探討麻竹葉甲醇萃取物對LPS誘發巨噬細胞發炎之抗發炎機轉

背景脂多醣體 (LPS) 是格蘭氏陰性菌外膜的組成之ㄧ，當細菌侵入人體時，LPS會誘導體內一連串的發炎反應。過去文獻研究指出，無論是竹葉水萃物、甲醇萃取物或精油，具有抗氧化、抑制發炎等作用，其抑制路徑仍不清楚；而且研究的竹葉品種在台灣不常見，因此，本研究的目的是說明麻竹竹葉的甲醇萃取物 (DLE) 如何影響LPS刺激的巨噬細胞RAW 264.7導致的發炎路徑中iNOS、COX-2、IL-1β、 IL-6及TNF-α表達。方法我們研究了以下內容：(1) 不同濃度的麻竹竹葉甲醇萃取物以MTT assay 測定對巨噬細胞RAW 264.7的細胞毒性。(2) RT-PCR測定DLE對LPS刺激RAW 264.7產生的iNOS、COX-2、IL-1β、IL-6及TNF-α mRNA之抑制作用。(3) Western blot測定DLE對LPS刺激RAW 264.7細胞的磷酸化AKT、JNK、ERK和p38之抑制作用。(4) DLE對LPS刺激RAW 264.7細胞產生NO的影響。(5) DLE對LPS刺激RAW 264.7後細胞核內NF-κB的影響。本篇實驗分別以不同濃度的DLE (0.05、0.1、0.25、0.5 μg/mL) 前處理RAW 264.7細胞後再以1 μg/mL的LPS刺激。結論DLE對於LPS (1 μg/mL) 刺激RAW 264.7細胞所產生iNOS、COX-2、IL-1β、IL-6及TNF-α mRNA的影響，DLE濃度越高，其mRNA表現越少。而對LPS刺激RAW 264.7細胞的磷酸化AKT、JNK、ERK和P38蛋白質表現也有抑制作用。故DLE抑制發炎機制可能是抑制RAW 264.7細胞iNOS、COX-2、IL-1β、IL-6及TNF-α mRNA表達，抑制NF-κB進入細胞核內及抑制LPS誘導發炎的MAPK路徑。對於NO雖然有抑制作用，但是並不明顯。本實驗是第一篇針對麻竹竹葉抗發炎的研究，結果證明麻竹竹葉甲醇萃取物有抗發炎作用，未來可以考慮併用非類固醇藥物以降低非類固醇藥物用量而降低藥物帶來的副作用，或運用在保健及化妝品上以提高麻竹的經濟價值。

BackgroundLipopolysaccharide (LPS) is part of the outer membrane of the cell wall of Gram-negative bacteria. LPS induced a series of inflammation when the bacteria into the body. In past studies, whether bamboo leaves water extract, methanol extract or essential oils which possess antioxidant activity and anti-inflammatory. But the mechanism of action still unknown, and the varieties of bamboo leaves in those studies are not common in Taiwan. Therefore, the objective of this study is to elucidate the anti-inflammatory effects of methanol extract of Dendrocalamus latiflorus Munro (DLE) on the expression of iNOS, COX-2, IL-1β, IL-6, and TNF-α via blocking of the pathway in LPS-stimulated macrophage RAW 264.7 cells.MethodsWe investigated the following: (1) Cytotoxicity effect of different concentrations DLE on RAW 264.7 cells by MTT assay. (2) The inhibitory effect of DLE on the LPS-induced iNOS, COX-2, IL-1β, IL-6, and TNF-α at the mRNA levels by RT-PCR. (3) The inhibitory effects of DLE on LPS-induced phosphorylation of AKT, JNK, ERK and P38 in RAW 264.7 cells by Western blot. (4) Effect of DLE on NO production in LPS-induced RAW 264.7 cells. (5) Effect of DLE on the activation of nucleus NF-κB in LPS-stimulated RAW 264.7 cells. The RAW 264.7 cells were pretreated with various doses (0.05、0.1、0.25、0.5 μg/mL) of DLE followed by a LPS (1 μg/mL) treatment. ConclusionThe inhibitory effects of DLE on iNOS, COX-2, IL-1β, IL-6 and TNF-α mRNA in LPS (1 μg/mL) -stimulated RAW 264.7 cells dependent on the DLE concentration . LPS-induced phosphorylation of AKT, JNK, ERK and P38 in RAW 264.7 cells expression is also inhibited by DLE. Therefore, the mechanism of DLE anti-inflammatory may inhibit the iNOS, COX-2, IL-1β, IL-6 and TNF-αmRNA expression、nuclear NF-κB and the of LPS-induced MAPKs pathway. Inhibition of NO is not obvious. This is the first experiment about anti-inflammatory for D. latiflorus bamboo leaves, the results prove that the DLE have anti-inflammatory effects. Future the methanol extract of D. latiflorus bamboo leaves could be combined with one of the NSAIDs for reduce non-steroidal drugs dosage and the side effects of drugs; or the DLE use for health care and cosmetics in order to improve the D. latiflorus economic value.