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題 名 | 從基因體層面探討臺灣赤楊Diarylheptanoid成分之抗發炎作用=Oregonin Inhibits Lipopolysaccharide-Induced INOS Gene and NO Expression in Macrophages and Microglia |
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作 者 | 何豐名; | 書刊名 | 中醫藥年報 |
卷 期 | 25:2 2007.10[民96.10] |
頁 次 | 頁411-425 |
專 輯 | 中醫藥基因體及免疫學研究 |
分類號 | 414.33 |
關鍵詞 | 脂多醣; 可誘導型一氧化氮合成酶; 血基質氧化酶; 吞噬細胞; 微神經膠細胞; Oregonin; Alnus formosana; Lipopolysaccharide; LPS; iNOS; Ho-1; Macrophage; Microglia; |
語 文 | 中文(Chinese) |
中文摘要 | 在此計畫中將進一步證實這類化合物是否可能透過影響血球細胞的附著及趨化作用而達到抗發炎的目的。希望這些天然來源豐富,不同結構diarylheptanoid化合物藥物作用機轉與活性的比較及研究,有助於釐清療效的根源及將來更廣泛臨床運用及新藥的開發。 RAW274.7巨噬細胞及BV-2微神經膠細胞經過濃度越高的Oregonin前處理之後,其NO產量就低(圖一、a)。RAW274.7巨噬細胞及BV-2微神經膠細胞經過濃度越高的Oregonin前處理之後,其iNOS表現量就越低(圖一、b),並以actin作為對照。當Oregonin濃度越高時,HO-1表現量就越高,反之,iNOS的表現量就越少(圖二、a)。HO-1在處理後的0-24小時中,從處理後2小時開始表現,以處理後4小時的表現量達到最大(圖二、b)。由圖可看出,當Oregonin濃度越高時,HO-1表現量就越高,(圖二、c),由此可推測,HO-1基因會隨著Oregonin的濃度而影響其表現量。 在此研究中,我們探索Oregonin在RAW274.7巨噬細胞及BV-2微神經膠細胞中,iNOS基因表現的調控機制。我們證實Oregonin確實在RAW274.7巨噬細胞中扮演了NO的抑制者角色,也證實了Oregonin確實有對抗LPS所引發的發炎反應,但是Oregonin調控iNOS的機制仍是未知。總之,在這項研究的過程中,我們證實天然產物Oregonin具有能抑制LPS所引發的iNOS基因的轉錄及增加HO-1的表現。 |
英文摘要 | Oregonin isolated from Alnus formosana is a diarylheptanoid derivative, which appears to have antioxidative and anti-inflammatory activities. In this study, our data demonstrated inhibitory actions of oregonin on the LPS-induced iNOS protein in RAW264.7 macrophages and BV-2 microglial cells. We also suggested that HO-1 induction by oregonin might contribute to this action. In this study, we examined the effects of oregonin on LPS stimulated iNOS gene expression in murine RAW264.7 macrophages and BV-2 microglia. Dulbecco's modified Eagle's medium (DMEN) with high glucose, FBS, penicillin, and streptomycin were obtained from Life Technologies. Rabbit polyclonal antibodies for ß-actin, iNOS and HO-1, and protein A/G aparose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The methaolic extract of the dry powder of leaves of A. formosana Burk (Betulaceae) (4.1 kg) was divided into fractions soluble in hexane, chloroform, n-butanol (293 g) and water by liquid-liquid partitioning. Murine RAW264.7 macrophages and BV-2 microglia were grown at 37℃ in 5% CO₂ using DMEN as the culture medium. DMEM was supplemented with 10% FBS, 100U/mlpenicillin, and 100 μg/mL streptomycin. In most conditions for NO measurement and iNOS protein assay, cells were 30 min pretreated with 10-100 μM oregonin, followed by 0.1 μg/mL (in BV-2) or 1μg/mL (in RAW264.7) LPS for 24h, unless otherwise indicated. Nitrite production, as an assay of NO release, was measured. Accumulation of nitrite in the medium was determined by a colorimetric assay with Griess reagent. Nitrite concentrations were determined by compcarison with the OD550 using standard solutions of sodium nitrite prepared in cell culture medium. Each experiment was performed in duplicate and repeated at least three times. After incubation with agents, cells were washed twice in icecold PBS and then solubilized in buffer. Samples of equal amounts of protein (80 mg) were separated through 8-12% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose paper by semidry electrophoretic transfer. Membranes were blocked in TBS containing 0.1% Tween 20 (TBST) and 5% nonfat mild for 1h. Proteins were visualized by specific primary antibodies, followed by peroxidase-labeled secondary antibodies. In RAW264.7 macrophages and BV-2 microglia, cotreatment with oregonin (10-100 μM) led to a concentration-dependent inhibition of both NO production and iNOS protein level stimulated by LPS. Results revealed that oregonin was able to decrease LPS-induced iNOS reporter activity in both cell types. As HO-1 induction participates in the negative regulation of iNOS expression, we attempted to determine whether oregonin has such action. oregonin (10-100μM)-treated RAW264.7 and BV-2 cells, HO-1 protein was dose-dependently induced. LPS also induced a moderate increase of HO-1 protein level and this response was additive to oregonin. Time course analysis in RAW264.7 cells revealed that the onset for HO-1 induction by 30 mM oregonin was around 2 h and this response achieved maximum at 4h. Oregonin, a natural diarylheptanoid compound isoated from A. formosana, acts as an inhibitor of NO production in RAW264.7 macrophages, the mechanism by which oregonin regulates iNOS expression is still unknown. In this study, we explored the effect of oregonin in the regulation of iNOS gene expression in RAW264.7 macrophages and BV-2 microglia. |
本系統中英文摘要資訊取自各篇刊載內容。