羟基酪醇生物学作用的细胞与分子机制研究
发布时间：2018-08-20 15:41
【摘要】： 前言:富含抗氧化物质的“地中海膳食”可降低癌症、动脉粥样硬化、心血管疾病以及炎症等疾患的发病率。橄榄油是地中海膳食的重要食物。近年来研究者关注较多的羟基酪醇(hydroxytyrosol)是从橄榄中提取的一种酚类物质,化学名为3,4,-二羟基苯基乙醇,属两性分子,既脂溶也水溶。羟基酪醇有很强的抗氧化作用,能阻止多不饱和脂肪酸的自氧化,羟基酪醇对自由基的清除能力比其他合成的和天然的抗氧化物质都高,可有效地清除内源性和外源性的自由基和氧化物,包括过氧化氢、超氧化物阴离子以及次氯酸等。 环境介质(水、食物、空气)中食物被污染对人类健康的危害不仅广泛而且直接,因而备受关注。近年来,陆续报道了多起食物污染而引起社会对致癌危险的恐慌。例如,食品染料苏丹红I号(Sudan I)事件,不粘锅特富龙事件,和油炸薯条丙烯酰胺(Acrylamide,AA)事件。这些遗传物质的潜在致癌性受到广泛重视。 国际癌症研究机构(International Agency for Research on Cancer, IARC)将Sudan I归类为第三类致癌物,这类物质虽缺乏足够的使人类致癌的证据,但它的遗传毒性使其具有潜在的致癌危险。IARC将AA划分为2A类的致癌物,即很可能对人类致癌的物质,因此对人类也具有潜在的致癌危险。肝脏是Sudan I和AA的代谢器官,同时也是酚类化合物的代谢场所;本研究选用人类来源的肝脏肿瘤细胞HepG2细胞系作为体外试验系统,研究羟基酪醇对Sudan I和AA所致的遗传毒性的化学预防作用及可能机制。HepG2细胞来源于人类肝胚细胞瘤,所含生物转化代谢酶与人正常肝实质细胞具有同源性。因其保留了较完整的生物转化代谢I相和II相酶,是检测外来化学物遗传毒性的理想细胞系。 最近有研究还发现,羟基酪醇能阻止核转录因子(NF-kB)和蛋白激酶-1的活化以致降低血管细胞黏附因子-1的基因转录。天然的和合成的抗氧化剂能通过调控转录因子,包括转录因子核因子(NF-kB)、信号传导以及转录激活剂-1α(STAT- 1α)以及干扰素调控因子(IRF-1),抑制促炎因子的基因表达。转录因子的活性依赖于细胞内的氧化还原状态。因此,本研究采用人的单核细胞系THP-1细胞,以脂多糖(LPS)刺激THP-1细胞产生炎症,来探讨羟基酪醇抗炎症的作用及可能机制。 方法:(1)以HepG2细胞系作为试验系统的试验:采用单细胞凝胶电泳(SCGE)试验和微核试验(MNT)分别检测细胞DNA损伤和染色体损伤。用噻唑蓝(MTT)法检测细胞存活率。为探讨机制,以2’,7’—二氢二氯荧光素二乙酸酯(DCFH-DA)为荧光探针检测细胞内活性氧(ROS)水平;以邻苯二甲醛荧光素(OPT)比色法测定细胞内还原型谷胱甘肽(GSH)水平;用硫代巴比妥酸反应物(TBARS)测定法检测细胞内脂质过氧化水平;以免疫组化方法检测细胞内8-羟基脱氧鸟苷(8-OHdG)的表达水平,以Western blot法检测细胞内的谷氨酰半胱氨酸合成酶(r-GCS)表达水平。 (2)采用THP-1细胞系作为炎症模型的试验:以LPS刺激THP-1制作炎症模型。以ELISA法测定肿瘤坏死因子α(TNF-α)水平;以RT-PCR法检测诱导性一氧化氮合酶(iNOS)和环氧合酶-2(COX2)以及TNF-α的基因表达以及Western blot法检测iNOS和COX2蛋白表达。为进一步探讨羟基酪醇的抗炎症作用与细胞的氧化还原电位关系,以OPT比色法测定细胞内GSH水平以及以Western blot测定r-GCS的蛋白表达水平。结果:(1)对Sudan I和AA遗传毒性的影响。100 uM Sudan I引起HepG2细胞的DNA链断裂程度以及微核形成率较对照组明显增加;不同浓度的羟基酪醇(0、25、50、100 uM)预处理HepG2细胞30min后,再加入100 uM Sudan I后,羟基酪醇预处理组的DNA链断裂程度以及MN形成率较单独接触Sudan I组明显减轻,并且存在剂量依赖关系。进一步研究发现,100 uM Sudan I能引起HepG2细胞的ROS水平明显升高、细胞内GSH水平明显降低、细胞内TBARS形成明显增多及8-OHdG表达水平明显增强。羟基酪醇预处理后再接触Sudan I,细胞内的GSH水平较单独接触Sudan I组明显升高;而ROS水平、细胞内TBARS及8-OHdG表达水平较单独接触Sudan I组明显降低,并且高浓度的羟基酪醇(100uM)几乎完全抑制上述各项指标的升高。不同浓度的羟基酪醇预处理30 min再接触AA,能明显降低单独接触5mM和10mMAA所致的细胞毒性。不同浓度的羟基酪醇(0、25、50、100um)预处理HepG2细胞30min,然后与10mMAA温育1h,结果显示SCGE试验各项指标明显减轻,提示DNA链断裂减轻并呈剂量依赖关系。利用MNT检测羟基酪醇对AA所致微核形成率的影响,结果发现,AA致HepG2微核形成率明显增高;羟基酪醇预处理再接触AA能降低由AA所引起的HepG2细胞微核形成率的增高。在羟基酪醇的试验浓度范围内,这种抑制作用呈剂量依赖关系,即羟基酪醇浓度越大,抑制作用越强。进一步研究发现,羟基酪醇能明显降低AA所致的细胞内的ROS水平、阻止AA所致的GSH的降低,并呈现剂量依赖关系;Western blot结果显示,25uM羟基酪醇能明显增强AA所致的r-GCS蛋白表达的降低。 (2)对LPS诱导THP-1细胞炎症的影响。羟基酪醇能明显降低LPS诱导THP-1细胞分泌的TNF-α增多,抑制iNOS以及COX-2基因表达和蛋白表达水平增高。本研究还发现,LPS刺激的THP-1细胞内GSH水平和r-GCS蛋白表达水平较对照组明显降低;羟基酪醇预处理再用LPS刺激THP-1细胞后,细胞内GSH水平和r-GCS蛋白表达水平明显增加,并且存在剂量依赖关系。 结论:本文首次利用HepG2细胞研究羟基酪醇对Sudan I和AA遗传毒性的影响,结果显示,羟基酪醇能够降低由Sudan I和AA所致的遗传毒性;羟基酪醇降低细胞内的ROS水平和升高GSH水平,从而调控氧化应激状态,预防氧化性DNA损伤,可能是其防护Sudan I和AA的遗传毒性的机制。羟基酪醇能明显抑制LPS刺激的炎症反应,可能机制是通过增强细胞r-GCS蛋白表达而增加细胞内GSH水平,从而降低了炎症相关因子的基因表达致使炎症减轻。
[Abstract]:Olive oil is an important food in the Mediterranean diet. In recent years, researchers have paid more attention to hydroxytyrosol, a phenolic substance extracted from olives. Its chemical name is 3,4. Dihydroxyphenyl ethanol, an amphoteric molecule, is both lipid-soluble and water-soluble. Hydroxytyrosol has a strong antioxidant effect and can prevent the autoxidation of polyunsaturated fatty acids. Hydroxytyrosol has a higher free radical scavenging capacity than other synthetic and natural antioxidants. It can effectively scavenge endogenous and exogenous free radicals and oxides. It includes hydrogen peroxide, superoxide anion and hypochlorite.
Food contamination in environmental media (water, food, air) is not only a widespread and direct hazard to human health, so it has attracted much attention. In recent years, a number of food contaminations have been reported, causing social panic about the risk of cancer. For example, the food dye Sudan I (Sudan I) incident, the non-stick pot Teflon incident, and the fried potato acrylic acid. Amine (Acrylamide, AA) events. The potential carcinogenicity of these genetic materials has attracted wide attention.
The International Agency for Research on Cancer (IARC) classifies Sudan I as a third class of carcinogens. Although there is insufficient evidence for carcinogens in humans, it is genetically toxic and potentially carcinogenic. The liver is the metabolic organ of Sudan I and A A, and also the metabolic site of phenolic compounds. In this study, human hepatoma cell line HepG2 was used as an in vitro experimental system to study the chemopreventive effect and possible mechanism of hydroxy tyrosol on the genetic toxicity of Sudan I and A A. HepG2 cells are derived from human hepatic embryo cell tumors and have the homology with human normal hepatic parenchymal cells. It is an ideal cell line for detecting the genotoxicity of exogenous chemicals because it retains relatively complete enzymes of biotransformation metabolism phase I and phase II.
Natural and synthetic antioxidants can regulate transcription factors, including transcription factor nuclear factor (NF-kB), signal transduction and activator of transcription-1 alpha (STAT-1 alpha), and so on. Interferon-regulated factor-1 (IRF-1) inhibits the expression of pro-inflammatory factors. The activity of transcription factors depends on the redox state of cells. Therefore, this study used human monocyte THP-1 cells, lipopolysaccharide (LPS) stimulated THP-1 cells to produce inflammation, to explore the anti-inflammatory effect of hydroxybutyrol and its possible mechanism.
Methods: (1) Using HepG2 cell line as the experimental system, single cell gel electrophoresis (SCGE) and micronucleus assay (MNT) were used to detect DNA damage and chromosome damage respectively. Cell viability was measured by MTT assay. To explore the mechanism, 2', 7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect fine cells. Intracellular reactive oxygen species (ROS) level; intracellular reduced glutathione (GSH) level was measured by phthalaldehyde fluorescein (OPT) colorimetry; intracellular lipid peroxidation level was measured by thiobarbituric acid reactant (TBARS); intracellular 8-hydroxydeoxyguanosine (8-OHdG) expression level was detected by immunohistochemistry and Western blot. The expression level of glutaminyl cysteine synthetase (r-GCS) in cells was detected.
(2) THP-1 cell line was used as the model of inflammation: the model of inflammation was made by stimulating THP-1 with LPS; the levels of tumor necrosis factor-alpha (TNF-alpha) were measured by ELISA; the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2) and TNF-alpha were detected by RT-PCR; and the expressions of iNOS and COX2 were detected by Western blot. To investigate the relationship between the anti-inflammatory effect of hydroxy-tyrosol and the redox potential of HepG2 cells, the levels of GSH and the expression of r-GCS were determined by OPT colorimetry and Western blot respectively. After pretreatment of HepG2 cells with different concentrations of hydroxy-tyrosol (0,25,50,100 uM) for 30 minutes and adding 100 uM Sudan I, the degree of DNA strand breakage and MN formation in the hydroxy-tyrosol pretreated HepG2 cells were significantly reduced compared with those in the single exposure group. The levels of ROS, GSH, TBARS and 8-OHdG were significantly higher in the cells pretreated with hydroxy-tyrosol than those in the cells exposed to Sudan I, while the levels of ROS, TBARS and 8-OHdG were significantly higher in the cells pretreated with hydroxy-tyrosol than those in the cells exposed to Sudan I alone. After pretreatment with different concentrations of hydroxy-tyrosol for 30 minutes, exposure to AA could significantly reduce the cytotoxicity induced by exposure to 5mM and 10mMAA alone. HepG2 cells were pretreated with different concentrations of hydroxy-tyrosol (0,25,50,100um) for 30 minutes, then with 10mMAA. After incubation for 1 h, the results showed that the indexes of SCGE test were significantly reduced, suggesting that DNA strand breakage was reduced in a dose-dependent manner. MNT was used to detect the effect of hydroxy tyrosol on the micronucleus formation rate induced by AA. The results showed that the micronucleus formation rate of HepG2 cells induced by AA was significantly increased. The micronucleus formation of HepG2 cells induced by AA could be reduced by hydroxy tyrosol pretreatment and re-exposure to AA. In the range of hydroxy tyrosol concentration, the inhibitory effect was dose-dependent, that is, the higher the concentration of hydroxy tyrosol, the stronger the inhibitory effect. The results showed that 25uM hydroxytyramol could significantly enhance the decrease of r-GCS protein expression induced by AA.
(2) The effect of hydroxy-tyrosol on LPS-induced THP-1 cell inflammation. Hydroxytyrosol could significantly reduce the increase of TNF-a secreted by LPS-induced THP-1 cells, inhibit the expression of iNOS and COX-2 gene and protein, and the expression of GSH and r-GCS protein in LPS-stimulated THP-1 cells was significantly lower than that in control group. After stimulating THP-1 cells with LPS, the levels of GSH and r-GCS protein in THP-1 cells increased significantly, and there was a dose-dependent relationship.
CONCLUSION: Hydroxytyrosol can reduce the genotoxicity induced by Sudan I and A A, decrease the ROS level and increase the GSH level in HepG2 cells, thereby regulating the oxidative stress state and preventing oxidative DNA damage, which may be protective against Su. The mechanism of genetic toxicity of Dan I and AA. Hydroxytyrosol can significantly inhibit LPS-stimulated inflammation, possibly by enhancing the expression of r-GCS protein and increasing the level of GSH in cells, thereby reducing the expression of inflammatory related factors and alleviating inflammation.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
本文编号：2194152
[Abstract]:Olive oil is an important food in the Mediterranean diet. In recent years, researchers have paid more attention to hydroxytyrosol, a phenolic substance extracted from olives. Its chemical name is 3,4. Dihydroxyphenyl ethanol, an amphoteric molecule, is both lipid-soluble and water-soluble. Hydroxytyrosol has a strong antioxidant effect and can prevent the autoxidation of polyunsaturated fatty acids. Hydroxytyrosol has a higher free radical scavenging capacity than other synthetic and natural antioxidants. It can effectively scavenge endogenous and exogenous free radicals and oxides. It includes hydrogen peroxide, superoxide anion and hypochlorite.
Food contamination in environmental media (water, food, air) is not only a widespread and direct hazard to human health, so it has attracted much attention. In recent years, a number of food contaminations have been reported, causing social panic about the risk of cancer. For example, the food dye Sudan I (Sudan I) incident, the non-stick pot Teflon incident, and the fried potato acrylic acid. Amine (Acrylamide, AA) events. The potential carcinogenicity of these genetic materials has attracted wide attention.
The International Agency for Research on Cancer (IARC) classifies Sudan I as a third class of carcinogens. Although there is insufficient evidence for carcinogens in humans, it is genetically toxic and potentially carcinogenic. The liver is the metabolic organ of Sudan I and A A, and also the metabolic site of phenolic compounds. In this study, human hepatoma cell line HepG2 was used as an in vitro experimental system to study the chemopreventive effect and possible mechanism of hydroxy tyrosol on the genetic toxicity of Sudan I and A A. HepG2 cells are derived from human hepatic embryo cell tumors and have the homology with human normal hepatic parenchymal cells. It is an ideal cell line for detecting the genotoxicity of exogenous chemicals because it retains relatively complete enzymes of biotransformation metabolism phase I and phase II.
Natural and synthetic antioxidants can regulate transcription factors, including transcription factor nuclear factor (NF-kB), signal transduction and activator of transcription-1 alpha (STAT-1 alpha), and so on. Interferon-regulated factor-1 (IRF-1) inhibits the expression of pro-inflammatory factors. The activity of transcription factors depends on the redox state of cells. Therefore, this study used human monocyte THP-1 cells, lipopolysaccharide (LPS) stimulated THP-1 cells to produce inflammation, to explore the anti-inflammatory effect of hydroxybutyrol and its possible mechanism.
Methods: (1) Using HepG2 cell line as the experimental system, single cell gel electrophoresis (SCGE) and micronucleus assay (MNT) were used to detect DNA damage and chromosome damage respectively. Cell viability was measured by MTT assay. To explore the mechanism, 2', 7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect fine cells. Intracellular reactive oxygen species (ROS) level; intracellular reduced glutathione (GSH) level was measured by phthalaldehyde fluorescein (OPT) colorimetry; intracellular lipid peroxidation level was measured by thiobarbituric acid reactant (TBARS); intracellular 8-hydroxydeoxyguanosine (8-OHdG) expression level was detected by immunohistochemistry and Western blot. The expression level of glutaminyl cysteine synthetase (r-GCS) in cells was detected.
(2) THP-1 cell line was used as the model of inflammation: the model of inflammation was made by stimulating THP-1 with LPS; the levels of tumor necrosis factor-alpha (TNF-alpha) were measured by ELISA; the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2) and TNF-alpha were detected by RT-PCR; and the expressions of iNOS and COX2 were detected by Western blot. To investigate the relationship between the anti-inflammatory effect of hydroxy-tyrosol and the redox potential of HepG2 cells, the levels of GSH and the expression of r-GCS were determined by OPT colorimetry and Western blot respectively. After pretreatment of HepG2 cells with different concentrations of hydroxy-tyrosol (0,25,50,100 uM) for 30 minutes and adding 100 uM Sudan I, the degree of DNA strand breakage and MN formation in the hydroxy-tyrosol pretreated HepG2 cells were significantly reduced compared with those in the single exposure group. The levels of ROS, GSH, TBARS and 8-OHdG were significantly higher in the cells pretreated with hydroxy-tyrosol than those in the cells exposed to Sudan I, while the levels of ROS, TBARS and 8-OHdG were significantly higher in the cells pretreated with hydroxy-tyrosol than those in the cells exposed to Sudan I alone. After pretreatment with different concentrations of hydroxy-tyrosol for 30 minutes, exposure to AA could significantly reduce the cytotoxicity induced by exposure to 5mM and 10mMAA alone. HepG2 cells were pretreated with different concentrations of hydroxy-tyrosol (0,25,50,100um) for 30 minutes, then with 10mMAA. After incubation for 1 h, the results showed that the indexes of SCGE test were significantly reduced, suggesting that DNA strand breakage was reduced in a dose-dependent manner. MNT was used to detect the effect of hydroxy tyrosol on the micronucleus formation rate induced by AA. The results showed that the micronucleus formation rate of HepG2 cells induced by AA was significantly increased. The micronucleus formation of HepG2 cells induced by AA could be reduced by hydroxy tyrosol pretreatment and re-exposure to AA. In the range of hydroxy tyrosol concentration, the inhibitory effect was dose-dependent, that is, the higher the concentration of hydroxy tyrosol, the stronger the inhibitory effect. The results showed that 25uM hydroxytyramol could significantly enhance the decrease of r-GCS protein expression induced by AA.
(2) The effect of hydroxy-tyrosol on LPS-induced THP-1 cell inflammation. Hydroxytyrosol could significantly reduce the increase of TNF-a secreted by LPS-induced THP-1 cells, inhibit the expression of iNOS and COX-2 gene and protein, and the expression of GSH and r-GCS protein in LPS-stimulated THP-1 cells was significantly lower than that in control group. After stimulating THP-1 cells with LPS, the levels of GSH and r-GCS protein in THP-1 cells increased significantly, and there was a dose-dependent relationship.
CONCLUSION: Hydroxytyrosol can reduce the genotoxicity induced by Sudan I and A A, decrease the ROS level and increase the GSH level in HepG2 cells, thereby regulating the oxidative stress state and preventing oxidative DNA damage, which may be protective against Su. The mechanism of genetic toxicity of Dan I and AA. Hydroxytyrosol can significantly inhibit LPS-stimulated inflammation, possibly by enhancing the expression of r-GCS protein and increasing the level of GSH in cells, thereby reducing the expression of inflammatory related factors and alleviating inflammation.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
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