【作者】 姚平；

【导师】 孙秀发； 刘烈刚；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2005， 博士

【摘要】 酒精滥用引发的多器官、多系统功能障碍和严重的精神及社会问题，已引起世界各国的普遍关注，成为全球范围内重要公共卫生问题。酒精作为兼具亲脂和亲水双重特性的高渗透性小分子和强烈的氧化应激源，在机体代谢过程中产生大量的自由基与活性氧，导致脂质过氧化、蛋白质氧化、DNA损伤和加合物的形成，是酒精引起肝脏及多器官损伤的重要机制。然而，对于酒精性氧化损伤的防治，孤立地依赖于传统的抗氧化剂，临床上并没有取得预期效果，而内源性抗氧化系统的激活，可能才是防治酒精性氧化损伤的关键。在机体各种内源性抗氧化蛋白中，Ⅰ型血红素氧化酶(HO-1)因其良好的可诱导性、强大的抗氧化活性在沉寂数十年后受到了空前的关注，HO-1的诱导对于多种疾病和应激性损伤具有良好的保护效应，甚至被认为是防御氧化应激的首选靶基因。然而，“经典”的HO-1诱导剂如血红素(heme)、钴原卟啉(cobalt protoporphyrin，CoPP)等具有潜在的过氧化毒性，基因转染也存在安全和效率等方面的问题，使HO-1的诱导仅限于实验室研究而难以得到实际应用。近年来人们兴奋地发现，一些天然的黄酮类或多酚类抗氧化剂，如姜黄素、槲皮素、银杏黄酮等可诱导HO-1表达，并由此展示出协同而增强的抗氧化效应和更为广泛的药理活性。有鉴如此，本研究以具有优良抗氧化和护肝效应的银杏黄酮作为干预物，采用酶学与生化分析、RT-PCR、流式细胞术、Western Blot、病理及超微病理等研究手段，通过动物试验，系统探讨急、慢性酒精暴露及银杏黄酮干预对血清和肝、心、肾、睾丸抗氧化系统及血清脂质水平的影响，在此基础上，以肝损伤为重点，进一步研究长期酒精摄入与不同剂量银杏黄酮干预对肝脏结构与功能、线粒体与微粒体抗氧化系统以及HO-1 mRNA、蛋白表达与酶活性(HO-1及CYP 2E1)水平的影响，主要内容如下：第一部分银杏黄酮对小鼠急性酒精暴露后抗氧化系统的保护作用尽管对酒精性损伤的氧化应激机制己开展了大量研究，但基于生物体的复杂性，乙醇摄入对机体抗氧化系统的具体影响至今报道不一。因此，本部分采用酶学与生化分析手段，探讨小鼠急性酒精暴露及银杏黄酮干预后血清与肝脏抗氧化系统的变化，以探寻相应的时间-效应关系。实验结果显示：小鼠急性酒精暴露(4.8 g／kg)后1～15h内，血清与肝脏GSH水平与抗氧化酶活性呈“U”形变化，而MDA水平呈明显的倒“U”形变化，且恢复较慢。其中血清GSH水平与SOD活性在2h降至最低，GSH-Px活性4h最低，降低幅度分别为23.5％、20.3％和20.2％，MDA水平4h最高(升幅32.4％)；肝脏GSH水平与GSH-Px、SOD活性在4h降至最低，降低幅度分别为29.3％、18.4％和23.6％，而MDA水平在6h升至最高(升幅27.5％)。预先经银杏黄酮(96 mg／kg)干预，可增强机体在急性乙醇暴露初期的代偿反应，减轻小鼠酒精摄入所致的GSH消耗、抗氧化酶失活和脂质过氧化，促使受损的抗氧化系统尽快恢复。以上结果表明，小鼠急性酒精暴露后2～6h，尤其是4h，可明显影响到血清与肝脏的抗氧化水平，而银杏黄酮干预可在一定程度上缓解这种影响而表现出良好的保护效应。第二部分大鼠酒精摄入后血清抗氧化系统与脂质水平的动态变化及银杏黄酮的保护效应流行病学调查资料显示，饮酒量不同，心血管疾病与糖尿病的发生风险也各异，提示不同水平的酒精摄入可能对机体健康有不同的影响。因此，本部分以大鼠为研究对象，探讨不同剂量的酒精持续摄入不同时间后，血清抗氧化水平与脂质水平的动态变化，在此基础上，研究不同剂量的黄酮干预对不利变化的影响。实验结果显示：大鼠摄入低剂量的酒精(0.8g／kg)30、60、90天后，血清GSH、MDA、TG与TC水平无明显变化，但SOD、CAT与GSH-Px活性则不断升高，HDL-C在90天明显升高；大鼠摄入中剂量的酒精(1.6g／kg)30、60、90天后，血清GSH水平逐渐下降，SOD与GSH-Px活性先升高后下降，CAT活性与MDA、TG、TC水平则持续升高，HDL-C轻微升高后逐渐回落；大鼠摄入高剂量的酒精(2.4g／kg) 30、60、90天后，血清GSH与HDL-C水平及SOD、GSH-Px活性不断下降，CAT活性轻微升高后明显下降，而MDA、TG、TC水平不断上升。对高剂量酒精摄入大鼠进行银杏黄酮(48及96mg／kg)干预，尤其高剂量(96mg／kg)干预，在不同时间均可部分抑制或降低酒精所致的抗氧化酶失活程度、GSH与HDL-C水平的下降及TG、TC与MDA水平的上升幅度，缓解高剂量酒精摄入对血清脂质水平及抗氧化系统的影响。以上结果表明，低剂量酒精摄入，可在一定程度上激活机体的抗氧化系统和升高HDL-C水平，从而产生相应的保护效应；中剂量的酒精摄入，尤其是长时间摄入后，血清脂质水平及过氧化程度不断升高，抗氧化水平总体呈先轻微升高后明显下降的趋势，氧化应激不断累积和加重；高剂量酒精摄入则进一步加重机体的氧化应激和脂质代谢紊乱程度。对高剂量酒精摄入大鼠进行银杏黄酮干预，尤其是高剂量银杏黄酮干预，可以缓解酒精的影响，显示出明显的调节血脂和拮抗酒精性氧化应激的作用。第三部分长期酒精摄入对大鼠组织抗氧化系统的影响及银杏黄酮的干预作用长期嗜酒不仅严重影响到肝功能，还可诱发心肌受损、肾功能不全、睾丸生精异常等多器官的病变和损伤。乙醇的高度弥散性及在代谢过程中产生过量的自由基与活性氧，继而引起多组织的氧化损伤，是酒精相关性疾病的重要发病机制。因此，本部分在观察长期高剂量的酒精摄入对肝、心、肾、睾丸抗氧化系统影响的基础上，进一步探讨银杏黄酮干预对酒精诱导的多组织氧化损伤的保护作用。实验结果显示：大鼠摄入高剂量酒精(2.4g／kg)90天后，除肾SOD及心CAT活性无明显变化外，肝、心、肾、睾丸GSH水平、抗氧化酶活性及总抗氧化能力明显下降，而MDA与ROS水平明显升高。经不同剂量的银杏黄酮干预(48及96mg／kg)，尤其是高剂量(96mg／kg)干预，可明显减轻或部分抑制因酒精染毒所致的GSH消耗、抗氧化酶失活及总抗氧化能力下降，降低ROS的产生水平和脂质过氧化程度，从而在一定程度上缓解了酒精对肝、心、肾、睾丸抗氧化系统的影响。以上结果表明，长期高剂量的酒精摄入可导致肝、心、肾、睾丸多器官的氧化-抗氧化失衡，而银杏黄酮干预，尤其是高剂量银杏黄酮干预，可以减轻或抑制酒精对多器官抗氧化系统的影响，对多组织酒精性氧化应激具有良好的保护作用。第四部分银杏黄酮防御酒精性肝损伤相关机制的研究肝脏是酒精吸收后代谢的主要场所，也是酒精中毒的主要靶器官。本部分采用酶学与生化分析手段，并借助于RT-PCR、流式细胞术、Western Blot及超微病理等方法，进一步探讨高剂量酒精摄入后，大鼠肝脏结构与助能、肝线粒体与微粒体氧化-抗氧化系统、CYP 2E1活性及肝HO-1表达与酶活性的变化，在此基础上，研究不同剂量银杏黄酮的干预效果及相关机制。实验结果显示：长期的酒精摄入(2.4g／kg，90天)导致肝细胞严重脂变性、浊肿和水变性，肝功能异常，线粒体、微粒体出现明显的肿胀、变形与退化等病理性改变，氧化-抗氧化失衡，CYP 2E1活性升高近4倍，而HO-1 mRNA、蛋白表达及酶活性水平分别下降55.0％、49.3％和45.5％；银杏黄酮(48及96mg／kg)干预，尤其高剂量(96mg／kg)干预，可明显减轻乙醇所致的病理性改变和线粒体及微粒体的氧化损伤，抑制CYP 2E1的活化，并在mRNA、蛋白与酶活性水平对HO-1具有明显的诱导作用，而对其它抗氧化蛋白无明显激活作用。以上结果表明，长期的酒精摄入削弱了肝细胞线粒体与微粒体抗氧化水平，抑制了HO-1的表达与活性；银杏黄酮可能通过对HO-1的诱导而展示出协同和增强的抗氧化效应，从而更有效清除乙醇代谢过程中形成的大量ROS，保护机体的抗氧化系统，减轻乙醇所致的细胞和细胞器结构改变与功能损伤。综上所述，低剂量的酒精摄入，可能通过抗氧化系统的代偿性活化和HDL-C水平的升高而发挥协同的保护作用，但过量(尤其是长期过量)的酒精摄入，可导致严重的脂质代谢紊乱和多组织的氧化损伤。而银杏黄酮干预可以明显减轻酒精对机体脂质代谢和抗氧化系统的不利影响，降低肝脏等多组织的氧化应激程度，银杏黄酮对HO-1的诱导可能介导了其拮抗酒精性氧化应激的作用。当然，HO-1的介导作用有待在细胞水平利用HO-1特异性的抑制剂与诱导剂加以进一步证实。此外，银杏黄酮对HO-1诱导的信号通路，以及HO-1介导的保护效应究竟是对底物的及时清除，还是某种代谢产物的作用，亦或是综合作用的结果还有待进一步深入研究。创新点：1)一般认为适量饮酒对机体的健康效应可能与HDL-C有关，本研究发现抗氧化系统代偿性活化可能也参与了这种保护作用。2)长期过量的酒精摄入使机体逐步失代偿而导致脂代谢紊乱和多组织氧化-抗氧化失衡。除传统的抗氧化酶与GSH外，本研究首次发现，HO-1表达与活性的下降也可能与这种失代偿状态有关。3)本研究深入到线粒体与微粒体水平，并利用多种分子生物学手段系统探讨了银杏黄酮对HO-1的诱导防御酒精性肝损伤的作用，首次揭示了HO-1介导银杏黄酮拮抗酒精性氧化应激的机制。更多还原

【Abstract】 As an important global issue on public health, alcohol abuse has been provoked widespread solicitude in the world because of its outcome including functional impairment of multiorgans or multisystems, as well as severe psychiatric disorder and social problem. Alcohol is an intensive oxidative stressor and small hyperosmotic molecule possessing both hydrophilia and lipophilicify. Excessive free radical and reactive oxygen species (ROS) produced during ethanol metabolism result in lipid peroxidation, protein oxidation. DNA damage and adducts formation, which is key mechanism of alcohol induced injury to liver and other organs. However, classic antioxidants medication, i.e. vitamin A, vitamin E, vitamin C, GSH, selenite, zinc and etc, plays little role on preventing ethanol-induced oxidative damage clinically, indicating that endogenous antioxidant system mediated and activated by exogenous antioxidant is crucial for the prophylaxis of alcoholic oxidative damage.Among various genes encoding for proteins in possession of antioxidant characteristics, heme oxygenase-1 (HO-1) has been attracted particular interest as it is finely up-regulated by stress conditions and generates metabolites exhibiting important biological activities and antioxidant capacity after decades quiescence. HO-1 as a potential preferred target gene for preventing oxidative stress, increasing evidences support that its induction represents an adaptive response or enhanced resistance to various oxidative stresses and diseases. However, these research outcomes have not been taken into consideration for practical application owing to potential oxidative toxicity of "classic inductor", i.e. heme or cobalt protoporphyrin, and the safety and efficiency of gene transfection.Of note, some natural flavoniods or polyphenols antioxidant. e.g. curcumin.quercetin, Ginkgo biloba extract (EGB) and ginkgetin, have been found recently to inducethe expression of HO-1 via antioxidant-response element or electrophile response elementin the upstream of gene promoter. These antioxidants might amplify antioxidant effect justby virtue of its induction on HO-1 and exert extensive pharmacological activity. On thebasis of these researches and hypothesis, the effect on serum and hepatic, renal, cardiac.and testicular antioxidant systems and serum lipid level were investigated with ginkgetinas preventer in possession of substantial antioxidant activity and hepatoprotective effect.Additionally, hepatic structure and function, antioxidant system of hepatic mitochondriaand microsomes, and mRNA, protein expression and enzymatic activity of hepatic HO-1were studied after chronic alcohol administration and ginkgetin pretreatment for rats.Enzymatic and biochemical assay, reverse transcriptase-ploymerase chain reaction(RT-PCR), flow cytometry (FCM), Western Blot, ultramicropathology and etc wereemployed in the present study. The details are as following:Part 1 The prophylaxis of ginkgetin on antioxidant system of mice after acute alcohol exposureAlthough the mechanism of alcoholic oxidative stress has been well documented, there is still some dispute on the specific influence or alteration after alcohol administration. Therefore, the dynamic change of serum and hepatic antioxidant systems, i.e. time-response effect, was investigated in mice after acute ethanol intake and ginkgetin pre-administration by enzymatic and biochemical assay in the present part.The results showed that GSH level and enzymatic activity of serum and liver presented U-shape whereas reverse U-shape was found with torpid restoration in MDA level of serum or liver from 1 hour to 15 hours after acute ethanol exposure (4.8 g/kg) for mice intragastrically. Serum GSH level and SOD activity dropped to minimum 2 hours after ethanol administration while GSH-Px activity reduced to the lowest and MDA level climbed to maximum 4 hours after administration. The altering extent of serum GSH, SOD, GSH-Px and MDA was 23.5%, 20.3%, 20.2% and 32.4% respectively. Hepatic GSH level, as well as GSH-Px or SOD activity descended to the minimum 4 hours whereas MDA level increased to the highest 6 hours after ethanol treatment. The altering extent of hepatic GSH, SOD, GSH-Px and MDA was 29.3%, 18.4%, 23.6% and 27.5% respectively. Pretreatment with ginkgetin at a dose of 96 mg/kg, increased the compensation effect at the early stage of ethanol administration and attenuated ethanol-induced GSH depletion, antioxidant enzymes inactivation and lipid peroxidation, by which ginkgetin promoted the restoration of antioxidant systems damaged by ethanol.From the results it could be concluded that serum and hepatic antioxidant capacity had been affected 2-6 hours, especially 4 hours after ethanol administration. Ginkgetin exerted evident protection by alleviating the influence of alcohol on antioxidant system.Part 2 The dynamic variation of serum lipid level and antioxidant system after ethanol administration and the protection of ginkgetin pretreatmentEpidemiological investigations show that ethanol intake with different dose might prognosticate different risk for cardiovascular disease and diabetes milletus, indicating diverse response of drink to health. Therefore, we assayed the serum antioxidant system, as well as serum lipid level of rats treated with different dose of ethanol for different endurance. The effect of ginkgetin on ethanol-fed rats with high dose was also investigated in the present part.The results showed that rats treated with low dose of ethanol (0.8 g/kg) had no obvious change in serum GSH, MDA, TG and TC level for 30, 60. 90 days. Serum SOD, GSH-Px and CAT activity gradually increased during experimental peroid and HDL-C significantly increased on the 90th day. Ethanol treatment with middle dose (1.6 g/kg) resulted in evident accumulation in serum MDA, TG and TC level, and gradual depletion in GSH. Serum HDL-C content, as well as the activity of SOD and GSH-Px declined after temporal and slight increase. High dose of ethanol (2.4 g/kg) led to conspicuous decrease in GSH and HDL-C level, as well as the activity of SOD and GSH-Px. but evident augument in MDA, TG and TC content. CAT activity declined rapidly after slight increase. Pretreatment of ginkgetin, especially high dose of ginkgetin (96 ing/kg), alleviated or counteracted the alteration of serum antioxidant system and lipid level induced by ethanol with high dose.From the results we could find that low dose of ethanol might activate antioxidant system and increase HDL-C level by which alcohol accordingly exerted protective effect. Serum lipid and lipid peroxidation aggravated, but antioxidant level evidently declined after trivial increase at the first stage overall after ethanol exposure with middle dose, especially for a long endurance. High dose of ethanol resulted in exacerbation in the disorder of lipid metabolism and impairing antioxidant system. Ginkgetin medication, especially high gingetin ptetreatment, alleviated the influences induced by ethanol, indicating evident lipid regulating and prophylaxis on alcoholic oxidative damage.Part 3 The effect of chronic ethanol administration on antioxidant system in rat tissues and prophylaxis of ginkgetinChronic alcohol binge not only impacted on liver function severely, but also induced pathological changes and dysfunction of multiorgans, including myocardium damage, renal inadequacy, spermatiferous disorder and etc. As a small molecule with high diffusibility, ethanol induced massive free radical and ROS during metabolism, by which ethanol impacts intensive oxidative stress on multiorgan and accordingly leads to alcohol-related diseases. Therefore, in the present part we investigated the effect of chronic ethanol administration on antioxidant system of liver, kidney, heart and testes, and explored the prophylactic effect of ginkgetin on ethanol-induced injury in the tissues.The results showed that MDA and ROS accumulated in all measured tissues. whereas GSH level, total antioxidant capacity (T-AOC) and the activity of SOD. GSH-Px and CAT in measured tissues significantly decreased except renal SOD and cardiac CAT in rats treated with ethanol at a dose of 2.4 g/kg for 90 days. Pretreatment with ginkgetin. especially high dose of ginkgetin (96 mg/kg), alleviated or counteracted the alteration of antioxidant system in tissues induced by high dose of ethanol.The results indicated that chronic ethanol intake might result in evident redox disbalance in liver, heart, kidney and testis. Ginkgetin medication, especially at high dose, alleviated the influences on antioxidant system of multiorgans induced by ethanol, exerting excellent prophylaxis on alcoholic oxidative damage.Part 4 The study on preventive mechanism of ginkgetin on alcoholic liver damageAs main organ for ethanol metabolism, liver becomes critical target of alcohol intoxication. Alcoholic liver disease (ALD) is the second biggest category ranking after infectious hepatopathy. In the present part we further explored prophylactic effect and related mechanism of ginkgetin on alcohol-induced liver damage. Hence, enzymatic or biochemical assay, pathological and ultra-micropathological method were employed to investigate the changes of liver structure and function, and of antioxidant system in hepatic mitochondria and microsomes of ethanol-fed and ginkgetin-intervention rats. Additionally, RT-PCR, FCM, Western Blot and enzymatic assay were deployed to determined the activity of CYP 2E1 and HO-1, and the expression of rat liver after ethanol administration and ginkgetin pretreatment.The results showed that chronic ethanol treatment (2.4 g/kg for 90 days) led to prevalent hepatocytes steatosis, watery and parenchymatous degeneration, and liver disorder. Mitochondria and rough endoplasmic reticulum (RER) in liver showed swollen, deformative and degenerative pathological changes, and redox disequilibrium. HO-1 level decreased by 55.0% in mRNA, 49.3% in protein, and 45.5% in activity, whereas CYP 2E1 activity increased by nearly 4 fold following chronic ethanol administration. Rat hepatic pathological change and oxidative damage in mitochondria and microsomes were attenuated in the presence of ginkgetin with different dose (48 and 96 mg/kg). especially with high dose (96 mg/kg). Of note, ginkgetin induced mRNA, protein expression and activity of HO-1 in ethanol plus ginkgetin group compared with ethanol-fed group, and in ginkgetin control versus normal control. No evident activation was found in other antioxidant enzymes after ginkgetin supplement.From above we could find that chronic ethanol administration impaired the antioxidant capacity of mitochondria and microsomes and inhibited the expression and inactivated the activity of HO-1. Ginkgetin induced HO-1 and exerted synergetic and enhanced antioxidant effect to scavenge ethanol-derived ROS, by which ginkgetin protected the antioxidant system from ethanol-induced oxidative stress.In summary, low ethanol might exhibit synergetic protection by promoting HDL-C synthesis and activating antioxidant system. However, excessive ethanol administration could result in disorder of lipid metabolism and oxidative stress in muhiorgans. Ginkgetin pretreatment attenuated or counteracted the adverse effects of ethanol and alleviated ethanol-induced oxidative damage. The induction of HO-1 by ginkgetin might mediate the prophylacitc effect. However, it is needed to elucidate the protection mediated by HO-1 via introduction of specific inhibitor or revulsivum on cellular level. In addition, it is still remained unclear about the signal pathways of ginkgetin induces HO-1 and whether the protective mechanism of HO-1 is related to the substrate eliminating, contribution of metabolism product, or their combination effect in the present study.更多还原