【作者】 周洪青；

【导师】 罗欣拉；

【作者基本信息】 湖北中医学院 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:晚期糖基化终末产物(AGEs)是指蛋白质、脂质或核酸等大分子在没有酶参与的条件下,自发地与葡萄糖或其他还原单糖反应所生成的稳定的共价化合物。近来研究认为肝脏在AGEs的代谢中起主要作用。其中肝硬化与AGEs关系密切,肝硬化患者血清AGEs水平是升高的。其发生机制在于:一方面,肝脏是AGEs主要的代谢部位,肝硬化时伴随的氧应激及内毒素血症、肝肾功能不全等可促进AGEs的产生和(或)抑制其降解;另一方面,血液和肝组织内AGEs增加可通过促进肝脏的氧化应激/脂质过氧化损伤、炎症反应以及肝纤维化等途径,诱发或加重肝脏损伤。肝硬化时普遍存在的肠源性内毒素血症(IETM)可能有助于AGEs的形成,因为内毒素可以通过激活单核巨噬细胞系统介导的细胞毒性作用,单核巨噬细胞系统包括单核细胞和枯否细胞,被脂多糖(LPS)激活后,可表达、分泌各种细胞因子和炎性介质,如肿瘤坏死因子-a(TNF-a)、白细胞介素(IL-1、IL-6、IL-8)、白三烯(LTs)、血小板活化因子(PAF),氧自由基、一氧化氮(NO)以及内皮素(ET-1)等,这些介质构成一个独特的网络系统,共同参与对肝组织及其它器官损伤,乃至出现多器官衰竭。这也可能与内毒素诱导氧应激,从而增加AGEs形成有关。降低或抑制AGEs水平,可以减少氧应激和促进炎症反应,缓解肝纤维化程度。而治疗内毒素血症,抑制内毒素的生成和对内毒素进行清除,减少氧应激,可以降低AGEs水平,减轻肝损伤,有利于肝硬化程度的好转。乳黄制剂是国家名老中医张赤志教授根据中医理论和临床经验总结出治疗肝性脑病的中药制剂,在前期防治急性肝性脑病和肝硬化IETM中的实验研究中发现乳黄制剂能降低血清内毒素水平,为了进一步研究该制剂对肝硬化模型大鼠AGEs的影响,我们将从细胞分子水平进行探讨,为肝硬化的防治提供新的理论依据。方法:SPF级雄性Wister大鼠88只,体重200～250克,随机分为6组:正常组、模型组、预防组、预防对照组、模型治疗组、模型治疗对照组。除正常组外,其余各组均采用CCL4复合因素法制造肝硬化模型,饲养10周。正常组、模型组生理盐水1ml/100g灌胃;预防组、预防对照组在造模同时分别给予乳黄制剂、乳果糖2ml/d灌胃;造模第9周开始模型治疗组、模型治疗对照组分别给予乳黄制剂、乳果糖2ml/d灌胃连续2周。第10周末处死大鼠取材。(1)酶联免疫分析法(ELISA)定量测定大鼠肝组织匀浆AGEs含量(2).在无热源条件下采右心室血5 ml检测血清ALT、AST、ALB、TP、TBil含量;(3)取肝左叶作病检。结果:(1)乳黄制剂可明显降低肝硬化大鼠AGEs含量水平(P＜0.05);(2)乳黄制剂可明显降低肝硬化大鼠血清ALT、AST、TBil水平(P＜0.05)尤以预防组效果明显;(3)乳黄制剂能改善预防组和模型治疗组肝组织的肝硬化以及纤维组织增生、炎性细胞浸润程度,其中预防组肝组织的病理变化改善明显。结论:乳黄制剂能有效降低肝组织中AGEs的水平,有利于肝功能的改善,肝硬化程度的好转,可能有下列机制实现:①乳黄制剂通过减少内毒素的生成,促进肝功能恢复,使肝细胞清除AGEs的能力增强;②加快肠道内毒素的清除,减少氧应激,从而减少AGEs的形成。更多还原

【Abstract】 Objective:Advanced glycation end products(AGEs) refers to protein,lipid or nucleic acid molecules in the absence of enzyme under the conditions of participation,spontaneous with glucose or other monosaccharides to restore the response generated by a stable covalent compounds.Recent studies suggest that the liver in the metabolism of AGEs play a major role.AGEs are closely related and one of liver cirrhosis,liver cirrhosis,the level of serum AGEs are elevated.Its mechanism is to happen:on the one hand,liver AGEs are the major site of metabolism,liver cirrhosis,when accompanied by oxidative stress and endotoxemia,liver and kidney dysfunction,such as AGEs can promote the generation and(or) inhibiting its degradation;another connection,the blood and liver tissue AGEs increase through the promotion of liver oxidative stress/lipid peroxidation injury,inflammatory response and liver fibrosis,such as ways to induce or aggravate liver damage.When the prevalence of cirrhosis intestinal endotoxemia(IETM) probably contribute to the formation of AGEs,because endotoxin can activate monocyte-macrophage system-mediated cytotoxicity,monocytemacr-ophage system,including mononuclear cells and Kupffer cells,the lipopolysaccharide(LPS) activated,can express and secrete various cytokines and inflammatory mediators,such as tumor necrosis factor-a(TNF-a),interleukin-(IL-1,IL-6,IL-8), leukotrienes(LTs),platelet-activating factor(PAF),oxygen free radicals,nitric oxide(NO) and endothelin(ET-1),which constitute a unique medium network system,the joint participation of the liver tissue and other organ damage,and even the emergence of multi-organ failure.This also may be associated with endotoxin-induced oxidative stress,thus increasing the formation of AGEs.Reduce or suppress the level of AGEs can reduce oxidative stress and promote the inflammatory response and alleviate the degree of liver fibrosis.And treatment of endotoxemia and suppress the generation of endotoxin and endotoxin removal,reducing oxidative stress,can reduce the level of AGEs to reduce liver injury and is conducive to the improvement of the degree of liver cirrhosis.RuHuang preparation is old country TCM Professor Zhang Chizhi According to Chinese medicine theories and clinical experience summarize the treatment of hepatic encephalopathy in Chinese medicine preparations,at early prevention and treatment of acute hepatic encephalopathy and cirrhosis IETM in the experimental study of RuHuang preparation is found to reduce serum endotoxin level in order to further study of the preparation on liver cirrhosis rat model the effects of AGEs,we will explore the cellular and molecular level,in order to provide a new prevention and treatment of cirrhosis of the theoretical basis.Methods:SPF level 88 male Wister rats,weighing 200 to 230 grams,were randomly divided into 6 groups:normal group,model group,prevention group,the prevention of the control group,model group,model control group.Except the normal group,the rest of the group factor method used to create composite CCL4 liver cirrhosis model,reared for 10 weeks.Normal group,model group distilled water 1ml/100g orally;prevention group,the prevention model in the control group were given RuHuang preparation at the same time,lactulose 2ml/d orally;model the first nine weeks beginning model of the treatment group,model control group Wong agents were given RuHuang preparation,lactulose 2ml/d orally for 2 weeks.No.10 rats were killed over the weekend based.(1) enzyme-linked immunosorbent assay(ELISA) quantitative determination of rat liver tissue homogenate AGEs content(2) in the absence of heat mining right ventricle under the conditions of 5 ml of serum ALT,AST,ALB,TP,TBil content;(3) check for diseases of liver left lobe seizure.Results:(1) RuHuang preparation can significantly reduce the concentration level of AGEs in rats with cirrhosis(P＜0.05);(2) RuHuang preparation can be significantly reduced in cirrhotic rat serum ALT,AST levels(P＜0.05) particularly effective prevention group;(3) RuHuang preparation can improve the preventive treatment group and model group cirrhotic liver tissue and fibrous tissue hyperplasia,the extent of inflammatory cell infiltration,one of the preventive group,the pathological changes in liver tissue to improve significantly.Conclusion:RuHuang preparation can effectively reduce the liver tissue levels of AGEs,there is conducive to the improvement of liver function,cirrhosis degree of improvement possible implementation has the following mechanisms:①to reduce the generation of endotoxin,and promote the restoration of liver function,so that removal of liver cells enhance the ability of AGEs;②to speed up the removal of intestinal endotoxin to reduce oxidative stress,thereby reducing the formation of AGEs.更多还原