【作者】 王志军；

【导师】 王丽颖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 酒精性肝病已经成为我国第二大肝病。有研究表明，酒精导致的肝损伤与天然免疫的激活及炎性细胞因子的产生有关。目前认为，Toll样受体（Toll-likereceptor, TLR）4与酒精性肝损伤关系密切，我们考虑，既然TLR4与其配体的相互作用在酒精性肝损伤中发挥着重要作用，那么其它TLR与其配体的作用是否也会影响酒精性肝损伤呢？而且，酒精会导致肝细胞的凋亡或坏死，那么肝细胞释放出来的DNA是否会通过与肝脏内细胞的TLR9结合从而影响酒精性肝损伤呢？酒精可致肠粘膜渗透性增加，肠道内的细菌DNA是否也会进入肝脏并通过TLR9对肝损伤有影响呢？基于以上考虑及本课题组在免疫调节方面积累的大量工作基础，我们通过给灌酒小鼠腹腔注射TLR9的配体——人工合成的含CpG基序的单链脱氧寡核苷酸（CpG ODN），来研究肝脏内TLR9与其配体的相互作用对酒精性肝损伤的影响。我们选用雄性ICR小鼠作为研究对象，通过给小鼠按7g/kg50%乙醇溶液灌胃的方法制造急性酒精性肝损伤模型，以血清中谷丙转氨酶（ALT）水平及肝脏病理变化作为衡量肝损伤的指标。我们首先选择了本科室自行研制的一种C型CpG ODN—YW002作为研究对象，研究发现，在灌酒的同时及灌酒后给予YW002均对小鼠的血清中ALT水平没有影响。之后，在继续研究YW002的同时，也对2216（A型CpG ODN）和200（6B型CpG ODN）对酒精性肝损伤的影响进行了研究。结果显示，2216和2006均可加重酒精性肝损伤，其中2216的作用更明显，而YW002可以一定程度的减轻灌酒小鼠肝脏的病理改变。在对A型CpG ODN加重酒精性肝损伤的可能机制进行研究时，我们发现2216可增加灌酒小鼠肝脏内TLR9及TNF-α的表达，而YW002可增加小鼠肝脏内IFN-α的表达。我们推测这可能就是这两种类型CpG ODN分别具有加重或减轻酒精性肝损伤的原因。在体外实验中，我们发现：2216可增加肝癌细胞系Hep G2细胞线粒体膜的通透性，这可能也与其能够加重酒精性肝损伤有关。随后我们又对另一种类型的ODN—抑制性ODN对酒精性肝损伤的影响进行了初步研究，我们所选用的ODN均为本科室自行研制。研究发现，灌酒同时给予抑制性ODN—MT01及免疫惰性ODN—MS19均可加重小鼠的急性酒精性肝损伤；灌酒前给予MT01也可加重小鼠的酒精性肝损伤，其作用具有剂量依赖性；但灌酒后给予抑制性ODN不但没有表现出加重肝损伤的作用，MT01还表现出减轻酒精性肝损伤的作用。综上，我们的研究表明，不同类型的CpG ODN通过对TLR9激活程度的不同在酒精性肝损伤中发挥不同的作用，A型和B型CpG ODN在灌酒后给予可加重小鼠的酒精性肝损伤，而C型CpG ODN对酒精性肝损伤表现出一定的保护作用。抑制性ODN在饮酒后应用具有减轻酒精性肝损伤的潜能，有望在酒精性肝损伤的预防及治疗中发挥重要的作用。更多还原

【Abstract】 Due to its unique location and function, liver is susceptible to various types ofinjury caused by viral infections, drugs, excessive free fatty acids and autoimmunereactions. In addition, liver injuries can be caused by alcohol abuse, which leads toalcoholic liver disease (ALD), a major liver disease in western countries, the secondliver disease in Asian countries next to viral hepatitis. The severity of ALD can rangefrom steatosis, steatohepatitis to cirrhosis and hepatocellular carcinoma. Nearly70%of alcoholic abusers develop steatosis,20%-30%develop steatohepatitis, and10%develop cirrhosis. In America, the liver failure evolved from ALD is the second mostcommon reason for liver transplantation. Up to now, the precise mechanism of howalcohol damages liver still remains unknown, but accumulating evidence suggests thatactivation of innate immunity and production of cytokine play an important role in thedevelopment of alcoholic liver injury. Synthetic ODN (oligodeoxynucleotide),including CpG ODN and suppressive ODN, has been proved immunomodulatory andshown great potential in the research of treatment of various deseases.According to reported methods and with preliminary experiments, we found away to establish the mouse model of acute alcohol-induced liver injury. We appliedODN to the model mice to investigate the influence of ODN on acute alcohol-inducedliver injury and upon the results, we conducted following experiments to explore thepossible mechanism.1Establishment of the mouse model of acute alcohol-induced liver injuryAn animal model is required to seek possible agents for lessening acutealcohol-induced liver injury caused by excessive alcohol drinking. In this study, toevaluate whether oligodeoxynucleotides (ODN) could intervene the development ofacute alcohol-induced liver injury, by following a method reported, mice wereadministered with50%alcohol at6g/kg (BW) intragastrically (i.g.) for three times at12hours interval. In the mice, the liver injury was monitored by the level of alamineaminotransferase (ALT) in their blood. At6h after the last administration of alcohol,the mice were bled and their sera were assayed. However, the assay showed no significant changes of ALT levels in the sera of the alcohol-induced mice. Toestablish the model, we tried to improve the method by administering the mice withalcohol once a day at7g/kg or8g/kg per mouse for several times. The tested micewere observed daily. In8g/kg group,5of7mice were deeply drunk and succumbedwithin4days after the alcohol giving. Comparatively, all of the mice in the7g/kggroup were drunk and survived, with the increased level of ALT in their blood whendetected in7or9days after alcohol giving. Next, we tried to administer the mice withalcohol at8g/kg once only and found that the mice couldn’t bear the alcohol andfailed to recover from deep drunk within12h. The failure could be due to the severedamage in stomach of the mice in addition to the liver injury. Severe hyperemia andeven hemorrhage were observed in the stomach of the mice received alcohol at8g/kg,compared with those of the mice received alcohol at7g/kg or H2O. To furtherimprove, mice were administered with alcohol at7g/kg once. At12h after theadministration, all of the mice recovered from drunk. At6and12h after theadministration, the mice were bled for detecting their serum ALT levels. Immediatelyafter the second bleeding, the mice were sacrificed and their livers werehistopathologically examined. The result showed within6hours, alcohol inducedsignificant elevation of serum ALT levels (p<0.01) and the elevation lasted to12hours (p<0.05) after alcohol intake, with a trend of declining. The histopathologicalexamination revealed dramatic steatosis and vacuolar degeneration caused by alcoholin liver of the mice. The results suggested that the mice received alcohol at7g/kgonce intragastrically could be used as an animal model for testing agents for theirefficacies on intervening acute liver injury caused by excessive alcohol intake.2Effect of CpG ODNs on acute alcohol-induced liver injury in miceIt is reported that alcoholic liver injury is tightly associated with activation ofinnate immunity and production of proinflammatory cytokine. It is now accepted thatTLR4plays an important role in alcoholic liver injury. We wonder whether otherTLRs are also linked to alcoholic liver injury besides TLR4, such as TLR9. Based onthe above consideration, we applied CpG ODN to the alcohol-feeding mice for theresearch of the influence the interaction between TLR9and its ligand on alcoholicliver injury.2.1Effect of simultaneous treatment of C-type CpG ODN on acutealcohol-induced liver injury in mice As C-type CpG ODN possesses the properties of both A-type and B-type CpGODN, so we firstly choose C-type CpG ODN as the subject of our research andYW002, a C-type CpG ODN designed by our lab, was used. We inject YW002i.p.immediately after the alcohol administration and the mice were bled at6h and12hafter alcohol intake for ALT detection. The result showd that YW002could neitherelevate nor lower the serum ALT levels of the alcohol-feeding mice, indicating thesimultaneous treatment of C-type CpG ODNs has no obvious effect on acutealcohol-induced liver injury.2.2Effect of pre-treatment of C-type CpG ODN on acute alcohol-induced liverinjury in miceMice were injected with YW002i.p. at25μg per mouse.40min later, the micewere treated with alcohol. The mice were bled at6h and12h after alcohol intake forALT detection. The result showd that YW002could neither elevate nor lower theserum ALT levels of the alcohol-feeding mice, indicating the pre-treatment of C-typeCpG ODNs has no obvious effect on acute alcohol-induced liver injury.2.3Effect of post-treatment of CpG ODN on acute alcohol-induced liver injury inmice40min after alcohol intake, the mice were intraperitoneally injected with2216(A-type CpG ODN),2006(B-type CpG ODN) or YW002at dose of25μg/mouse andtheir blood was collected at6and12h after the alcohol feeding for detecting serumALT levels. After the second bleeding, the mice were sacrificed and their livers werehistopathologically examined. The results showed that2216induced a dramaticincrease in serum ALT levels in alcohol-feeding mice at6h and12h after the feedingcompared with that in the mice fed with alcohol only (P=0.013and P=0.020). Smallincrease in ALT level at6h and obvious increase in ALT level at12h was observed in2006treated alcohol-feeding mice. YW002, however, still could not result in obviouschange in serum ALT level in the alcohol-feeding mice. The pathologicalexaminations revealed that2216treatment could cause more severe pathologicalchanges and the pathological score was much higher than that in alcohol-feeding mice(P<0.05), whereas the liver pathological changes in2006-or YW002-treatedalcohol-feeding mice were similar to that in alcohol-feeding mice and YW002seemedable to lessen pathological changes in the liver. The results demonstrate that A-typeCpG ODN and B-type CpG ODN could aggravate acute alcohol-induced liver injury in mice, and that C-type CpG ODN could not. Comparatively,2216displayed moredramatic aggravating effect than2006on acute alcohol-induced liver injury.To exclude the possibility that CpG ODN themselves could cause acute liverinjury, water-feeding mice were injected i.p with2216,2006or YW002at25μg/mouse and their blood was collected at6h and12h after water administration forserum ALT detection. The result showed that2216,2006or YW002could not elevateserum ALT level in the mice at6h. However, the serum ALT levels were increased at12h in2216-and2006-treated mice with similar levels as that in alcohol-feedingmice. Notably, at12h, YW002displayed no effect on elevating serum ALT level inmice.2.4Dose effect of2216on aggravating acute alcohol-induced liver injury in miceTo confirm its aggravating effect, mice were administered with2216at differentdoses at40min after alcohol feeding via i.p. and their blood was collected at6and12h after alcohol feeding for detecting serum ALT levels. The results showed that2216at12.5,25and50μg/mouse, not at3.125μg/mouse, caused a significant increase ofserum ALT levels in the mice at6h and12h. After being standardized by mean levelof serum ALT in alcohol-feeding mice, the positive rate of serum ALT levels was2/8(25%),6/9(66.67%),8/10(80%) and9/10(90%) of the mice treated with2216at3.125,12.5,25and50μg/mouse at6h, respectively. The data indicated that2216could aggravate acute alcohol-induced liver injury in mice in a dose-dependentmanner.2.5Route effect of2216on aggravating acute alcohol-induced liver injury in miceAlcohol-feeding mice were administered with25μg/mouse of2216at40minafter alcohol intake via i.p., s.c. or i.g. administration and their blood was collected forALT detection. The results showed that2216injected i.p or s.c caused significantelevation of serum ALT levels in the alcohol-feeding mice at6h and12h (P<0.05),whereas i.g. administration of2216only caused a slight elevation of the serum ALTlevel in the mice, with no statistical significance. Histopathologically, i.p. or s.cadministration of2216could cause more severe pathological changes in the liversthan that in alcohol-feeding mice and no obvious exacerbation or lessening wasobserved in the livers of the mice when the2216was administered i.g. The resultsindicated that2216via i.p and s.c could aggravate acute alcohol-induced liver injuryin mice. 3Mechanism exploration of A-type CpG OCDN on aggravating acutealcohol-induced liver injury in mice3.1Influence of recombinant IFN-α on alcohol-induced liver injury in miceConsidering the IFN-α inducing property of A-type CpG ODN, we firstspeculated that IFN-α produced by immune cells upon the stimulation of2216mightcontribute to the acute alcohol-induced liver injury. To verify this assumption, weadministered the model mice with recombinant IFN-α at10,000IU/mouse afteralcohol intake. The result showed that injection of IFN-α could not significantlyelevate the serum ALT levels in model mice, suggesting that2216-aggravatedalcohol-induced liver injury might not be associated with IFN-α production.3.2Effect of CpG ODN on expression of TLR9, TLR4, TNF-α, IFN-α and IFN-βin the livers of alcohol-feeding miceTo explain the mechanism of different effect of CpG ODN on aggravating acutealcohol-induced liver injury, we firstly measured the levels of TNF-α in the seracollected at1.5h and6h after alcohol or water intake from the tested mice and foundthat the levels of the serum TNF-α are inconsistent with the severity of acute liverinjury in the mice. This implies that systemically produced TNF-α might not beinvolved in the acute alcohol-induced liver injury. Thus, we moved to detect theexpression of TNF-α in the liver tissue with Real-time RT-PCR to find whether thelocally expressed TNF-α was involved in the development of acute alcohol-inducedliver injury in the model mice. At the same time, the expression of TLR-9, IFN-α andIFN-β in the liver tissue was also detected. The mice were administered with alcoholfollowed by2216,2006and YW002, respectively, at40min after alcohol feeding.The mice were sacrificed at1.5h after alcohol feeding and their livers were isolatedfor extracting total RNAs. The total RNAs were analyzed with Quantitative RT-PCRfor levels of mRNA of TLR9, IFN-α, IFN-β and TNF-α. The result showed that2216could significantly up-regulate TLR9expression in liver of alcohol-feeding mice(P<0.05), whereas2006and YW002couldn’t, hinting that2216-caused aggravationon acute alcohol-induced liver injury in mice could be associated with TLR9up-regulation. Both2216or YW002could significantly up-regulate IFN-α mRNAexpression in liver of the mice (p<0.05). Noticeably, the level of IFN-α mRNAinduced by2216was significantly lower than that induced by YW002(P=0.05). As toIFN-β mRNA expression, no significant difference was found in livers of the alcohol-feeding mice received2216,2006or YW002. Both2216and2006couldsignificantly elevate the levels of TNF-α mRNA expression in livers of thealcohol-feeding mice (P<0.05) whereas YW002only induced a slight elevation inTNF-α expression. Comparatively, the TNF-α mRNA expression induced by2216was significantly higher than that induced by YW002(P=0.05). Since TLR4is tightlyrelated to TNF-α production in the liver, we also detected its expression in the liver ofalcohol-feeding mice treated with2216and found that2216could not elevate theTLR4expression in liver tissues of the mice. The data suggest that the effect of2216on acute alcohol-induced liver injury may not be associated with the TLR4expression.Collectively, these findings suggest that the aggravating effect of2216on acutealcohol-induced liver injury in mice could be associated with the up-regulation ofTNF-α mRNA, presumably, via signaling pathway of up-regulated TLR9.3.3In vitro effect of A-type CpG ODN on liver cellsBesides the possible mechanism mediated by immne responses and production ofcytokine, we wonder whether A-type CpG ODN could damage the hepatocyte directly,that is to say, the direct action of A-type CpG ODN on hepatocyte. To investigate this,we cultured Hep G2cells, human hepatocellular carcinoma cell line cells, in mediumcontaining alcohol at0,10,20,40,80,160,320,640mmol/L or alcohol plus2216at2μg/ml for24h, and then the cytoviability was determined by MTT assay. The resultshowed that the cells treated with2216plus alcohol at low concentration in a range of0-80mmol/L displayed higher OD value compared with those treated with alcoholalone (p<0.05).To confirm the findings, Hep G2cells were cultured in the presence of2216at2μg or4μg/ml with alcohol at0,40,80or160mmol/L and assayed by following thesimilar procedure as above. The result showed that the cells treated with2216at2μgor4μg/ml plus alcohol at each concentration displayed higher OD value (p<0.05)compared with those treated with alcohol only (p<0.05). With alcohol at160mmol/L,2216at4μg/ml was more potent than that of2216at2μg/ml (p<0.05).In the experiments above, we have only tested2216. To verify that this effect issequence-specific, we cultured Hep G2cells (2×104/well in96-well plate) in mediumcontaining alcohol (40mmol/L) with or without ODN (2216,2006, YW002, MT01,MS19or SAT05f) at2μg/ml for24h and then underwent MTT assay. The resultshowed that the cells treated with2216displayed the highest OD value compared with those treated with other ODNs(p<0.05), implying that the above effect induced by2216is sequence-specific.To confirm whether2216could indeed increase the number of living cells,crystal violet staining and cell counting under microscope were adopted. Hep G2cellswere cultured (2×104/well in96-well plate) in medium containing different amount ofalcohol with or without2216(2μg/ml) for24h and then the number of living cellswas detected. The data detected by these two methods indicated that treatment of2216could not increase the number of living cells. As MTT assay is based onmitochondrion, we deduce that the higher OD value detected by MTT assay maymean that2216increase the permeability of mitochondrion.Considering mitochondrial involvement in apoptosis and association of apoptosiswith alcoholic liver injury, we cultured Hep G2cells in medium containing alcohol oralcohol plus2216.24h later, the cells were harvested, and their genomic DNA wasisolated and subjected to agarose gel electrophoresis for revealing DNA ladders, asign of apoptosis. No DNA ladders were observed in the cells treated with2216or2216plus alcohol.3.4Effect of A-type CpG ODN on the content of MDA of alcohol-feeding miceIt is well established that the metabolism of alcohol generates reactive oxygenspecies (ROS). If ROS is not eliminate promptly, the excessive ROS would react withcellular macromolecules, causing the damage to the cells and tissue. This is one of theputative mechanisms about alcoholic liver injury. To investigate whether theaggravation effect of2216is mediated by the induction of ROS, we detected thecontent of MDA, a product of lipid reacted with ROS, in both the sera and liver tissueof mice. The result showed that the content of MDA was not increased by2216inboth the sera and liver tissue of the model mice.4Effect of suppressive ODN on acute alcohol-induced liver injury in miceBased on the effect of CpG ODNs on acute alcohol-induced liver injury andnegative regulatory property of suppressive ODN on the activation of innate immunity,we wonder whether suppressive ODN could prevent or cure alcoholic liver injury bysuppressing innate immunity or production of inflammatory cytokine.4.1Effect of simultaneous treatment of suppressive ODN on acutealcohol-induced liver injury in miceImmediately after the alcohol administration, mice were intraperitoneally injected with MT01, a suppressive ODN designed by our lab, or with MS19, an inertODN, at25μg per mouse. The mice were bled at both6h and12h after alcoholintake for serum ALT detection. It was found that MT01or MS19significantlyelevated serum ALT levels in the alcohol-feeding mice at6h (p<0.01) and12h(p<0.05) after alcohol intake, compared with that in the alcohol-feeding mice receivedPBS, suggesting that both MT01and MS19could aggravate the acute alcohol-inducedliver injury.4.2Effect of pre-treatment of suppressive ODN on acute alcohol-induced liverinjury in miceMice were intraperitoneally injected with MT01at12.5μg,25μg and50μg permouse, respectively.40min later, the mice were treated with alcohol and bled at both6h and12h after alcohol intake for serum ALT detection. The result showed that50μg of MT01could significantly elevate serum ALT levels of model mice at both6h(p<0.05) and12h (p<0.01) after alcohol intake, while12.5μg or25μg of MT01could not.4.3Effect of post-treatment of suppressive ODN on acute alcohol-induced liverinjury in miceUpon that the simultaneous treatment and pre-treatment of MT01could notalleviate acute alcohol-induced liver injury in mice, we next designed to investigatethe effect of post-treatment of suppressive ODN on acute alcohol-induced liver injuryin mice and another suppressive ODN titled SAT05f which is designed in our lab wasalso involved in the experiment. At40min after the alcohol administration, the micewere intraperitoneally injected with MT01, MS19, or SAT05f at25μg per mouse. At6h and12h after the alcohol intake, the mice were bled for detecting their serum ALTlevels. After the second bleeding, the mice were sacrificed and their livers werehistopathologically examined. The result showed that none of the used ODN couldeither elevate or lower the serum ALT levels of the model mice. Histopathologicalexamination revealed that MT01could alleviate the pathological changes of the liversof the alcohol-feeding mice and the pathological score also reflects this trend. As faras MS19and SAT05f are concerned, there were no obvious pathological changes inthe livers of the alcohol-feeding mice when treated with MS19or SAT05f.5Possible mechanism of the effect of MT01on acute alcohol-induced liver injuryin mice In order to clarify why simultaneous treatment and post-treatment of MT01havedifferent effect on acute alcohol-induced liver injury in mice, we analyzed theexpression of TNF-α and IFN-α,2cytokines supposed tightly associated withalcoholic liver injury. The alcohol-feeding mice were treated with MT01immediatelyor40minutes after alcohol intake.1.5h after alcohol feeding, the mice weresacrificed and livers were isolated for extracting total RNA. The total RNAs weredetected with Quantitative RT-PCR for levels of mRNA of IFN-α and TNF-α. TheTNF-α detection showed that there was no obvious differences in the level of TNF-αamong different groups and IFN-α detection showed that simultaneous treatment ofMT01could significantly increase the expression of IFN-α in the livers of thealcohol-feeding mice (P＜0.05), whereas the post-treatment of MT01could not.To sum up, we established the mouse model of acute alcohol-induced liver injuryby administering the mice with alcohol i.g. at7g/kg once. Either2216（A-type CpGODN） or2006（B-type CpG ODN） could aggravate acute alcohol-induced liverinjury in mice and2216displayed displayed more dramatic aggravating effect than2006. Comparatively, YW002（C-type CpG ODN） could alleviate the liver injurylightly. As far as MT01(suppressive ODN) is concerned, simultaneous treatment ofMT01could aggravate acute alcohol-induced liver injury and post-treatment of MT01could alleviate it to some extent.更多还原