以小鼠初代巨噬細胞之ex vivo和in vitro研究模式探討台灣土肉桂葉精油及其活性成分枷羅木醇及肉桂醛之抗發炎作用

Ex vivo and in vitro studies on the anti-inflammatory effect of the leaf essential oil of Cinnamomum osmophloeum Kanehira and its active ingredients linalool and cinnamaldehyde with primary macrophage from mouse

洪宜葶(Yi-Ting Hung)

指導教授 ： 劉承慈

中山醫學大學/健康管理學院/營養學系碩士班/碩士(2017年)

摘要

已知灌食台灣土肉桂葉精油（TC）及其活性成分枷羅木醇（linalool；Lin）和肉桂醛（cinnamaldehyde；Cin）能夠抑制小鼠因LPS（lipopolysaccharide）而誘發的系統性發炎反應，改善迴腸絨毛損傷，並能抑制小鼠因尿酸而誘發慢性高尿酸血症中腎臟之發炎反應。本研究目的為利用ex vivo及in vitro模式，以小鼠初代腹腔巨噬細胞觀察TC及其活性成分是否能夠透過調節巨噬細胞發炎活性，而改善LPS及尿酸鈉（monosodium urate；MSU）結晶所導致的組織發炎性損傷，並探討其作用機制。Ex vivo模式之實驗以十週齡雄性C57BL/6小鼠隨機分為六組，分別隔日灌食載劑玉米油（4 ml/kg BW）、土肉桂葉精油（13 mg/kg BW）、低劑量枷羅木醇（2.6 ml/kg BW）、高劑量枷羅木醇（5.2 mg/kg BW）、低劑量肉桂醛（0.4 ml/kg BW）和高劑量肉桂醛（0.9 mg/kg BW），共灌食八次，於最後一次灌食後隔日腹腔注射Brewer thioglycollate medium（4%，2 ml），四天後犧牲，收集腹腔巨噬細胞，而in vitro模式則於十週齡正常雄性C57BL/6小鼠如上述腹腔注射之方法收集腹腔巨噬細胞。Ex vivo模式於培養液中加入LPS，而in vitro模式先在培養液中加入TC（5、25、75 μg/ml）、Lin（2.0、10.1、30.2 μg/ml）及 Cin（0.3、1.7、5.2 μg/ml）預處理半小時，再於LPS或MSU刺激下同時添加上述各劑量TC、Lin和Cin，培養24小時。然後收集培養液分析其中nitrate/nitrite、促發炎細胞激素IL-1β及TNF-α、抗發炎細胞激素IL-10濃度，並且收集巨噬細胞以Western blotting分析細胞中TLR4和NLRP3訊號路徑相關分子之表現量。結果顯示，不論是在ex vivo模式中以灌食的方式或是in vitro模式中添加於培養液中給予TC、Lin及Cin，均可抑制LPS誘發巨噬細胞產生nitrate/nitrite及IL-1β，並且在ex vivo模式中還可抑制細胞產生TNF-α。TC抗LPS誘發之發炎作用在ex vivo模式中，主要可歸因於其抑制細胞TLR4蛋白質表現量，而在in vitro模式中則歸因於其抑制細胞MD2和NLRP3蛋白質表現量。TC主要活性成分Lin和Cin在ex vivo模式中，對於TLR4和NLRP3路徑訊號分子表現量的調控雖與TC並不一致，但在in vitro模式中則具有一致性，推論在小鼠灌食TC及其活性成分會因對於巨噬細胞以外的生理系統具有差異性的影響，而間接造成巨噬細胞ex vivo活性之差異性。此外，在以MSU刺激巨噬細胞的in vitro模式中，發現於培養基中添加TC、Lin及Cin，均可降低培養液中的IL-1β及TNF-α之濃度，且可歸因於TC及這些活性成分能夠抑制細胞NLRP3蛋白質表現量。由本研究結果得知，TC及其活性成分Lin及Cin不論是在LPS或MSU誘發之巨噬細胞發炎反應，均具有改善的效果，顯示對於感染性的革蘭氏陰性菌或非感染性的痛風性發炎病症的預防或治療上，均具有應用潛力。

關鍵字

巨噬細胞 ； LPS ； MSU ； 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) and its active ingredients linalool (Lin) and cinnamaldehyde (Cin) on LPS-induced systemic inflammatory response syndrome and the intestinal damage and on inflammatory condition in chronic hyperuricemia in mice. The aim of the present study is to investigate the anti-inflammatory effect of Lin, Cin, and TC on LPS- or monosodium urate (MSU)-stimulated macrophages and the underlying mechanisms ex vivo and in vitro. In the ex vivo study, 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), Lin (2.6 mg/kg BW) or Cin (0.9 mg/kg BW) for eight times. At the day after the last treatment, all mice were injected peritoneally with 4% Brewer thioglycollate medium (2ml/ mouse) followed by the collection of peritoneal macrophages four days after the injection. The cells were cultured for 24h with LPS followed by the determination of the concentrations of nitrate/nitrite, IL-1β, TNF-α and IL-10 in culture medium and cellular expression of TLR4, MD2, MyD88, NLRP3, ASC, and caspase-1. The same analysis was carried out in the in vitro study but with peritoneal macrophages collected from normal male C57BL/6 mice and were preincubated with TC (5, 25, 75 μg/ml), Lin (2.0, 10.1, 30.2 μg/ml) or Cin (0.3, 1.7, 5.2 μg/ml) for half an hour, before the treatment with LPS or MSU for 24 h. The present study found that TC, Lin and Cin inhibited the secretion of nitrate/nitrite and IL-1β ex vivo and in vitro, and inhibited the generation of TNF-α ex vivo by LPS-induced macrophages. Such anti-inflammatory activity of TC can be attributed to its inhibitory effect on TLR4 expression ex vivo, and its inhibitory effect on MD2 and NLRP3 expression in vitro. In addition, TC, Lin and Cin were found to lower the secretion of IL-1β and TNF-α by MSU-induced macrophages. This can be attributed to the inhibitory effect of TC, Lin and Cin on the expression of NLRP3 protein. In summary, the present study demonstrated that TC and its active compositions Lin and Cin, can reverse inflammatory response of macrophages induced by LPS and MSU and suggesting the prophylactic and therapeutic use of them on Gram-negative bacteria-induced and gouty inflammation.

並列關鍵字

macrophages ； LPS ； MSU ； Cinnamomum osmophloeum Kanehira ； linalool ； cinnamaldehyde ； TLR4 ； NLRP3

參考文獻

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