa ； Cinnamomum osmophloeum Kanehira ； Linalool ； cinnamaldehyde ； TLR4 ； NLRP3

參考文獻

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