海兔素改善大鼠酒精性肝损伤的效果及其机制研究

发布时间：2018-04-16 23:30

  本文选题：海兔素 + 氧化应激　； 参考：《青岛大学》2014年博士论文

【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(P0.05)；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(P0.05)。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(P0.05),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(P0.05)；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(P0.05),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(P0.05),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(P0.05)。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(P0.05)。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(P0.05)。 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。
[Abstract]:Objective : To study the effects of rabbit on liver ethanol metabolism , antioxidant capacity , DNA damage and repair , hepatocyte apoptosis , oxidation / nitration stress , mitochondrial function and endogenous apoptosis signal pathway .

Method :

1 . Animal grouping and model were established : 100 male Wistar rats weighing 180 - 220g were randomly divided into 5 groups according to body weight , 20 in each group .
At the same time , 50 , 100 , 150 mg 路 kg - 1 路 d - 1 in rabbits were given orally at the same time .
The rats were perfused with normal saline for 6 weeks . After the last 12 hours , the rats were anesthetized with 7 % chloral hydrate , blood from the abdominal aorta was taken , the liver tissue was removed , the erythrocyte membrane was isolated , the mitochondria and microsomes of hepatocytes were extracted , and the liver index was calculated .

2 . Liver pathology examination : The pathological changes of liver tissues were observed by H - E staining , and the ultrastructural changes of liver cells were observed by transmission electron microscope .

3 . Evaluation of liver function and lipid metabolism : The activity of alanine aminotransferase ( ALT ) and aspartate aminotransferase ( AST ) in serum was determined by the method of Wright .
The activity of alkaline phosphatase ( ALP ) was detected by microplate method .
Determination of TC in serum by COD - PAP method
Serum and liver TG contents were determined by GPO - PAP method .

4 . Evaluation of the degree of nitrification stress : the activity of nitric oxide synthase ( NOS ) was detected by chemical colorimetry .
Determination of nitric oxide ( NO ) in serum by nitric acid reduction method
Western blotting was used to detect the expression of iNOS in rat liver .

5 . Determination of hepatic alcohol metabolism enzyme activity : 10 % liver tissue homogenate was prepared , and the activity of liver ethanol dehydrogenase ( ADH ) was detected by colorimetry .
The activity of cytochrome P450 subunit 2E1 was detected by differential centrifugation and calcium precipitation method .

6 . Evaluation of the degree of DNA oxidative damage : In situ two - step collagenase type IV collagenase perfusion digestion and separation of hepatocytes , the rat hepatocyte suspension was prepared , and the degree of DNA damage was determined by comet assay ( single cell gel electrophoresis ) .
The content of 8 - OHdG in plasma was measured by enzyme linked immunosorbent assay ( ELISA ) , and the degree of DNA oxidative damage was evaluated .

7 . Analysis of the comprehensive ability of anti - oxidation : the ratio of lactic acid / pyruvate to the ratio of NAD + / NADH was determined by enzyme method .
The fluidity of erythrocyte membrane was detected by low - permeability one - step hemolysis method and red cell membrane fluorescence labeling method .
The level of lipid peroxide ( LPO ) in plasma was detected by microplate method .
The content of malondialdehyde ( MDA ) in liver and plasma was determined by TBA method .
Determination of superoxide dismutase ( SOD ) activity in serum by xanthine oxidase method
The activity of glutathione peroxidase ( GSH - Px ) was determined by the colorimetry of dithio - 2 - nitrobenzoic acid ( DTNB ) .
The activity of catalase ( CAT ) was determined by colorimetry .

8 . Mitochondrial function evaluation : mitochondria were prepared by differential centrifugation , and the activity of Mn - SOD and GSH content in mitochondria were determined .
The activity of mitochondrial respiratory chain complex ( MRC ) was determined by colorimetry .

9 . The apoptosis of hepatocytes was assessed by annexin V - FITC / PI double staining method .

10 . Western blotting was used to detect the changes of the key proteins ( Bcl - 2 , Bax , cytochrome C , caspase - 3 ) in rat liver .

11 . Total RNA in liver was extracted with Trizol reagent and cDNA was obtained by reverse transcription . The mRNA expression level of CYP2E1 mRNA and the mRNA expression level of endogenous apoptosis related genes ( Bcl - 2 , Bax , cytochrome C , caspase - 9 , caspase - 3 ) were detected by real - time quantitative PCR .

Results :

1 . Evaluation of the effect of hepidine on alcoholic liver injury

Compared with the normal control group , the body weight of the alcohol model group decreased slightly , and the liver index increased significantly ( P0.05 ) .
In this study , the levels of ALT , AST and ALP in the serum of the rats were significantly decreased ( P0.05 ) .

2 . The effects of hepidine on nitrating stress and hepatic alcohol metabolism in rats exposed to alcohol

Compared with the normal group , the activity of serum TNOS and iNOS in alcohol model group increased significantly , NO content increased , liver ADH and CYP2E1 activity increased ( P0.05 ) .
In addition , the levels of NOS , iNOS and NO in serum TNOS , iNOS and NO were significantly lower than those in alcohol model group .

3 . Effects of Rabbit on Hepatic Oxidative Damage and Oxidative Stress in Rats Exposed to Alcohol

The concentration of NAD + / NADH and erythrocyte membrane fluidity in alcohol model group were significantly lower than those in normal control group ( P0.05 ) . GSH - Px and CAT activity .

4 . Role of mitochondria - mediated endogenous apoptosis pathway in alcoholic liver injury in rabbits

Compared with alcohol model group , the expression of Bax , cytochrome C , caspase - 9 and caspase - 3 mRNA in liver decreased gradually , and the expression level of Bax , cytochrome C , caspase - 9 and caspase - 3 decreased gradually , and the difference was significant ( P0.05 ) .

Conclusion :

1 . According to the observation of liver index , liver pathology , the evaluation of liver function enzyme and the detection of lipid metabolism , it is preliminarily confirmed that the rabbit liver injury has a significant improvement effect on alcohol - induced liver injury .

2 . The mechanism of protective action of hepidine on alcoholic liver injury may be as follows : 鈶，

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