土肉桂葉精油對於內毒素誘發小鼠全身性發炎反應症候群中腸黏膜損傷之影響

The effect of leave essential oil of Cinnamomum osmophloeum Kaneh. on intestinal mucosal damage in LPS-induced systemic inflammatory response syndrome in mice

10.6834/CSMU.2013.00132

許捷盛

小鼠 ； LPS ； 腸道 ； 土肉桂 ； NALP3 ； TLR4 ； 促發炎細胞激素 ； mice ； LPS ； intestinal ； Cinnamomum osmophloeum Kaneh. ； NALP3 ； TLR4 ； proinflammatory cytokines

中山醫學大學營養學系碩士班學位論文

2013年

碩士

李健群

繁體中文

本研究目的為觀察灌食土肉桂葉精油 (TC)是否可改善由LPS誘發系統性發炎反應 (SIRS)之腸黏膜損傷。實驗以C57BL/6雄性小鼠分別灌食載劑玉米油C組 (4 ml/kg BW)，低、中、高劑量TC，分別為6.5、13、26 mg/kg BW，每隔一日灌食一次，共八次，於最後一次灌食後隔日，再將灌食載劑組再分為二組，分別經由腹腔注射生理食鹽水 (4 ml/kg BW)或LPS (10 mg/kg BW)誘發系統性發炎反應，所有灌食TC之小鼠亦注射如上劑量之LPS，並於注射後並禁食12小時犧牲，然後以二氧化碳窒息法犧牲。收集血液、肝臟、迴腸黏膜及組織分析NO、或腸繫膜淋巴結中促發炎細胞激素含量，分析脾臟、迴腸黏膜、腸繫膜淋巴結中NF-κB的含量、Caspase-1酵素活性及迴腸黏膜中xanthine Oxidase酵素活性，並以western blotting分析脾臟肝臟、迴腸黏膜、腸繫膜淋巴結中TLR4及NALP3訊號路經相關分子之表現量，另以H&E染色觀察腸道組織形態學。結果顯示，給予不同劑量的TC均可降低血漿、肝臟及迴腸黏膜NO含量及血漿中促發炎激素IFN-γ、IL-18濃度，此外，中、高劑量TC可顯著降低血漿中IL-1β、TNF-α濃度，而高劑量TC可顯著降低血漿中HMG-1濃度。所有測試劑量的TC可顯著降低LPS誘發之脾臟IL-1β、迴腸黏膜IL-1β和IFN-γ及腸繫膜淋巴結中IL-1β和TNF-α的含量，而中、高劑量TC可顯著降低脾臟TNF-α、腸繫膜淋巴結IFN-γ的含量，中劑量TC也顯著降低脾臟IFN-γ的含量，而TC在高劑量可降低迴腸黏膜TNF-α的含量 (P<0.05)。LPS在脾臟、迴腸黏膜及腸繫膜淋巴結中所誘發之NF-κB含量，顯著因灌食中、高劑量之TC而獲得改善 (P<0.05)。所有測試劑量的TC均降低脾臟及腸繫膜淋巴結中Caspase-1的活性及降低迴腸黏膜xanthine oxidase的活性，而高劑量TC可降低迴腸黏膜Caspase-1的活性 (P<0.05)。所有劑量測試下，TC呈現劑量依賴性降低TLR4及NALP3訊號分子表現，又以高劑量達顯著效果，腸道組織形態學發現以LPS誘發之小鼠與未誘發組相較下，其絨毛變短水腫，排列不太一致。由以上結果在注射LPS的小鼠，灌食TC可透過降低TLR4路徑分子表現量，減少NF-κB含量，進而減少周邊血及組織中NO的產生與促發炎激素合成。而其降低之NO至少可部分說明迴腸黏膜中xanthine oxidase活性之下降。 此外，在注射LPS的小鼠，灌食TC也可透過降低NALP3路徑分子表現量，降低組織Caspase-1的活性，因而減少促發炎細胞激素IL-1β及IL-18之釋放。因而TC可以有效改善由LPS誘發小鼠系統性發炎所造成的腸道絨毛損傷及減緩系統性發炎的嚴重程度。

The aim of the present study is to investigate the effect of leave essential oil of Cinnamomum osmophloeum Kaneh. (TC) on intestinal mucosal damage in systemic inflammatory response syndrome (SIRS) and the underling mechanisms with LPS-injected mice. Male C57BL/6 mice received every other day for eight times by gavage corn oil (4 ml/kg BW) or various doses of TC (6.5, 13, and 26 mg/kg BW). One day after the last treatment, corn oil group was randomly divided into control and SIRS groups and injected intraperitioneally with saline (4 ml/kg BW) or LPS (10 mg/kg BW), respectively. Animals received TC were all injected with LPS. All animals were sacrificed by CO2 at 12h after the injection, followed by the collection of blood, and tissues/organs for the determination of levels of nitrate/nitrite, proinflammatory cytokines, activities of caspase-1 and xanthine oxidase, and nuclear NF-κB activation index. Western blotting was also carried out to analyze the expression of TLR4 and NALP3 signaling pathway related molecules in tissues/organs. Morphological investigation on intestinal tissue was carried out with H&E staining. The results showed that in LPS-injected mice, all tested doses of TC significantly reduced nitrate/nitrite, IFN-γ, IL-18 concentrations in plasma; nitrate/nitrite content in liver and ileum; IL-1β content in spleen, ileum, and MLN; IFN-γ content in ileum; TNF-α content in MLN; activity of caspase-1 in spleen and MLN; and activity of xanthine oxidase in ileum, concomitantly with significantly reduced expression of TLR4 signaling pathway related molecules, MD2 and MyD88, and NALP3 signaling pathway related molecules, ASC, NALP3, and caspase-1, in a dose-dependent manner (P<0.05). In addition, 13 and 26 mg/kg BW of TC significantly reduced plasma IL-1β and TNF-α concentrations; spleen TNF-α content; and MLN IFN-γ content, concomitantly with reduced NF-κB activation index in spleen, ileum, and MLN of LPS-injected mice (p<0.05). Furthermore, 13 mg/kg BW of TC significantly reduced IFN-γ content in spleen (p<0.05), while 26 mg/kg BW of TC significantly reduced plasma concentration of HMG-1 and ileum content of TNF-α, and reduced caspase-1 activity in ileum of LPS-injected mice (p<0.05). Morphological study on intestinal mucosa observed that compared with saline-injected mice, LPS induced shorter villus and caused edema in small intestine while was reversed by TC treatment. In conclusion, the present study demonstrated anti-inflammatory activity of TC and its beneficial effect on morphology of intestinal mucosa in LPS-induced SIRS. This is at least partly through the reduced expression of TLR4 signaling pathway related molecules thus reduced NF-κB activation, thereby reduced the production of NO and proinflammatory cytokines in these animals. Reduced NO content can at least partially explain why xanthine oxidase activity was reduced in ileum. In addition, the beneficial effect of TC in LPS-induced SIRS was also demonstrated to be at least partly through reduced expression of NALP3 signaling pathway related molecules that reduced tissue caspase-1 activity, thereby reduced the release of IL-1β and IL-18.

1. 洪宜葶(2017)。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。中山醫學大學營養學系碩士班學位論文。2017。1-117。