- 學位論文
人參皂苷Rb1 對U-937細胞發炎物質調控機制之探討
Regulatory mechanisms of ginsenoside Rb1 on inflammatory substances in U-937 cells
摘要
本研究目的主要探討在以脂多醣刺激發炎反應下，給予類人類巨噬細胞U-937人參皂苷Rb1，對細胞發炎反應之影響及其調節機制。實驗係以人類單核/類巨噬細胞(human monocytes/macrophages-like cells- U-937)為體外實驗的模式。將細胞培養於含10% 胎牛血清之RPMI 1640培養液中，並置於37℃、含5% CO2的細胞培養箱中，以phorbol 12-myristate 13-acetate (PMA)誘導細胞分化為巨噬細胞，之後更換為0.1%牛血清蛋白之新鮮培養液，並添加不同濃度的人參皂苷Rb1，而培養液中人參皂苷Rb1最終濃度分別為5, 10, 25, 50, 100 μg/ml，於1小時後再給予脂多醣(lipopolysacchrides; LPS)刺激細胞產生發炎反應。細胞培養1小時後，收集細胞懸浮液，進行NF-κB路徑相關物質等分析。培養23小時後，收集培養液與細胞懸浮液，分析培養液中腫瘤壞死因子-α(tumor necrosis factor; TNF-α)、可溶性細胞間黏著分子-1 (soluble intercellular adhesion molecule-1; sICAM-1)分泌量、細胞內環氧化酶-2 (cyclooxygenase-2; COX-2)蛋白質表現量。結果顯示：給予細胞人參皂苷Rb1於10-100 μg/ml劑量下可降低LPS所誘導之TNF-α分泌量(p < 0.05)，於5-100 μg/ml劑量下可抑制sICAM-1分泌量(p < 0.05)，於25-100 μg/ml劑量可抑制細胞內COX-2蛋白質表現量(p < 0.05)。在NF-κB路徑指標方面，在50-100 μg/ml劑量下可顯著增加NF-κB p65表現量(p < 0.05)，而在100 μg/ml 劑量下可顯著增加IκBα表現量(p < 0.05)。由結果得知，人參皂苷Rb1可藉由抑制U-937細胞中 NF-κB路徑活化，降低促發炎物質的生成，而具有抗發炎之功能。
並列摘要
This study investigated the effects of ginsenoside Rb1 (98.8% purity) on cell regulation of inflammatory responses in human monocytes/macrophages-like U-937 cells after lipopolysacchrides (LPS) stimulation. The U-937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. The cells were incubated at 37℃and 5% CO2 atmosphere. The U-937 cells were treated with phorbol 12-myristate 13-acetate (PMA) for differentiation into macrophages. Subsequently, the medium was replaced by fresh medium containing 0.1% bovine serum albumin and added different concentrations of ginsenoside Rb1 (5, 10, 25, 50, 100 μg/ml). After 1-hour incubation, LPS was added to induce the inflammatory response. After 1 hour, cells were collected for NF-kB assays. After 23- hour incubation, the medium was collected for TNF-α, soluble intercellular adhesion molecule-1 (sICAM-1) assays and cells were collected for the determination of COX-2 protein expression. The results showed that ginsenoside Rb1 at 10-100 μg/ml inhibited LPS-induced TNF-a (p < 0.05) and suppressed sICAM-1 scretion and COX-2 expression at 5-100 μg/ml and 5-100 μg/ml, respectively (p < 0.05). The expression of NF-kB p65 subunit was also increased when treated with ginsenoside Rb1 (50 and 100 mg/ml), and ginsenoside Rb1 at the dose of 100 mg/ml increased IkBa levels. Therefore, ginsenoside Rb1 might inhibit pro-inflammatory substances through suppressing the activation of NF-kB pathway in U-937 cells.