海兔素改善大鼠酒精性肝损伤的效果及其机制研究

【作者】 戈娜；

【导师】 梁惠；

【作者基本信息】 青岛大学 ， 营养与食品卫生学， 2014， 博士

【摘要】 目的：海兔素是一种溴代倍半萜,主要来源于红藻凹顶藻属海藻以及海兔中,具有抑菌、抗炎、抗肿瘤、免疫增强、抗氧化等生物学活性,对酒精暴露大鼠亦具有一定的肝脏保护作用。本研究通过探讨海兔素对酒精暴露大鼠肝脏乙醇代谢酶、抗氧化能力、DNA损伤与修复、肝细胞凋亡、氧化/硝化应激、线粒体功能以及内源性凋亡信号通路等的影响,以此阐明海兔素保肝效果及其可能作用机制。方法：1.动物分组及模型建立：两月龄健康雄性Wistar大鼠100只,体重180-220g,按体重随机分为5组,每组20只。酒精模型组以50%酒精8m1·kg1·d-1灌胃2w后,12ml·kg-1·d-1灌胃4w；海兔素低、中、高剂量组酒精剂量同模型组,同时每日分别给予海兔素50、100、150mg·kg-1·d-1灌胃；正常组以等体积生理盐水灌胃,持续6w。末次灌胃12h后,大鼠称重后给予7%水合氯醛麻醉,腹主动脉取血,剥取肝组织,分离红细胞膜,提取肝细胞线粒体及微粒体,并计算肝指数。2.肝脏病理学检查：H-E染色观察肝脏组织的病理学改变,透射电镜观察大鼠肝细胞超微结构变化。3.肝功能及脂质代谢水平评价：用赖氏法检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性；微量酶标法检测碱性磷酸酶(ALP)的活性；用COD-PAP法测定血清中TC含量；GPO-PAP法测定血清及肝脏TG含量。4.硝化应激程度的评价：用化学比色法检测血清中—氧化氮合酶(NOS)活性；硝酸还原法测定血清中—氧化氮(NO)含量；采用Western blotting检测大鼠肝脏中iNOS蛋白表达水平的变化。5.肝脏乙醇代谢酶活性的测定：制备10%肝组织匀浆,应用比色法检测肝脏乙醇脱氢酶(ADH)的活性；差速离心结合钙沉淀法制备微粒体,硝基酚法检测肝微粒体细胞色素P450亚酶2E1活性。6.DNA氧化损伤程度的评价：采用原位两步Ⅳ型胶原酶灌注消化分离肝细胞,制备大鼠肝细胞悬液,通过彗星实验(单细胞凝胶电泳实验)测定肝细胞DNA损伤程度；利用酶联免疫吸附测定法(ELISA法)检测血浆中8-OHdG含量,评价DNA氧化损伤程度。7.抗氧化综合能力的分析：酶法测定肝脏胞浆乳酸/丙酮酸比值,反映NAD+/NADH比值；利用低渗一步溶血法和红细胞膜荧光标记法检测红细胞膜流动性；微板法检测血浆中脂质过氧化物(LPO)的水平；硫代巴比妥酸法(TBA法)测定肝脏和血浆中丙二醛(MDA)的含量；黄嘌呤氧化酶法测定血清超氧化物歧化酶(SOD)活性；二硫代-2-硝基苯甲酸(DTNB)比色法测定血清谷胱甘肽过氧物酶(GSH-Px)的活性；采用比色法测定肝脏过氧化氢(CAT)的活性。8.线粒体功能评价：差速离心法制备线粒体,测定线粒体悬液中Mn-SOD的活性和GSH含量；比色法测定线粒体呼吸酶链复合物(MRC)活性。9.肝细胞凋亡的评估：采用Annexin V-FITC/PI双染法检测肝细胞凋亡情况。10. Western blotting法检测大鼠肝脏中iNOS、CYP2E1以及线粒体介导的内源性凋亡通路关键蛋白(Bcl-2、Bax、细胞色素C、caspase-3)的变化。11.用Trizol试剂提取肝脏中总RNA,反转录获得cDNA,用实时定量PCR检测肝组织中CYP2E1mRNA表达水平以及内源性凋亡相关基因(Bcl-2、Bax.细胞色素C、caspase-9、caspase-3)的mRNA表达水平。结果：1.海兔素改善酒精性肝损伤的效果评价与正常对照组相比,酒精模型组大鼠周体重有轻微下降,肝指数明显增加(P<0.05)；与酒精模型组相比,各剂量海兔素干预组大鼠周体重均有所提高,但海兔素中、高剂量组大鼠肝指数均显著降低(P<0.05)。HE染色病理观察结果显示,海兔素各剂量组肝脏脂肪变性明显得到改善,炎性细胞浸润减少,与酒精模型组相比较,中、高剂量海兔素干预组,肝索排列逐渐恢复整齐,组织结构趋向正常。透射电镜下观察发现,各剂量海兔素组胞浆脂滴数量减少,线粒体病变明显减轻且数目有所增加,粗面内质网退化与排列紊乱程度有所改善。在本研究中,酒精模型组大鼠血清中ALT、AST和ALP的活性显著增加(P<0.05),而海兔素可有效抑制酒精诱导的血清ALT、AST和ALP的活性升高。此外,长期大量酒精灌胃能使大鼠体内脂质代谢紊乱,血清TC、TG以及肝脏TG水平升高,而海兔素干预抑制了酒精摄入引起的这些变化,同时表现出良好的血脂调节作用。2.海兔素对酒精暴露大鼠硝化应激和肝脏酒精代谢酶的影响与正常组比较,酒精模型组大鼠血清TNOS和iNOS的活性明显增加,NO含量升高,肝脏ADH及肝微粒体CYP2E1活性都有所增强(P<0.05)；而不同剂量的海兔素组和酒精模型组相比,血清TNOS、iNOS活性以及NO含量均有不同程度地降低,且成剂量依赖关系,但ADH和CYP2E1活性仅150mg/kg海兔素组同时抑制了它们活性的变化。此外,中、高剂量海兔素可明显抑制大鼠肝组织中iNOS的蛋白表达(P<0.05),且海兔素的摄入明显降低了酒精诱导的CYP2E1的蛋白和mRNA过表达。3.海兔素对酒精暴露大鼠肝脏氧化损伤及氧化应激的影响酒精模型组胞浆NAD+/NADH比值、红细胞膜流动性均较正常对照组显著降低(P<0.05),而海兔素干预组可显著拮抗酒精所致的NAD+/NADH的变化以及改善红细胞膜流动性,尤其是100和150mg/kg剂量组。海兔素干预后可以明显缓解因酒精诱导的血浆8-OHdG的升高,同时彗星实验表明海兔素组大鼠肝脏分离细胞DNA损伤程度明显减轻,其尾部DNA%、尾长、尾距和Olive尾距值显著性低于酒精模型组组(P<0.05)。研究显示,酒精模型组大鼠血/肝脏LPO和MDA的水平都显著升高,相比之下,各海兔素干预组LPO和MDA水平均显示出明显的降低,并且海兔素干预组明显抑制酒精诱导的GSH的下降,恢复SOD. GSH-Px以及CAT的活性。4.线粒体介导的内源性凋亡通路在海兔素干预的酒精性肝损伤中的作用荧光显微镜Annexin V-FITC/PI双染法检测发现,酒精模型组大鼠凋亡的肝细胞数目明显增加,且多为晚期凋亡细胞。而在海兔素处理组,肝细胞凋亡数目明显减少,且多以早期凋亡为主。线粒体呼吸酶链复合物(MRC)活性检测中,可见海兔素干预逆转了酒精所致的MRC I, Ⅲ和Ⅳ活性降低,与酒精模型组相比,差异具有显著性(P<0.05)。此外,Western blotting结果显示,海兔素可上调Bcl-2的蛋白表达,下调Bax的表达,减少细胞色素C的释放,抑制caspase-3和caspase-9的激活。同时,real-time PCR的结果也显示,与酒精模型组相比,随着海兔素作用剂量的增加,肝脏内Bax、细胞色素C、caspase-9和caspase-3的mRNA的表达量逐渐下降,而Bcl-2基因mRNA表达的量逐渐增加,且差异具有显著性(P<0.05)。结论：1.根据对肝脏指数、肝脏病理学的观察、肝功能酶的评价以及脂质代谢的检测,初步证实了海兔素对酒精诱导的大鼠肝损伤具有显著的改善作用。2.海兔素对酒精性肝损伤的保护作用机制可能与①抑制iNOS活性,下调肝脏iNOS蛋白表达,减少NO的过量生成；②抑制大鼠肝脏ADH和微粒体CYP2E1活性,下调过表达的CYP2E1蛋白和：mRNA表达水平有关。然而海兔素作为一种具有iNOS抑制作用且可调节肝脏酒精代谢酶的物质能否成为有效预防和逆转酒精性肝损伤的药物尚需进一步的实验研究来佐证。3.海兔素的补充可提高机体抗氧化能力抵抗酒精所致的氧化应激,从而减轻脂质过氧化以及DNA的氧化损伤,这可能是海兔素拮抗酒精性肝损伤的机制之一4.过量的酒精摄入可导致肝细胞凋亡的发生,而海兔素可通过调控Bcl-2家族mRNA和转录蛋白的表达水平,抑制线粒体细胞色素C的释放,阻断caspase-3的活化,降低细胞色素C、caspase-9以及caspase-3mRNA的表达,抑制线粒体介导的凋亡通路的激活,从而达到对酒精性肝损伤的保护作用。更多还原

【Abstract】 Objective Aplysin, belonging to one of the bromine-sesquiterpene compounds and mainly extracted from red alga Laurencia tristicha, had attracted much attention due to its potent biological activities, including anti-bacteria, anti-inflammatory, anti-tumor, immunopotentiation and antixdiant. This study intended to explore the hepatic protective effects of Aplysin against alcohol-induced liver injury in rats through regulating ethanol metabolic enzyme system, antioxidant capacity, DNA damage and repair, hepatocyte apoptosis, oxidative/nitrative stress, mitochondrial function and endogenous apoptotic signaling pathways. Thus, the potential of liver injury preventive activity and possibly related mechanism of Aplysin will be illustrated in this paper.Methods1. Animal grouping and Model building:there were100healthy male Wistar rats aged two months, weighing180-220g, which were randomly divided into five equal groups with20in each by weight:control group, normal diet with normal saline; alcohol-model group, normal diet with alcohol administration; and three low-, medium-, and high-dose Aplysin plus alcohol treatment groups. Except the control group, all the alcohol-treated group were initially given with50%(v/v) alcohol8ml·kg-1·day-1for two weeks by intragastric administration. Then the dose of alcohol would be increased to12ml·kg-1·day-1for the remaining four weeks. In addition, three low-, medium-, and high-dose Aplysin groups were respectively given Aplysin50,100,150mg·kg-1·day-1for entire six weeks by intragastric administration. Control group would be given normal saline with the same volume for whole six weeks by intragastric administration. The rats were weighed and anesthetized with7%chloral hydrate at the twelve hours after the last treatment. Then blood samples were collected by aorta ventralis puncture to determine biochemical parameters. Liver tissue was rapidly dissected, then cut and fixed in formaldehyde saline (10%) solution for histopathological analysis and liver index. Mitochondria and microsome were extracted from isolated erythrocyte membranes.2. Pathological evaluation of liver:Pathological changes of liver tissue were evaluated by hematoxylin-eosin (H-E) staining; the changes of hepatocellular ultrastructure were observed by using transmission electron microscopy.3. Evaluation of hepatic function and level of lipid metabolism:serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities were measured by using Yoriuji method; serum alkaline phosphatase (ALP) activity was tested by using enzyme linked immunosorbent assay (ELISA); serum level of total cholesterol (TC) was measured by using COD-PAP method; serum and liver tissue level of triglyceride (TG) was tested by using GPO-PAP method. 4. Evaluation of the extent of nitrative stress:serum nitric oxide synthase (NOS) activity was tested by using chemical colorimetry; protein expression of iNOS was measured by using western blotting.5. Measurement of hepatic ethanol metabolic enzymes activity:prepared10%liver homogenate was used to test the activity of alcohol dehydrogenase (ADH); microsome was prepared by using differential centrifugation Ca2+precipitation method; the activity of CYP2E1in liver microsome was measured by using p-nitrophenol hydroxylase (PNPH) method.6. Evaluation of the extent of DNA oxidative damage:an in situ Ⅳ collagenase two-step perfusion method were applied to isolate hepatocytes which were used to prepare liver cell suspension; the extent of DNA oxidative damage was measured by using the alkaline comet assay (single cell gel electrophoresis, SCGE); plasma level of8-OHdG was determined by using an8-hydroxy-desoxyguanosine (8-OHdG) ELISA kit.7. Analysis of comprehensive antioxidant abilities:cytoplasmic redox state ([NAD+]/[NADH] ratio) was estimated from the corresponding [lactate]/[pyruvate](L/P) ratio and the equation of the chemical equilibrium; the fluidity of erythrocyte membrane was performed by the fluorescence polarization technique with1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe; the concentration of lipid peroxide (LPO) was determined by using lipid peroxidation assay kit; the concentration of malondialdehyde (MDA) in plasma and liver tissue was measured by using thiobarbituric acid (TBA); the activities of serous superoxide dismutase (SOD) was tested by using xanthine oxidase method; the activities of plasmic glutathione peroxidase (GSH-Px) was determined by using DTNB method; the activity of hepatic catalase (CAT) in liver tissue was tested by using visible spectrophotometry method.8. Evaluation of mitochondrial function:mitochondria were prepared by using differential centrifugation method and were used to determine the activity of Mn-SOD and the concentration of GSH in mitochondrial suspension; colorimetric method was utilized to measure the activity of mitochondrial respiratory chain complex (MRC).9. Evaluation of hepatocyte apoptosis:the extent of hepatocyte apoptosis was determined by using Annexin V-FITC/PI Apoptosis Detection Kit.10. Western blotting technology was utilized to determine the changes of protein expression of iNOS, CYP2E1and critical protein of mitochondria-induced endogenous apoptosis pathway in liver tissue, such as Bcl-2, Bax, cytochrome c and caspase-3.11. Total RNA was isolated from liver tissues using TRIzol reagent and cDNA was prepared via reverse transcription. Then the real-time PCR (qPCR) method was utilized to determine the changes of mRNA expression of CYP2E1and endogenous apoptosis related genes in liver tissue, such as Bcl-2, Bax, cytochrome c, caspase-9and caspase-3.Results 1. Evaluation of protective effect of Aplysin improving alcohol-induced liver damageCompared with normal group, weekly-weight in alcohol-model group was slightly decreased and liver index was siginificantly increased (P<0.05), compared with alcohol-model group, weekly-weight in three low-, medium-, and high-dose Aplysin groups all went up, but liver index among medium-, and high-dose Aplysin groups was significantly decreased (P<0.05). The results from HE staining pathological observation revealed that hepatic steatosis in three low-, medium-, and high-dose Aplysin groups was obviously improved, such as reducing of inflammatory cell infiltration. In addition, compared with alcohol-model group, hepatic cords arranged orderly and hepatocyte morphology was normal in medium-and high-dose Aplysin groups. Through the observation of transmission electron microscope, among medium-and high-dose Aplysin groups, the number of lipid droplets within cytoplasm was decreased; mitochondrial pathological changes was significantly alleviated in the liver and the number of mitochondria was obviously increased; degeneration of rough endoplasmic reticulum and disorder arrangement were both improved. In this study, serum activities of ALT, AST and ALP in alcohol-model group were all prominently increased (P<0.05), but Aplysin could effectively inhibit the increasing of serum activities of ALT, AST and ALP. In addition, long-term massive alcohol-treatment could disorder lipid metabolism via intragastric administration and make the level of TC, TG within serum and liver tissue increase. Aplysin-treatment successfully inhibited these changes caused by alcohol and seemed to have a positive moderating effect of blood lipid.2. The effect of Aplysin against alcohol-exposed nitrative stress and ethanol metabolic enzymes in ratsCompared with the normal group, serum activities of TNOS and iNOS in alcohol-model group was significantly increased, the concentration of NO went up, and liver activity of ADH and liver microsome activity of CYP2E1was increased (P<0.05); compared with alcohol-model group, serum activities of TNOS, iNOS and NO within three low-, medium-, and high-dose Aplysin groups were all decreased and had dose-dependent effect. However, activity of ADH and CYP2E1was only inhibited in high-dose Aplysin group. In addition, medium-, and high-dose Aplysin group could obviously inhibit protein expression of iNOS in liver tissue (P<0.05); intaking of Aplysin significantly reduce the protein or mRNA expression of CYP2E1.3. The effect of Aplysin against alcohol-exposed liver oxidative damage and oxidative stressCompared with the normal group, cytoplasm NAD+/NADH ratio and the fluidity of erythrocyte membrane in alcohol-model group were significantly decreased (P<0.05); Aplysin-treatment groups could inhibit the changes of NAD"/NADH and the fluidity of erythrocyte membrane caused by alcohol, especially in medium-, and high-dose Aplysin groups. Aplysin-treatment could significantly relieve the increasing of plasma8-OHdG induced by alcohol. On the other hand, the results of comet assay test indicated that the extent of DNA damage isolated from liver was significantly attenuated, for example, tail DNA percent, tail length, tail moment and olive tail moment were all significantly decreased compared with alcohol-model group(P<0.05) Concentration of LPO and MDA in serum or liver was both obviously increased. In contrast, concentration of LPO and MDA in all Aplysin-treatment groups significantly went down. In addition, Aplysin-treatment groups could obviously inhibit the decreasing of GSH and recover the activity of SOD, GSH-Px and CAT.4. The role of mitochondria-mediated endogenous apoptosis pathway for alcohol-induced liver damage interfered by AplysinAccording to the measurement of Annexin V-FITC/PI method by using fluorescence microscope, the number of liver apoptosis was significantly increased, and most of them were the late stage apoptotic cells. In contrast, the number of liver apoptosis in Aplysin-treatment groups was obviously decreased, and most of them were the early stage apoptotic cells. During the test of activity of mitochondrial respiratory chain complex (MRC), it could be seen that Aplysin-treatment reverse the decreasing of activities of MRC I, III and IV resulted from alcohol. Compare with the alcohol-model group, the differences had significance (P<0.05). In addition, the results of Western blotting demonstrated that Aplysin could up-regulate protein expression of Bcl-2, down-regulate expression of Bax, reduce the release of cytochrome C, and inhibit the activation of caspase-3and caspase-9. The results of real-time PCR indicated that mRNA expression of Bax, cytochrome C, caspase-3, caspase-9in the liver was gradually decreased with the development of dose of Aplysin, compared with alcohol-model group. But mRNA expression of Bcl-2was gradually increased (P<0.05).Conclusion1.Based on the results of liver index, observation of hepatic histopathology, the evaluation of related enzymes of liver function, and measurement of lipid metabolism, it was initially proved that Aplysin had significantly protective effect on the improvement of alcohol-induced liver damage in rats.2.The mechanism of hepatic protective effect of Aplysin against alcohol-induced liver damage in rats could be relevant with some factors, such as:1. Inhibit the activity of iNOS, down-regulate protein expression of iNOS, reduce the formation of NO;2. Inhibit the activity of ADH in liver and the activity of CYP2E1in microsome, down-regulate protein or mRNA expression of CYP2E1. But whether Aplysin would effectively prevent alcohol-induced liver damage, as one of drugs which had inhibiting effect against iNOS and regulate ethanol metabolic enzymes in liver, still needed more and further experiments to confirm. 3.Supplement of Aplysin could enhance the ability of antioxidant to resist alcohol-induced oxidative stress, and alleviate lipid peroxidation and DNA oxidative damage. This could be one of the mechanisms of Aplysin against alcohol-induced liver damage.4.Overdose of intaking alcohol could lead to the liver apoptosis, but Aplysin would protect alcohol-induced liver damage, through regulating protein or mRNA expression of Bcl-2family, inhibiting the release of cytochrome C, inhibiting the activation of caspase-3, reducing the mRNA expression of cytochrome C, caspase-9and caspase-3, inhibiting the activation of mitochondria-mediated apopsis pathway.更多还原