本文選題：海兔素 + 氧化應(yīng)激��； 參考：《青島大學(xué)》2014年博士論文

【摘要】：目的：海兔素是一種溴代倍半萜,主要來源于紅藻凹頂藻屬海藻以及海兔中,具有抑菌、抗炎、抗腫瘤、免疫增強(qiáng)、抗氧化等生物學(xué)活性,對酒精暴露大鼠亦具有一定的肝臟保護(hù)作用。本研究通過探討海兔素對酒精暴露大鼠肝臟乙醇代謝酶、抗氧化能力、DNA損傷與修復(fù)、肝細(xì)胞凋亡、氧化/硝化應(yīng)激、線粒體功能以及內(nèi)源性凋亡信號通路等的影響,以此闡明海兔素保肝效果及其可能作用機(jī)制。 方法： 1.動物分組及模型建立：兩月齡健康雄性Wistar大鼠100只,體重180-220g,按體重隨機(jī)分為5組,每組20只。酒精模型組以50%酒精8m1·kg1·d-1灌胃2w后,12ml·kg-1·d-1灌胃4w；海兔素低、中、高劑量組酒精劑量同模型組,同時每日分別給予海兔素50、100、150mg·kg-1·d-1灌胃；正常組以等體積生理鹽水灌胃,持續(xù)6w。末次灌胃12h后,大鼠稱重后給予7%水合氯醛麻醉,腹主動脈取血,剝?nèi)「谓M織,分離紅細(xì)胞膜,提取肝細(xì)胞線粒體及微粒體,并計(jì)算肝指數(shù)。 2.肝臟病理學(xué)檢查：H-E染色觀察肝臟組織的病理學(xué)改變,透射電鏡觀察大鼠肝細(xì)胞超微結(jié)構(gòu)變化。 3.肝功能及脂質(zhì)代謝水平評價：用賴氏法檢測血清中谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的活性；微量酶標(biāo)法檢測堿性磷酸酶(ALP)的活性；用COD-PAP法測定血清中TC含量；GPO-PAP法測定血清及肝臟TG含量。 4.硝化應(yīng)激程度的評價：用化學(xué)比色法檢測血清中—氧化氮合酶(NOS)活性；硝酸還原法測定血清中—氧化氮(NO)含量；采用Western blotting檢測大鼠肝臟中iNOS蛋白表達(dá)水平的變化。 5.肝臟乙醇代謝酶活性的測定：制備10%肝組織勻漿,應(yīng)用比色法檢測肝臟乙醇脫氫酶(ADH)的活性；差速離心結(jié)合鈣沉淀法制備微粒體,硝基酚法檢測肝微粒體細(xì)胞色素P450亞酶2E1活性。 6.DNA氧化損傷程度的評價：采用原位兩步Ⅳ型膠原酶灌注消化分離肝細(xì)胞,制備大鼠肝細(xì)胞懸液,通過彗星實(shí)驗(yàn)(單細(xì)胞凝膠電泳實(shí)驗(yàn))測定肝細(xì)胞DNA損傷程度；利用酶聯(lián)免疫吸附測定法(ELISA法)檢測血漿中8-OHdG含量,評價DNA氧化損傷程度。 7.抗氧化綜合能力的分析：酶法測定肝臟胞漿乳酸/丙酮酸比值,反映NAD+/NADH比值；利用低滲一步溶血法和紅細(xì)胞膜熒光標(biāo)記法檢測紅細(xì)胞膜流動性；微板法檢測血漿中脂質(zhì)過氧化物(LPO)的水平；硫代巴比妥酸法(TBA法)測定肝臟和血漿中丙二醛(MDA)的含量；黃嘌呤氧化酶法測定血清超氧化物歧化酶(SOD)活性；二硫代-2-硝基苯甲酸(DTNB)比色法測定血清谷胱甘肽過氧物酶(GSH-Px)的活性；采用比色法測定肝臟過氧化氫(CAT)的活性。 8.線粒體功能評價：差速離心法制備線粒體,測定線粒體懸液中Mn-SOD的活性和GSH含量；比色法測定線粒體呼吸酶鏈復(fù)合物(MRC)活性。 9.肝細(xì)胞凋亡的評估：采用Annexin V-FITC/PI雙染法檢測肝細(xì)胞凋亡情況。 10. Western blotting法檢測大鼠肝臟中iNOS、CYP2E1以及線粒體介導(dǎo)的內(nèi)源性凋亡通路關(guān)鍵蛋白(Bcl-2、Bax、細(xì)胞色素C、caspase-3)的變化。 11.用Trizol試劑提取肝臟中總RNA,反轉(zhuǎn)錄獲得cDNA,用實(shí)時定量PCR檢測肝組織中CYP2E1mRNA表達(dá)水平以及內(nèi)源性凋亡相關(guān)基因(Bcl-2、Bax.細(xì)胞色素C、caspase-9、caspase-3)的mRNA表達(dá)水平。 結(jié)果： 1.海兔素改善酒精性肝損傷的效果評價 與正常對照組相比,酒精模型組大鼠周體重有輕微下降,肝指數(shù)明顯增加(P0.05)；與酒精模型組相比,各劑量海兔素干預(yù)組大鼠周體重均有所提高,但海兔素中、高劑量組大鼠肝指數(shù)均顯著降低(P0.05)。HE染色病理觀察結(jié)果顯示,海兔素各劑量組肝臟脂肪變性明顯得到改善,炎性細(xì)胞浸潤減少,與酒精模型組相比較,中、高劑量海兔素干預(yù)組,肝索排列逐漸恢復(fù)整齊,組織結(jié)構(gòu)趨向正常。透射電鏡下觀察發(fā)現(xiàn),各劑量海兔素組胞漿脂滴數(shù)量減少,線粒體病變明顯減輕且數(shù)目有所增加,粗面內(nèi)質(zhì)網(wǎng)退化與排列紊亂程度有所改善。在本研究中,酒精模型組大鼠血清中ALT、AST和ALP的活性顯著增加(P0.05),而海兔素可有效抑制酒精誘導(dǎo)的血清ALT、AST和ALP的活性升高。此外,長期大量酒精灌胃能使大鼠體內(nèi)脂質(zhì)代謝紊亂,血清TC、TG以及肝臟TG水平升高,而海兔素干預(yù)抑制了酒精攝入引起的這些變化,同時表現(xiàn)出良好的血脂調(diào)節(jié)作用。 2.海兔素對酒精暴露大鼠硝化應(yīng)激和肝臟酒精代謝酶的影響 與正常組比較,酒精模型組大鼠血清TNOS和iNOS的活性明顯增加,NO含量升高,肝臟ADH及肝微粒體CYP2E1活性都有所增強(qiáng)(P0.05)；而不同劑量的海兔素組和酒精模型組相比,血清TNOS、iNOS活性以及NO含量均有不同程度地降低,且成劑量依賴關(guān)系,但ADH和CYP2E1活性僅150mg/kg海兔素組同時抑制了它們活性的變化。此外,中、高劑量海兔素可明顯抑制大鼠肝組織中iNOS的蛋白表達(dá)(P0.05),且海兔素的攝入明顯降低了酒精誘導(dǎo)的CYP2E1的蛋白和mRNA過表達(dá)。 3.海兔素對酒精暴露大鼠肝臟氧化損傷及氧化應(yīng)激的影響 酒精模型組胞漿NAD+/NADH比值、紅細(xì)胞膜流動性均較正常對照組顯著降低(P0.05),而海兔素干預(yù)組可顯著拮抗酒精所致的NAD+/NADH的變化以及改善紅細(xì)胞膜流動性,尤其是100和150mg/kg劑量組。海兔素干預(yù)后可以明顯緩解因酒精誘導(dǎo)的血漿8-OHdG的升高,同時彗星實(shí)驗(yàn)表明海兔素組大鼠肝臟分離細(xì)胞DNA損傷程度明顯減輕,其尾部DNA%、尾長、尾距和Olive尾距值顯著性低于酒精模型組組(P0.05)。研究顯示,酒精模型組大鼠血/肝臟LPO和MDA的水平都顯著升高,相比之下,各海兔素干預(yù)組LPO和MDA水平均顯示出明顯的降低,并且海兔素干預(yù)組明顯抑制酒精誘導(dǎo)的GSH的下降,恢復(fù)SOD. GSH-Px以及CAT的活性。 4.線粒體介導(dǎo)的內(nèi)源性凋亡通路在海兔素干預(yù)的酒精性肝損傷中的作用 熒光顯微鏡Annexin V-FITC/PI雙染法檢測發(fā)現(xiàn),酒精模型組大鼠凋亡的肝細(xì)胞數(shù)目明顯增加,且多為晚期凋亡細(xì)胞。而在海兔素處理組,肝細(xì)胞凋亡數(shù)目明顯減少,且多以早期凋亡為主。線粒體呼吸酶鏈復(fù)合物(MRC)活性檢測中,可見海兔素干預(yù)逆轉(zhuǎn)了酒精所致的MRC I, Ⅲ和Ⅳ活性降低,與酒精模型組相比,差異具有顯著性(P0.05)。此外,Western blotting結(jié)果顯示,海兔素可上調(diào)Bcl-2的蛋白表達(dá),下調(diào)Bax的表達(dá),減少細(xì)胞色素C的釋放,抑制caspase-3和caspase-9的激活。同時,real-time PCR的結(jié)果也顯示,與酒精模型組相比,隨著海兔素作用劑量的增加,肝臟內(nèi)Bax、細(xì)胞色素C、caspase-9和caspase-3的mRNA的表達(dá)量逐漸下降,而Bcl-2基因mRNA表達(dá)的量逐漸增加,且差異具有顯著性(P0.05)。 結(jié)論： 1.根據(jù)對肝臟指數(shù)、肝臟病理學(xué)的觀察、肝功能酶的評價以及脂質(zhì)代謝的檢測,初步證實(shí)了海兔素對酒精誘導(dǎo)的大鼠肝損傷具有顯著的改善作用。 2.海兔素對酒精性肝損傷的保護(hù)作用機(jī)制可能與①抑制iNOS活性,下調(diào)肝臟iNOS蛋白表達(dá),減少NO的過量生成；②抑制大鼠肝臟ADH和微粒體CYP2E1活性,下調(diào)過表達(dá)的CYP2E1蛋白和：mRNA表達(dá)水平有關(guān)。然而海兔素作為一種具有iNOS抑制作用且可調(diào)節(jié)肝臟酒精代謝酶的物質(zhì)能否成為有效預(yù)防和逆轉(zhuǎn)酒精性肝損傷的藥物尚需進(jìn)一步的實(shí)驗(yàn)研究來佐證。 3.海兔素的補(bǔ)充可提高機(jī)體抗氧化能力抵抗酒精所致的氧化應(yīng)激,從而減輕脂質(zhì)過氧化以及DNA的氧化損傷,這可能是海兔素拮抗酒精性肝損傷的機(jī)制之一 4.過量的酒精攝入可導(dǎo)致肝細(xì)胞凋亡的發(fā)生,而海兔素可通過調(diào)控Bcl-2家族mRNA和轉(zhuǎn)錄蛋白的表達(dá)水平,抑制線粒體細(xì)胞色素C的釋放,阻斷caspase-3的活化,降低細(xì)胞色素C、caspase-9以及caspase-3mRNA的表達(dá),抑制線粒體介導(dǎo)的凋亡通路的激活,從而達(dá)到對酒精性肝損傷的保護(hù)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，

本文編號：1761080