- 學位論文
β-胡蘿蔔素及類黃酮對激發之HL-60細胞發炎反應的抑制作用
The anti-inflammatory effect of β-carotene and flavonoids on stimulated HL-60 cells
摘要
本研究的目的為探討：1）β-胡蘿蔔素單獨或與類黃酮共同作用下，對激活的monocytes/ macrophages發炎反應之影響；2）β-胡蘿蔔素單獨或合併quercetin是否影響人類肺細胞（A549 cells）對抗激活的monocytes/ macrophages的傷害。首先，HL-60細胞與β-胡蘿蔔素單獨或合併類黃酮共同預培養1小時，再與phorbol-12-myristate-13-acetate (PMA)或PMA +4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)培養兩天，收集培養液進行IL-8, TNF-α及thiobarbituric acid reactive substances (TBARS)分析，並收集HL-60細胞，分析DNA股斷裂情形及細胞內活性氧含量，其中一部份則與A549細胞共同培養1天；其次，A549細胞預先以不同濃度β-胡蘿蔔素單獨或與quercetin合併預培養，再與PMA＋NNK激活的HL-60細胞共同培養，觀察A549細胞抵抗發炎反應的效應。 結果顯示: 20 μM β-胡蘿蔔素促進PMA或PMA＋NNK活化的HL-60細胞分泌IL-8及TNF-α，並增加TBARS和細胞內活性氧的生成。此外，20 μM β-胡蘿蔔素預培養亦使得PMA＋NNK激活的HL-60細胞對A549細胞DNA的傷害增加。然而，低濃度（2 μM）β-胡蘿蔔素預培養下，能顯著抑制PMA或PMA＋NNK誘發HL-60細胞之發炎反應，同時，2 μM β-胡蘿蔔素預培養亦可顯著降低PMA＋NNK激活的HL-60細胞對A549細胞造成的DNA傷害。HL-60進行20 μM β-胡蘿蔔素預處理時，若合併使用20 μM quercetin, naringenin或vitamin E，均可抑制20 μM β-胡蘿蔔素促進HL-60細胞產生的發炎反應及對A549細胞DNA的傷害；比較三者關係與效果quercetin及naringenin與vitamin E相當或較好， quercetin同時相加性的增強2 μM β-胡蘿蔔素的抗發炎效果。 而20 μM β-胡蘿蔔素預培養的A549細胞，與激活的HL-60細胞共培養下，培養基中發炎激素含量及A549細胞DNA傷害有增加的傾向。另一方面，A549細胞以2 μM β-胡蘿蔔素預培養則傾向抑制這些反應。20 μM quercetin與2 μM β-胡蘿蔔素共同與A549細胞預培養則顯著增加A549細胞對激活的HL-60細胞的抗性。這些體外實驗顯示，高濃度的β-胡蘿蔔素可能促進發炎而增加肺上皮細胞的傷害，反之，低劑量的β-胡蘿蔔素可能具抗發炎作用。類黃酮則可能影響高劑量及低劑量β-胡蘿蔔素的作用。
並列摘要
The aims of this study were to investigate 1) the effects of beta-carotene alone or in combination with flavonoids on the inflammatory reaction of stimulated-monocytes/ macrophages; and 2) whether beta-carotene alone or in combination with quercetin affect the resistance of human lung cell line, A549 cells, toward stimulated-monocytes/ macrophages. First, HL-60 cells were pre-incubated with 2 or 20 μM beta-carotene alone or in combination with each of 20 μM flavonoid (quercetin and naringenin) or vitamin E for 1 h, followed by incubated with phorbol-12-myristate-13-acetate (PMA) or PMA +4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) for 2 days. The cultured media were collected for IL-8, TNF-α and thiobarbituric acid reactive substances (TBARS) assay. The HL-60 cells were collected to determine DNA strand breaks and the formation of cellular ROS or to be coincubated with A549 cells for 1 day. Second, A549 cells were preincubated with different concentrations of beta-carotene alone or in combination with quercetin, followed by incubated with PMA+NNK stimulated-HL-60 cells, observing the resistance of A549 cells toward inflammation. The results demonstrated that 20 μM of beta-carotene enhance the release of IL-8, TNF-α and TBARS as well as the formation of ROS in PMA or PMA+NNK-activated HL-60 cells. In addition, preincubation with 20 μM of beta-carotene, PMA+NNK stimulated-HL-60 cells induced an increase of DNA damage in A549 cells by 30% (p<0.05) as compared with the stimulated HL-60 cells without preincubation with beta-carotene. By contrast, preincubation with 2 μM of beta-carotene tended to inhibit the inflammation reaction of HL-60 cells induced by PMA or PMA+NNK. Moreover, the DNA damage in A549 cells induced by PMA+NNK-stumulated HL-60 cells with preincubation with 2 μM beta-carotene was significantly lower than that induced by stimulated-HL-60 cells without preincubation with beta-carotene. Pre-incubation of HL-60 cells with 20 μM beta-carotene in combination with each of 20 μM quercetin, naringenin or vitamin E tended to suppress the enhancing effect of high dose of beta-carotene on proinflammatory reaction and on the DNA damage of A549 cells. The effects of quercetin and naringenin were comparable to or better than that of vitamin E. Quercetin also additively enhanced the anti-inflammatory effect of 2 μM beta-carotene. Preincubation of A549 cells with 20 μM of beta-carotene tended to increase the release of proinflammatory cytokines and the DNA damage of A549 cells induced by co-incubation with stimulated-HL-60 cells. On the other hand, preincubation of 2 μM of beta-carotene tended to decrease these reactions. Co-preincubation of 20 μM quercetin with 2 μM of beta-carotene enhanced the resistance of A549 cells toward stimulated HL-60 cells. These in vitro studies demonstrated that high dose of beta-carotene may act as a pro-inflammatory agent and enhances the damage in lung epithelial cells induced by inflammation reaction, while low dose of beta-carotene may be anti-inflammatory. Concomitant presence of flavonoids may influence the effects of high and low dose of beta-carotene.