【作者】 郭卉；

【导师】 齐俊英；

【作者基本信息】 华中科技大学 ， 内科学， 2013， 硕士

【摘要】 背景及目的重型肝炎发病率高，危害大，发病机制非常复杂，临床缺乏特异有效的治疗手段。经研究发现EETs具有抗炎、促进新生血管形成、调节离子通道、舒张血管以及促进肿瘤细胞的增殖、迁移等作用。EETs产生于花生四烯酸的第三条代谢途径，由CYP450表氧化酶的亚家族CYP2C和CYP2J的同工酶催化花生四烯酸生成具有较高生物活性的四种形式EETs:5,6-EETs、8,9-EETs、11,12-EETs和14,15-EETs。正常情况下生成的EETs在很短的时间内即被可溶性环氧化物水解酶(sEH)催化降解,产生DHET。通过抑制sEH（sEHI）可减慢EETs的降解速度，提高EETs的水平。目前关于EETs在病毒性暴发型肝炎中的研究在国内外未见报道。CYP450表氧化酶可催化多种物质的合成和代谢，亚家族以CYP2J和CYP2C家族为主，CYP2C9是CYP2C家族中的一员，主要分布在肝脏，约占肝微粒体CYP酶总量的20％，肝疾病对CYP2C9的影响比较明显。近十年来sEH抑制剂（sEHI）在活体和临床试验中的应用进步显著。研究表明注射sEHI可降低自发性高血压大鼠及血管紧张素介导的高血压大鼠的血压，并能减缓其肾脏的损害。近年来sEHI作为心血管疾病的新的治疗靶点已进入临床Ⅱ期试验。本研究通过建立MHV-3病毒诱导的小鼠病毒性暴发型肝炎动物模型，研究花生四烯酸CYP450表氧化酶代谢物EETs在病毒性暴发型肝炎中的作用及其可能机制，同时加用sEH抑制剂AUDA减慢EETs降解，升高体内内源性EETs水平，观察小鼠肝脏及血清内EETs有无升高，对小鼠生存率有无影响，可否作为病毒性暴发型肝炎抗炎治疗的新靶点。方法建立Balb/c小鼠经MHV-3病毒诱导的病毒性暴发型肝炎模型，用ELISA法检测不同时间点肝脏、血清内11,12-EETs、14,15-EETs含量变化；用免疫组化、western blot等方法检测肝脏内EETs是否由花生四烯酸经CYP2C9产生；加用sEH抑制剂AUDA5mg/kg减慢EETs的降解速度,观察小鼠肝脏及血清内EETs有无增多，小鼠生存时间有无延长。结果1、在暴发型肝炎病程中，11,12-EETs、4,15-EETs增多，在72h时P<0.05，具有统计学意义。提示在MHV-3诱导的小鼠暴发型肝炎的病理进程中，肝脏EETs水平发生改变，可能参与了该病理过程。2、在MHV-3病毒诱导的小鼠暴发型肝炎肝组织切片中，免疫组化显示CYP2C9无表达；在MHV-3病毒诱导的小鼠暴发型肝炎肝组织提取的蛋白中，western blot未见到阳性结果。证明肝组织内EETs并非由花生四烯酸经酶CYP2C9产生。3、加用sEH抑制剂AUDA后小鼠肝脏内11,12-EETs增多，但P>0.05，无统计学意义，且小鼠的生存时间无明显延长。结论1.EETs参与了MHV-3诱导的小鼠病毒性暴发型肝炎的病理过程，但不能阻断暴发型肝炎的进展，不能提高病毒性暴发型肝炎小鼠的生存时间。2.sEH抑制剂AUDA未能使小鼠暴肝模型肝脏11,12-EETs明显增多，小鼠生存率无改善；同时在该模型中，肝脏CYP2C9无表达，提示在该模型中，可能肝脏CYP2C9受损，导致上游EETs生成减少，未能发挥抗炎的保护作用。11,12-EETs、14,15-EETs增多，可能与其他类型的表氧化酶有关。更多还原

【Abstract】 Background and objective High incidence of severe hepatitis, hazard, verycomplex pathogenesis, clinical lack of specific effective treatment. Studies EETshave to dilate blood vessels, anti-inflammatory, promoting the formation of new bloodvessels, promote endothelial cell proliferation, migration, inhibition of endothelial cellapoptosis, promote tumor proliferation and metastasis, and so on. The epoxygenaseCYP enzyme（sCYP450）generate epoxyeicosatrienoic acids (EETs), by catalysing theepoxidation of arachidonic acid olefin bonds, resulting in the production of fourregioisomeric EETs:5,6-EETs,8,9-EETs,11,12-EETs and14,15-EETs. The EETs aremetabolized to their di-hydroxyl derivatives (DHET) by the soluble epoxide hydrolase(sEH) Recently, sEH inhibitors (sEHIs) have been developed to enhance the actions ofEETs. CYP450is Ι metabolic enzymes, catalyzed many xenobiotics metabolism andsteroid hormone, arachidonic acid, etc. The synthesis and metabolism of endogenoussubstances. CYP2C9is one of the family of CYP2C, isozyme, mainly distributed inliver tissue, and accounts for about20%of the total liver microsomal CYP enzyme.The rapid development of sEHIs for in vivo use and clinical testing in the past decadeis remarkable. A landmark study showed that injection of a sEHI to the spontaneouslyhypertensive rat (SHR) and angiotensin-induced hypertension lowered blood pressure.Another breakthrough showed that an orally administered sEHI was antihypertensive and slowed the progression of renal damage. Following this, a number of studies haveprovided exciting findings on the broad potential for sEHIs as cardiovasculartherapeutic agents, and a first in class sEHI began clinical Phase IIa testing this year.This research through the MHV-3infection to establish animal model of viralfulminant hepatitis in mice study arachidonic acid oxide metabolites EETs CYP450table in the role of viral fulminant hepatitis and its possible mechanism. And thesurvival time of mice will extend or not when add the sEH inhibitors of MHV-3virusinfection,,whether the sEHI can be the new severe hepatitis therapeutic agents.Methods Establish Balb/c mice MHV-3virus infection model with ELISAmethod to detect different time points within the liver, serum11,12-EETs,14,15-EETs content changes; Using immunohistochemistry, western blot and other methodsto detect whether EETs in the liver by arachidonic acid by CYP2C9. Add with sEHinhibitors AUDA5mg/kg slows the degradation of EETs observe the survival time ofmice after the presence of extensionResults1, In the course of fulminant hepatitis,11,12-EETs and14,15-EETsincreased, at the time of72h (P <0.05, with statistical significance). Prompt EETs inMHV-3induced pathological process of fulminant hepatitis in mice.2, In MHV-3fulminant hepatitis virus infection mice liver tissue slices, rows of CYP2C9immunohistochemical not see positive results; In MHV-3fulminant hepatitis virusinfection mice liver tissue protein extraction, do western blot to see positive results.Show the EETs in the liver tissue is not produced by arachidonic acid via enzymeCYP2C9.3, add with sEH inhibitors of MHV-3virus infection after AUDA,11,12-EETs gradually increased, at the time of72h (P>0.05, without statisticalsignificance). But it does not extend survival time of mice.Conclusion1.EETs participation MHV-3-induced murine viral fulminant hepatitispathological process, but did not block the progress of fulminant hepatitis, can notimprove the survival time of mice of fulminant viral hepatitis. 2.sEH inhibitor AUDA failed to mice violence liver model liver11,12-EETs increasedsignificantly, no improvement in the survival rate of mice; In this model, liverCYP2C9expression, suggesting that in this model, possible liver CYP2C9damaged,leading to a reduction in upstream EETs generated, failed to play the role ofanti-inflammatory protection.11,12-EETs,14,15-EETs increase may be related toother types of table oxidase.更多还原