龍葵葉子水萃物作用於鼠類巨噬細胞株RAW 264.7之抗發炎機制探討

Solanum nigrum Linn (SNL)，中文名稱為龍葵，他是屬於茄科家族的一位成員，在野外常常可以很容易地見到他的蹤跡。在傳統上，龍葵常常被用來當作中藥來使用，並且主要是用來治療發炎與癌症等疾病。根據統計，有百分之二十五左右的癌症被認為與慢性感染及發炎有關。在文獻中普遍認為由慢性發炎組織中的巨噬細胞所產生出來的活性氧分子(Reactive Oxygen Species，ROS)及活性氮分子 (Reactive Nitrogen Species，RNS)會對細胞內的DNA造成損傷，進而造成癌症的產生。而在這過程中，一些與製造活性氧分子及二十碳烯酸(Eicosanoid)相關的酵素[e.g. 第二型煙醯胺腺嘌呤二核苷酸磷酸氧化酶(NADPH Oxidase-2，NOX-2)，黃嘌呤氧化酶(Xanthine Oxidase，XO)，誘導型一氧化氮合成酶(inducible Nitric Oxide Synthase，iNOS)，第二型環氧合成酶(Cyclooxygenase-2，COX-2)]其調節上的異常已經被證實對此過程有很重要的影響。在巨噬細胞中，與這些酵素有關的訊息傳導途徑主要包含核轉錄因子-κB(Nuclear Factor-κB，NF-κB)、轉錄激活蛋白-1 (Activator Protein-1，AP-1)、促分裂素原活化蛋白激酶(Mitogen-Activated Protein Kinase，MAPK)等途徑。因此，透過研究一些具有抗發炎潛力的物質可能對在發炎及癌化等方面上的化學預防會有很重要的幫助。為了探討龍葵水萃物其所具有的抗氧化及抗發炎效應，我們藉由XO活性測定法(Xanthine Oxidase activity assay)，發現到龍葵的葉子及綠果實水萃物部分對於黃嘌呤氧化酶的活性具有比較顯著的抑制能力，然而在莖的水萃物部分就沒有這樣的結果。另外也有使用亞硝酸鹽測定(nitrite assay)來檢測龍葵對於脂多醣 (Lipopolysaccharide，LPS)激活處理下的鼠類巨噬細胞RAW 264.7其一氧化氮釋放能力的影響。我們發現到僅有龍葵的葉子及綠果實水萃物具有抑制一氧化氮釋放的能力。除此之外我們還發現到僅有龍葵葉子的水萃物可以抑制脂多醣激活處理下的RAW 264.7細胞其活性氮分子產生的情形。從這裡我們可以推測龍葵葉子的水萃物可能就是藉由降低鼠類巨噬細胞RAW 264.7之一氧化氮、活性氮分子的生成並且抑制黃嘌呤氧化酶的活性來達到抗發炎的作用。另外還有使用西方墨點法(western blot)去分析龍葵葉子水萃物對於細胞內的發炎訊息傳導途徑影響情形。這邊的實驗結果顯示出龍葵葉子水萃物不但可以抑制細胞內的p38/JNK MAPK活性，同時也可以去降低NF-κB (p65)/AP-1 (phospho c-jun)的核移位(nucleus translocation)程度。在反轉錄聚合酶鏈鎖反應(RT-PCR)實驗中，我們還發現到龍葵葉子水萃物可以抑制脂多醣激活處理下的RAW 264.7細胞其iNOS mRNA的表現。

關鍵字

誘導型一氧化氮合成酶第二型環氧合成酶轉錄激活蛋白-1 ；核轉錄因子-κB ；一氧化氮；促分裂素原活化蛋白激酶

並列摘要

Solanum nigrum Linn (SNL), a member of the Solanaceae family , is a common herb that grows wildly and abundantly in open fields .Traditionally, it has been used in chinese medicine for curing inflammatory disease and various kinds of cancers. Approximately, 25 % of all cancers are somehow associated with chronic infection and inflammation. Chronic inflammation is considered as a possible mechanism in initiating tumorigenesis, resulting from the generation of ROS (Reactive Oxygen Species)/RNS (Reactive Nitrogen Species) by macrophages in the inflamed tissue and resulted in DNA damage. Chronic dysregulated activation of some cellular ROS/Eicosanoid generating enzymes, NOX-2 (NADPH Oxidase-2), XO (Xanthine Oxidase),iNOS (inducible Nitric Oxide Synthase) and COX-2 (Cyclooxygenase-2), have been shown to play pivotal roles during this process. The signal transduction pathways related to these enzymes in macrophages mainly including NF-κB/AP-1/MAPK pathway. Thus, to study the mechanism of the potential agents with anti-inflammatory activities might be important in the effects of chemoprevention of chronic inflammation and carcinogenesis. The main aim of this study is to evaluate the anti-oxidant and anti-inflammatory effects about Solanum nigrum Linn water extracts (SNE). Using the xanthine oxidase activity assay, we found that the stem water extracts of SNL didn't inhibit xanthine oxidase activity, but only the leaf and green fruit water extracts of SNL demonstrate the inhibitory activity. We also used the nitrite assay to investigate the inhibition ability of SNL on the nitrite released in culture medium of LPS-stimulated RAW 264.7 cells. We found that only the leaf and green fruit water extracts of SNL effectively inhibited the nitrite excretion. Additionally, the leaf water extracts of SNL effectively inhibited the ROS production in LPS-stimulated RAW 264.7 cells, but the stem and fruit water extracts of SNL didn’t. These results suggested that the leaf water extracts of SNL might exert the anti-inflammatory actions by suppressing NO and ROS production and xanthine oxidase activity. Moreover, we used western blot to investigate the inhibition ability of the leaf water extracts on the cellular inflammatory signal transduction pathway . The data demonstrated that the leaf water extracts greatly inhibited the p38/JNK MAPK activity , and also had the ability to decrease the NF-κB (p65)/AP-1 (phospho c-jun) nuclear translocation level. Then, we used RT-PCR to assay the iNOS mRNA expression, indicating that the leaf water extracts could inhibit iNOS mRNA expression dose-dependently.

並列關鍵字

iNOS ； COX-2 ； AP-1 ； I-κB α ； NF-κB ； NO ； MAPK

參考文獻

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龍葵葉子水萃物作用於鼠類巨噬細胞株RAW 264.7之抗發炎機制探討

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