羥基酪醇生物學(xué)作用的細(xì)胞與分子機制研究
發(fā)布時間：2018-08-20 15:41
【摘要】： 前言:富含抗氧化物質(zhì)的“地中海膳食”可降低癌癥、動脈粥樣硬化、心血管疾病以及炎癥等疾患的發(fā)病率。橄欖油是地中海膳食的重要食物。近年來研究者關(guān)注較多的羥基酪醇(hydroxytyrosol)是從橄欖中提取的一種酚類物質(zhì),化學(xué)名為3,4,-二羥基苯基乙醇,屬兩性分子,既脂溶也水溶。羥基酪醇有很強的抗氧化作用,能阻止多不飽和脂肪酸的自氧化,羥基酪醇對自由基的清除能力比其他合成的和天然的抗氧化物質(zhì)都高,可有效地清除內(nèi)源性和外源性的自由基和氧化物,包括過氧化氫、超氧化物陰離子以及次氯酸等。 環(huán)境介質(zhì)(水、食物、空氣)中食物被污染對人類健康的危害不僅廣泛而且直接,因而備受關(guān)注。近年來,陸續(xù)報道了多起食物污染而引起社會對致癌危險的恐慌。例如,食品染料蘇丹紅I號(Sudan I)事件,不粘鍋特富龍事件,和油炸薯條丙烯酰胺(Acrylamide,AA)事件。這些遺傳物質(zhì)的潛在致癌性受到廣泛重視。 國際癌癥研究機構(gòu)(International Agency for Research on Cancer, IARC)將Sudan I歸類為第三類致癌物,這類物質(zhì)雖缺乏足夠的使人類致癌的證據(jù),但它的遺傳毒性使其具有潛在的致癌危險。IARC將AA劃分為2A類的致癌物,即很可能對人類致癌的物質(zhì),因此對人類也具有潛在的致癌危險。肝臟是Sudan I和AA的代謝器官,同時也是酚類化合物的代謝場所;本研究選用人類來源的肝臟腫瘤細(xì)胞HepG2細(xì)胞系作為體外試驗系統(tǒng),研究羥基酪醇對Sudan I和AA所致的遺傳毒性的化學(xué)預(yù)防作用及可能機制。HepG2細(xì)胞來源于人類肝胚細(xì)胞瘤,所含生物轉(zhuǎn)化代謝酶與人正常肝實質(zhì)細(xì)胞具有同源性。因其保留了較完整的生物轉(zhuǎn)化代謝I相和II相酶,是檢測外來化學(xué)物遺傳毒性的理想細(xì)胞系。 最近有研究還發(fā)現(xiàn),羥基酪醇能阻止核轉(zhuǎn)錄因子(NF-kB)和蛋白激酶-1的活化以致降低血管細(xì)胞黏附因子-1的基因轉(zhuǎn)錄。天然的和合成的抗氧化劑能通過調(diào)控轉(zhuǎn)錄因子,包括轉(zhuǎn)錄因子核因子(NF-kB)、信號傳導(dǎo)以及轉(zhuǎn)錄激活劑-1α(STAT- 1α)以及干擾素調(diào)控因子(IRF-1),抑制促炎因子的基因表達(dá)。轉(zhuǎn)錄因子的活性依賴于細(xì)胞內(nèi)的氧化還原狀態(tài)。因此,本研究采用人的單核細(xì)胞系THP-1細(xì)胞,以脂多糖(LPS)刺激THP-1細(xì)胞產(chǎn)生炎癥,來探討羥基酪醇抗炎癥的作用及可能機制。 方法:(1)以HepG2細(xì)胞系作為試驗系統(tǒng)的試驗:采用單細(xì)胞凝膠電泳(SCGE)試驗和微核試驗(MNT)分別檢測細(xì)胞DNA損傷和染色體損傷。用噻唑藍(lán)(MTT)法檢測細(xì)胞存活率。為探討機制,以2’,7’—二氫二氯熒光素二乙酸酯(DCFH-DA)為熒光探針檢測細(xì)胞內(nèi)活性氧(ROS)水平;以鄰苯二甲醛熒光素(OPT)比色法測定細(xì)胞內(nèi)還原型谷胱甘肽(GSH)水平;用硫代巴比妥酸反應(yīng)物(TBARS)測定法檢測細(xì)胞內(nèi)脂質(zhì)過氧化水平;以免疫組化方法檢測細(xì)胞內(nèi)8-羥基脫氧鳥苷(8-OHdG)的表達(dá)水平,以Western blot法檢測細(xì)胞內(nèi)的谷氨酰半胱氨酸合成酶(r-GCS)表達(dá)水平。 (2)采用THP-1細(xì)胞系作為炎癥模型的試驗:以LPS刺激THP-1制作炎癥模型。以ELISA法測定腫瘤壞死因子α(TNF-α)水平;以RT-PCR法檢測誘導(dǎo)性一氧化氮合酶(iNOS)和環(huán)氧合酶-2(COX2)以及TNF-α的基因表達(dá)以及Western blot法檢測iNOS和COX2蛋白表達(dá)。為進(jìn)一步探討羥基酪醇的抗炎癥作用與細(xì)胞的氧化還原電位關(guān)系,以O(shè)PT比色法測定細(xì)胞內(nèi)GSH水平以及以Western blot測定r-GCS的蛋白表達(dá)水平。結(jié)果:(1)對Sudan I和AA遺傳毒性的影響。100 uM Sudan I引起HepG2細(xì)胞的DNA鏈斷裂程度以及微核形成率較對照組明顯增加;不同濃度的羥基酪醇(0、25、50、100 uM)預(yù)處理HepG2細(xì)胞30min后,再加入100 uM Sudan I后,羥基酪醇預(yù)處理組的DNA鏈斷裂程度以及MN形成率較單獨接觸Sudan I組明顯減輕,并且存在劑量依賴關(guān)系。進(jìn)一步研究發(fā)現(xiàn),100 uM Sudan I能引起HepG2細(xì)胞的ROS水平明顯升高、細(xì)胞內(nèi)GSH水平明顯降低、細(xì)胞內(nèi)TBARS形成明顯增多及8-OHdG表達(dá)水平明顯增強。羥基酪醇預(yù)處理后再接觸Sudan I,細(xì)胞內(nèi)的GSH水平較單獨接觸Sudan I組明顯升高;而ROS水平、細(xì)胞內(nèi)TBARS及8-OHdG表達(dá)水平較單獨接觸Sudan I組明顯降低,并且高濃度的羥基酪醇(100uM)幾乎完全抑制上述各項指標(biāo)的升高。不同濃度的羥基酪醇預(yù)處理30 min再接觸AA,能明顯降低單獨接觸5mM和10mMAA所致的細(xì)胞毒性。不同濃度的羥基酪醇(0、25、50、100um)預(yù)處理HepG2細(xì)胞30min,然后與10mMAA溫育1h,結(jié)果顯示SCGE試驗各項指標(biāo)明顯減輕,提示DNA鏈斷裂減輕并呈劑量依賴關(guān)系。利用MNT檢測羥基酪醇對AA所致微核形成率的影響,結(jié)果發(fā)現(xiàn),AA致HepG2微核形成率明顯增高;羥基酪醇預(yù)處理再接觸AA能降低由AA所引起的HepG2細(xì)胞微核形成率的增高。在羥基酪醇的試驗濃度范圍內(nèi),這種抑制作用呈劑量依賴關(guān)系,即羥基酪醇濃度越大,抑制作用越強。進(jìn)一步研究發(fā)現(xiàn),羥基酪醇能明顯降低AA所致的細(xì)胞內(nèi)的ROS水平、阻止AA所致的GSH的降低,并呈現(xiàn)劑量依賴關(guān)系;Western blot結(jié)果顯示,25uM羥基酪醇能明顯增強AA所致的r-GCS蛋白表達(dá)的降低。 (2)對LPS誘導(dǎo)THP-1細(xì)胞炎癥的影響。羥基酪醇能明顯降低LPS誘導(dǎo)THP-1細(xì)胞分泌的TNF-α增多,抑制iNOS以及COX-2基因表達(dá)和蛋白表達(dá)水平增高。本研究還發(fā)現(xiàn),LPS刺激的THP-1細(xì)胞內(nèi)GSH水平和r-GCS蛋白表達(dá)水平較對照組明顯降低;羥基酪醇預(yù)處理再用LPS刺激THP-1細(xì)胞后,細(xì)胞內(nèi)GSH水平和r-GCS蛋白表達(dá)水平明顯增加,并且存在劑量依賴關(guān)系。 結(jié)論:本文首次利用HepG2細(xì)胞研究羥基酪醇對Sudan I和AA遺傳毒性的影響,結(jié)果顯示,羥基酪醇能夠降低由Sudan I和AA所致的遺傳毒性;羥基酪醇降低細(xì)胞內(nèi)的ROS水平和升高GSH水平,從而調(diào)控氧化應(yīng)激狀態(tài),預(yù)防氧化性DNA損傷,可能是其防護(hù)Sudan I和AA的遺傳毒性的機制。羥基酪醇能明顯抑制LPS刺激的炎癥反應(yīng),可能機制是通過增強細(xì)胞r-GCS蛋白表達(dá)而增加細(xì)胞內(nèi)GSH水平,從而降低了炎癥相關(guān)因子的基因表達(dá)致使炎癥減輕。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R363
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