【作者】 张小梅；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2008， 博士

【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。更多还原

【Abstract】 Aim:Numerous epidemiological data have demonstrated an association between a diet rich in antioxidants, such as the“Mediterranean diet,”and a lower incidence of several diseases, such as cancers, atherosclerosis and coronary heart disease. Olive oil is the most important food of“Mediterranean diet,”and can modulate the diseases from studies in vivo and in vitro. Although the protective effect of such a diet is likely to be multifactorial, there is consistent evidence for an antioxidant activity of some selected polyphenolic compounds from extra virgin olive oil. Hydroxytyrosol (HT), an olive phenolic, chemiclally named (3, 4-dihydroxyphenyl) ethanol, is hydrosoluble and liposoluble moelcule. HT is an efficient scavenger of free radicals, which prevents the autooxidation of polyunsaturated fatty acid. The free radical scavenging activity of HT is higher than synthetic and natural antioxidant and such as vitamin E, vitamin C and butylated hydroxytoluenez (BHT).Environmental pollution includes water pollution, food contamination and air pollution, in which effects of pollutants in food on the health are not only extensive but also direct and cause widespread concern. In recent years, there are so many food contaminations that caused social panic about risk of cancer, such as Sudan I, Teflon, and acrylamide (AA) and so on. These potential carcinogenicity substances have been paid to attentions.The International Agency for Research on Cancer (IARC) assessed Sudan I as a Group 3 carcinogen. AA is neurotoxic in humans and laboratory animals, and was classified as“probably carcinogenic to humans”(Group 2A carcinogen) by a working group of the IARC. This might represent a potential threat to public health. The liver is not only the target site of Sudan I and AA metabolism but also the site of phenolic compound metabolism. HepG2 cells were used as the experimental system in vitro to investigate the chemoprotective effect of HT on the genotoxicity induced by Sduan I and AA in our study. The HepG2 cell line retained many of the functions of normal liver cells and expresses the activities of several phases I and II xenobiotic metabolizing enzymes that play key roles in the activation and/or detoxification of DNA-reactive carcinogens. It has been shown to be a suitable system for genotoxicity testing.A recent report also indicates that HT could reduce vascular cell adhesion molecule-1 mRNA expression by blocking the activation of transcription factors nuclear factor-kappaB (NF-κB) and activator protein-1. It has been demonstrated that natural and synthetic antioxidants inhibit pro-infla- mmatory gene expression regulated by transcription factors, including NF-κB, signal transducer and activator of transcription-1α(STAT-1α) and interferon regulatory factor-1(IRF-1). These transcription factors are dependent on the intracellular redox state. We selected THP-1 cells stimulated by LPS to study the anti-inflammatory effect of HT and the possible mechanisms.Methods: (1) Methods of HepG2 cells line as test system were as follows. The single cell gel electrophoresis assay (SCGE) in addition to the micronucleus test (MNT) to study the genotoxic effects was performed. The cell viability was examined using the methyl thiazol tetrazolium bromide (MTT) assay. In order to clarify the underlying mechanisms we measured the intracellular ROS formation using 2, 7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe and intracellular glutathione(GSH) level by fluorometric methods. The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (γ-GCS), and western blot forγ-GCS was applied in present study.(2) The THP-1 cell was stimulated by LPS as the inflammtory model. ELISA was used to detect the level of tumor necrosis factor-α(TNF-α). The gene expression of TNF-α, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by RT-PCR and the protein expression of iNOS and COX-2 was estimated by Western blot. To futher study the the relation between the anti-inflammtory effect of HT and intracellular redox state, intracellular GSH andγ-GCS were measured.Results: (1) The chemoprotective effects of HT on the genotoxicity induced by Sudan I and AA were as follows. We found that HepG2 cells treated with 100 uM Sudan I resulted in serious DNA strand breaks. In contrast, the DNA damage was significantly reduced in cells pretreatment with 25-100 uM HT in a concentration-dependent manner. The results of MNT showed that HepG2 cells treated with 100 uM Sduan I could induce the decrease of GSH and the increase of ROS, intracellular TBARS level and the increase of 8-OHdG expression. Pretreatment with HT could increase the level of GSH and decrease the level of ROS, TBARS and 8-OHdG in a concentration-dependent manner. Moreover, high dose of HT (100μM) could completely inhibit the increase the levels of ROS, TBARS and 8-OHdG. Pretreatment with HT could inhibit the cytotoxicity induced by 5mM and 10mM AA. The SCGE results showed that HepG2 cells treated with 10mM resulted in serious DNA damage. Pretreatment with doses of HT for 30 min then exposed to 10mM inhibited AA-induced DNA damage in a concentration-dependent manner. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 2.5 mM AA for 24 h. Pretreatment with doses of HT for 30 min decrease the frequencies of MN in a concentration-independent manner. Furthermore, HT was able to reduce intracellular ROS formation and attenuate GSH depletion caused by AA in a concentration-dependent manner. The futher study showed that 25μM HT enhanced the expression ofγ-GCS in HepG2 cells treated with 10 mM AA using immmnoblotting.(2) The effects of HT on the inflammation stimulated by LPS were as follows. HT could significantly decrease the increase of TNF-αlevel stimulated by LPS and inhibit the increases of iNOS and COX-2 gene expression stimulated by LPS. HT also could significantly decrease the levels of increase of iNOS and COX-2 protein expression stimulated by LPS. The level of GSH in THP-1 cells stimulated by LPS was significantly decreased and the level ofγ-GCS was significantly increased as compared to cells without LPS, and pretreatment with HT could increase the level of GSH and enhanced the level ofγ-GCS expression in a concentration-dependent manner.Conclusions: In the study, we are first to investigate the chemoprotective of HT on genotoxicity induced by Sudan I and AA in HepG2 cells. We found HT could decrease the genotoxicity induced by Sudan I and AA in HepG2 cells. Moreover, we found that HT could modulate the redox state and prevent the oxidative damage by decreasing the level of ROS and increasing the level of GSH to attenuate the the genotoxicity in HepG2 cells induced by Sudan I and AA. In addition, HT could inhibit the inflammtory response in THP-1 cells stimulated by LPS. It suggested that HT could inhibit inflammtion in THP-1 cells stimulated by LPS through decreasing the gene expression of the inflammtion-relative cytokines, which was related to increase of GSH and the enhancement ofγ-GCS expression.更多还原