探討紫草素低濃度作用影響THP-1巨噬細胞產生IL-1β的機制

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題名:	探討紫草素低濃度作用影響THP-1巨噬細胞產生IL-1β的機制 Low Doses of Shikonin Reduce the Production of IL-1β through Inhibiting the Activation of Caspase-1 in THP-1 Macrophage
作者:	周沛昌;Pei-Chang Chou
貢獻者:	免疫學研究所碩士班
關鍵詞:	紫草素;發炎體;Shikonin;inflammasome
日期:	2013-07-30
上傳時間:	2013-10-02 11:01:49 (UTC+8)
出版者:	中國醫藥大學
摘要:	背景：紫草素是來自中國傳統中藥材紫草萃取出的奈?化合物，過去已被證實具有廣泛的藥理作用，其中包含了抗發炎的作用。最近的研究發現在巨噬細胞產生並釋放慢性發炎因子IL-1β的過程中，發炎體的活化扮演關鍵的角色。而過去的研究也已證實，紫草素能經由不同的分子信號干擾而降低單核細胞和巨噬細胞中，TNF-α和IL-1β等促發炎因子的產生。然而，紫草素過去研究也指出對許多癌細胞具有細胞毒性的影響。因此，紫草素具有抗發炎的能力可能是由不同的紫草素的毒性作用影響。在本研究中，我們將探討紫草素對人類巨噬細胞的NLRP3發炎體的影響。材料與方法：我們利用PMA將THP單核球細胞分化成巨噬細胞，再加入酯多醣和不同濃度的紫草素處理，接著加入ATP使發炎體活化。而後分別收集培養的細胞與上清液，將一部分的上清液利用酵素免疫分析法來探討紫草素對IL-1β的影響，另一部分則利用丙酮將蛋白質沉澱。而上清液與細胞萃取出的蛋白質利用西方墨點法分析紫草素對NLRP3發炎體的影響。並利用CCK-8分析不同濃度的紫草素一到三天對細胞生存力之影響。結果: 紫草素劑量小於0.5μM時，對THP-1巨噬細胞不具有毒性。紫草素濃度超過0.5μM時對細胞生存力造成影響，而紫草素處理THP-1巨噬細胞之半影響劑量約為0.92μM。並發現低濃度的紫草素具有抑制ATP促進巨噬細胞分泌IL-1β 與IL-18的能力，且對細胞是不具毒性的。同時也發現caspase-1的活性受到紫草素的抑制。而這樣的現象主要不是因RNA和NF-κB pathway的影響導致。結論: 低濃度的紫草素可在不影響NF-κB活性的情況下，經由調降發炎體活性而抑制IL-1β 與IL-18的產生，這表明低濃度的紫草素能在比較不影響免疫細胞的狀態下，作為治療IL-1β相關自體免疫疾病的藥物。 Backgrounds: Shikonin, a naphthoquinone compound extracted from Lithospermum erythrorhizo, has been shown to exert a wide range of pharmacological properties, including anti-inflammatory activities. Recently, it has been shown that the inflammasome activation plays a crucial role in the production of macrophage-derived IL-1β, which mediated the chronic inflammation. Previous studies have been shown that Shikonin inhibits production of proinflammatory cytokines in monocytes and macrophages via interfering with different signaling molecules and thus has anti-inflammatory activities. However, shikonin has been also reported to have strong cytotoxic effects on many cancer cell lines. Therefore, actions of shikonin with anti-inflammation could be different from the toxic effect of shikonin. In the present study, we investigate the effect of Shikonin at low concentrations on the production of pro-inflammatory cytokine, IL-1β and activation of NLRP3 inflammasome in THP-1 macrophages. Materials and Methods: After being differentiated by phorbol myristate acetate (PMA), THP-1 macrophages were treated with LPS or LPS plus Shikonin for different time periods. The supernatants and cell pellets were collected separately for investigating the effect of Shikonin on the expression of IL-1β by ELISA. Proteins from cell media were precipitated by acetone and investigated the activation of NLRP3 inflammasome molecules by western blot analysis. Total RNA extracted was followed the manufacturer’s instructions of TRIzol reagent and used to investigated mRNA expression of IL-1β, caspase-1, TNF-α and NLRP3. The viability of THP-1 macrophages treated with Shikonin was analyzed by the cell counting kit 8 (CCK8) assay for 1-3 days. Results: In THP-1 macrophages, the concentration of Shikonin at less than 0.5 μM had little or no detectable toxicity. Minor toxicity was first noted at > 0.75 μM.the average of IC50 of shikonin for three days was approximately 0.92 μM in THP-1 macrophages. We found that, after treatment with the low concentration of Shikonin, the secretion of ATP-induced IL-1β and IL-18 were inhibited in LPS-primed macrophages. The decreasing levels of Procaspase-1 and caspase-1 were also detected. However, these phenomena are no impact on mRNA level and NF-κB signaling pathway. Conclusion: Low concentration of Shikonin can effectively suppressed IL-1β expression in THP-1 macrophages via downregulation of inflammasome activation but not affect NF-κB activity. Suggesting that low concentrations of Shikonin in less affect the state of the immune cells, and as a drug to treatment IL-1β-associated autoinflammatory diseases.
顯示於類別:	[免疫學研究所] 博碩士論文

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