發炎反應是血管及細胞受到傷害及感染所產生的反應。巨噬細胞的活化是這些發炎開始即增幅的主要關鍵。當巨噬細胞受到內毒素脂多醣LPS (lipopolysaccharide) 或免役刺激 (Interferon-gamma，IFN-γ) 時，會釋放ㄧ氧化氮 (NO)、PGE2、TNF-ㄐBIL-1、IL-6，誘發一系列的發炎反應。發炎訊號分子的產生與釵h發炎疾病有相關性。因此，抑制過量的發炎訊號分子產生是新藥物開發的一個重要目標。本研究主要以LPS刺激誘導的老鼠巨噬細胞株RAW 264.7作為反應模式，探討藥用貼布的正己烷萃取物(n-Hexane extract)、二氧化碳超臨界萃取物 (SFE-CO2)、甲醇萃取物(Methanol extract)、乙醇萃取物 (Ethanol extract)、水萃取物 (Aqueous extract)及乳劑配方對於促發炎的調節因子的釋放及相關調控基因的影響。並且和西藥indomethacin作相比較。
此論文第一部分主要探討n-hexane、SFE-CO2、methanol、ethanol、aqueous extract、indomethacin及乳劑配方對LPS刺激誘導的老鼠巨噬細胞株RAW 264.7的亞硝酸鹽產生的抑制效果。研究結果顯示，n-hexane extract (0.005-0.125mg/ml)、SFE-CO2 (0.005-0.05mg/ml)、methanol extract (0.005-0.25mg/ml)、ethanol extract (0.005-0.25mg/ml)、aqueous extract (0.25-1.5mg/ml)、indomethacin (0.01-0.5mM) 對一氧化氮產生的抑制作用，隨著濃度的增加遞增。 n-Hexane、methanol、ethanol萃取液在濃度0.125mg/ml可以100%抑制nitrite產生，而SFE-CO2萃取液在濃度0.05mg/ml完全100%抑制nitrite產生，抑制效果是SFE-CO2 > n-hexane > methanol, ethanol >aqueous萃取物。 Indomethacin在最高濃度0.5mM對nitrite有70%抑制能力。DMSO/Ethanol及Ethanol配方的乳劑有抑制nitrite產生的效果。利用西方吸漬法及RT-PCR分析誘導性一氧化氮合成酶 (iNOS) 的基因表達時發現，n-hexane萃取液隨著濃度的增加，iNOS基因的表達有顯著的被抑制的效果。因此n-hexane萃取液主要是借調控iNOS的轉錄來抑制一氧化氮合成酶的表達，進而抑制一氧化氮的生合成。Methanol、SFE-CO2 、ethanol及aqueous萃取液及indomethacin隨著濃度的增加，iNOS mRNA的表達並未被抑制，但iNOS蛋白的表達隨著濃度的增加卻隨之被抑制。SFE-CO2 萃取液經由iNOS mRNA 穩定性試驗結果證實，其抑制發炎機制是因為影響iNOS mRNA的穩定性，進而使iNOS蛋白質減少。而Methanol、ethanol及aqueous萃取液及indomethacin可能是在轉譯作用上影響iNOS蛋白質表現或是後轉譯的蛋白質穩定性的改變。DMSO/Ethanol及Ethanol配方的乳劑對iNOS基因的表達有顯著的被抑制的效果，因此DMSO/Ethanol及Ethanol配方的乳劑是借調控iNOS的轉錄來抑制一氧化氮合成酶的表達，進而抑制一氧化氮的生合成。 Inflammatory response was typically represented by a cascade of vascular and cellular reactions caused by injury and infection. The activation of macrophages was major key point for the initiation and propagation of these defensive reactions. Then macrophages stimulated with lipopolysaccharide or Interferon-gamma, macrophages release nitric oxide, PGE2, TNF-? IL-1 and IL-6. The sustained inflammatory mediator production has been implicated associated with the pathologic process of inflammatory disease. Thus, the inhibition of over production of inflammatory mediators stands as an important therapeutic goal for drug development. The goal of study is to design that lipopolysaccharide (LPS)-induced mouse macrophages RAW 264.7 cells as inflammatory reaction. To optimize the n-hexane, SFE-CO2, methanol, ethanol, aqueous extract of medical plasters and Lecithin-Based Oil-in-Water Microemulsions for the effect of pro-inflammatory mediators and regulated genes, and comparison with COX-2 inhibitor- indomethacin.
The first part of paper is to optimize that effect of n-hexane、SFE-CO2、methanol、ethanol、aqueous extract of medical plasters and indomethacin and microemulsions on LPS-induced nitrite formation in RAW 264.7 macrophage. In the present study demonstrated that n-hexane extract (0.005-0.125mg/ml)、SFE-CO2 (0.005-0.05mg/ml)、methanol extract (0.005-0.25mg/ml)、ethanol extract (0.005-0.25mg/ml)、aqueous extract (0.25-1.5mg/ml)、indomethacin (0.01-0.5mM) produced a significant and concentration-dependent decreases in NO production in a endotoxin-stimulated RAW 264.7 macrophage. 0.125mg/ml of n-hexane、methanol、ethanol extracts incubated with LPS-stimulated macrophages completely inhibited NO production, 0.05mg/ml of SFE-CO2 extract incubated with LPS-stimulated macrophages completely inhibited NO production. The inhibited result is SFE-CO2 > n-Hexane > Methanol, Ethanol >Aqueous extracts. Highest concentration (0.5mM) of indomethacin has 70% inhibited NO production. In addition, DMSO/Ethanol and Ethanol microemulsions showed inhibitory effect on the amounts of No released by LPS-stimulated macrophages. Western blot analysis and RT-PCR assay revealed that n-hexane extract inhibited the inducible nitric oxide synthase (iNOS) expression in dose-dependent manners. Therefore, n-hexane extract down-regulates LPS-induced iNOS expression primarily by blocking its transcription. Methanol、SFE-CO2 、ethanol、aqueous extracts and indomethacin not inhibited the inducible nitric oxide synthase (iNOS) mRNA in dose-dependent manners, but inhibited the inducible nitric oxide synthase (iNOS) protein in dose-dependent manners. INOS mRNA stability analysis revealed that SFE-CO2 extract effect iNOS mRNA expression. Methanol、ethanol、aqueous extracts and indomethacin results suggest that effect iNOS protein expression at the translational or chang at the post-translational level (protein stability). DMSO/Ethanol and Ethanol microemulsions inhibited the inducible nitric oxide synthase (iNOS) expression and down-regulates LPS-induced iNOS expression primarily by blocking its transcription.
