过氧化氢酶在内毒素诱导的肝炎中的作用及其与二甲双胍保肝效应的关联性研究

发布时间：2018-03-02 07:28

本文关键词： 二甲双胍过氧化氢酶腺苷酸活化蛋白激酶急性肝炎内毒素　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：二甲双胍（metformin, MET）是临床上治疗2型糖尿病最常见的药物，近年来也发现MET还具有抗炎、抗氧化等药理学效应，我们前期研究发现MET可有效减轻脂多糖/右旋半乳糖胺（lipopolysaccharide/D-galactosamine, LPS/D-Gal）诱导的急性肝炎，但其机制尚不清楚。一般认为MET发挥效应主要通过激活腺苷酸活化蛋白激酶（AMP-activedprotein kinase, AMPK），，但近年来也发现MET可通过AMPK非依赖的途径发挥作用。我们近期还发现MET可能通过直接与过氧化氢酶（catalase, CAT）结合而增强后者的活性。本研究在LPS/D-Gal诱导的小鼠肝炎模型中探讨CAT与MET保肝效应间的可能关联。本研究分两部分。第一部分中，我们复制了LPS/D-Gal诱导的小鼠急性肝炎模型，通过应用CAT抑制剂氨基三唑（aminotriazole, ATZ）限制CAT发挥效应后，再检测模型小鼠血浆丙氨酸氨基转移酶（alanineaminotransferase, ALT）、门冬氨酸氨基转移酶（alanine aminotransferase,AST）水平、肝组织CAT活性、过氧化氢（hydrogen peroxide, H2O2）含量、丙二醛（malondialdehyde, MDA）水平及毮过氧化物酶（myeloperoxidase, MPO）水平并观察了肝组织病理形态学改变。我们也通过TUNEL染色分析、激活型caspase-3水平检测及caspase-3，-8，-9活性检测评估了肝细胞凋亡情况。我们对模型动物的生存时间也予以了记录和分析。第二部分中，我们尝试应用ATZ去阻断或用AMPK激活剂5-氨基-4-咪唑甲酰胺核苷酸（5-amino-4-imidazolecarboxamideriboside, AICAR）去模拟MET对LPS/D-Gal诱导肝损伤的保护效应。肝内氧化应激水平及组织损伤的程度分别通过检测肝组织CAT活性、H2O2含量及MDA水平、血浆ALT、AST水平及肝组织病理形态改变来评价。本研究第一部分发现：（1）ATZ抑制CAT后导致LPS/D-Gal诱导的肝损伤明显加重，表现为模型小鼠血浆ALT和AST的水平进一步升高、肝内MPO水平进一步升高、肝组织坏死和充血进一步加重、动物生存率降低；（2）ATZ抑制CAT后增强肝内氧化应激，表现为模型小鼠肝内H2O2含量进一步增高、MDA水平进一步上升；（3）ATZ抑制CAT后促进肝细胞凋亡，表现为模型小鼠肝内TUNEL阳性细胞数目增加、激活型caspase-3含量进一步增高、caspase-3，-8，-9活性进一步提升。第二部分实验发现：（1）MET处理可提升正常小鼠和LPS/D-Gal模型小鼠肝内CAT活性，ATZ可明显解除MET对模型小鼠肝内CAT活性的提升作用；（2）MET可显著降低模型小鼠肝内H2O2及MDA含量，ATZ仅能部分解除MET对模型小鼠肝内H2O2含量的抑制作用，ATZ不能解除MET对模型小鼠肝内MDA生成的抑制效应；（3）ATZ不能消除MET对模型小鼠血浆ALT和AST水平的下调作用，也不能消除MET对模型小鼠肝组织损伤的改善效应；（4）AICAR处理可显著减低LPS/D-Gal模型小鼠血浆ALT和AST水平并减轻肝小叶结构破坏及淤血。本研究结果提示抗氧化酶CAT可能在LPS/D-Gal诱导的急性肝炎中发挥一定保护功效；MET虽可提升LPS/D-Gal模型小鼠肝组织内CAT活性，但这一效应可能在MET的保肝效应中不占有主要地位；MET的保肝效应可能与AMPK激活或其他抗炎、抗氧化机制有关。
[Abstract]:Metformin (metformin, MET) is a clinical medicine for the treatment of type 2 diabetes is the most common in recent years, also found that MET has anti-inflammatory, antioxidant and other pharmacological effects, our previous study found that MET can effectively attenuate lipopolysaccharide / D-galactosamine (lipopolysaccharide/ - D-galactosamine, LPS/D-Gal) - induced acute hepatitis, but the mechanism is not clear. It is believed that MET effect mainly through activation of AMP activated protein kinase (AMP-activedprotein kinase, AMPK), but in recent years also found that MET can play a role in AMPK dependent way. We have also found that MET can directly with catalase (catalase, CAT) and enhanced the activity of the latter. This study was to investigate the possible association of CAT MET and hepatoprotective effect among hepatitis model mice induced by LPS/D-Gal.
This study is divided into two parts. The first part, we established acute hepatitis mouse model induced by LPS/D-Gal, through the application of CAT inhibitor - three (aminotriazole, ATZ) with limited CAT play effect, then detection model of mouse plasma alanine aminotransferase (alanineaminotransferase, ALT), aspartate aminotransferase (alanine aminotransferase. AST), liver CAT activity, hydrogen peroxide (hydrogen peroxide, H2O2) content, malondialdehyde (malondialdehyde, MDA) and Sha peroxidase (myeloperoxidase, MPO) level and liver tissue pathological changes were observed by TUNEL staining. We also analysis, detection and activation of Caspase-3, caspase-3 -8 level, -9 activity detection assessment of liver cell apoptosis. We model animal survival time will be recorded and analyzed. In the second part, we try to use the ATZ to block Or with the AMPK activator 5- amino -4- imidazole carboxamide nucleotide (5-amino-4-imidazolecarboxamideriboside, AICAR) to simulate the protective effect of MET on LPS/D-Gal induced liver injury. The level of oxidative stress and tissue damage in the liver were detected by liver tissue CAT activity, H2O2 content and MDA level, plasma ALT, AST levels and morphological changes of liver tissue pathology to evaluate.
The first part of this study found that: (1) ATZ CAT resulted in the inhibition of LPS/D-Gal induced liver injury was aggravated, as ALT and AST in plasma of mice model level increased further, intrahepatic MPO levels increased further, liver necrosis and congestion aggravated, the animal survival rate decreased; (2) ATZ after inhibition of CAT enhanced oxidation the stress in the liver, as the content of H2O2 in model mice liver is further increased, the level of MDA increased further; (3) hepatocyte apoptosis promoting ATZ inhibition of CAT, increase the performance for the number of TUNEL positive cells in the liver of mice model, activation of the caspase-3 content further increased, Caspase-3, -8, -9 activity increased further.
The second part of the experiment found that: (1) MET can increase the activity of CAT in normal mice and LPS/D-Gal mice liver, ATZ can relieve the effect of MET on the activity of CAT in model mice liver; (2) MET can significantly reduce H2O2 and MDA content in mice liver, ATZ can only inhibit MET decomposition in the Department the content of H2O2 in model mice liver, ATZ does not relieve the inhibitory effect of MET on mice liver MDA generation; (3) ATZ can not eliminate the downregulation of MET on mouse model of plasma ALT and AST levels, also cannot eliminate MET on the mouse model of liver tissue injury improvement effect; (4) AICAR treatment LPS/D-Gal model mice significantly decreased plasma ALT and AST levels and reduce the damage and congestion of liver lobules.
The results of this study suggest that antioxidant CAT may play a protective effect on acute hepatitis induced by LPS/D-Gal; MET can enhance the activity of CAT in liver tissue of mice in LPS/D-Gal model, but this effect may not occupy a major position in the hepatoprotective effect of MET in MET; hepatoprotective effect can be activated with AMPK or other anti-inflammatory, antioxidant mechanism relevant.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【共引文献】