本文關(guān)鍵詞： 二甲雙胍 過氧化氫酶 腺苷酸活化蛋白激酶 急性肝炎 內(nèi)毒素　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：二甲雙胍（metformin, MET）是臨床上治療2型糖尿病最常見的藥物，近年來也發(fā)現(xiàn)MET還具有抗炎、抗氧化等藥理學(xué)效應(yīng)，我們前期研究發(fā)現(xiàn)MET可有效減輕脂多糖/右旋半乳糖胺（lipopolysaccharide/D-galactosamine, LPS/D-Gal）誘導(dǎo)的急性肝炎，但其機(jī)制尚不清楚。一般認(rèn)為MET發(fā)揮效應(yīng)主要通過激活腺苷酸活化蛋白激酶（AMP-activedprotein kinase, AMPK），，但近年來也發(fā)現(xiàn)MET可通過AMPK非依賴的途徑發(fā)揮作用。我們近期還發(fā)現(xiàn)MET可能通過直接與過氧化氫酶（catalase, CAT）結(jié)合而增強(qiáng)后者的活性。本研究在LPS/D-Gal誘導(dǎo)的小鼠肝炎模型中探討CAT與MET保肝效應(yīng)間的可能關(guān)聯(lián)。 本研究分兩部分。第一部分中，我們復(fù)制了LPS/D-Gal誘導(dǎo)的小鼠急性肝炎模型，通過應(yīng)用CAT抑制劑氨基三唑（aminotriazole, ATZ）限制CAT發(fā)揮效應(yīng)后，再檢測模型小鼠血漿丙氨酸氨基轉(zhuǎn)移酶（alanineaminotransferase, ALT）、門冬氨酸氨基轉(zhuǎn)移酶（alanine aminotransferase,AST）水平、肝組織CAT活性、過氧化氫（hydrogen peroxide, H2O2）含量、丙二醛（malondialdehyde, MDA）水平及毮過氧化物酶（myeloperoxidase, MPO）水平并觀察了肝組織病理形態(tài)學(xué)改變。我們也通過TUNEL染色分析、激活型caspase-3水平檢測及caspase-3，-8，-9活性檢測評估了肝細(xì)胞凋亡情況。我們對模型動物的生存時間也予以了記錄和分析。第二部分中，我們嘗試應(yīng)用ATZ去阻斷或用AMPK激活劑5-氨基-4-咪唑甲酰胺核苷酸（5-amino-4-imidazolecarboxamideriboside, AICAR）去模擬MET對LPS/D-Gal誘導(dǎo)肝損傷的保護(hù)效應(yīng)。肝內(nèi)氧化應(yīng)激水平及組織損傷的程度分別通過檢測肝組織CAT活性、H2O2含量及MDA水平、血漿ALT、AST水平及肝組織病理形態(tài)改變來評價。 本研究第一部分發(fā)現(xiàn)：（1）ATZ抑制CAT后導(dǎo)致LPS/D-Gal誘導(dǎo)的肝損傷明顯加重，表現(xiàn)為模型小鼠血漿ALT和AST的水平進(jìn)一步升高、肝內(nèi)MPO水平進(jìn)一步升高、肝組織壞死和充血進(jìn)一步加重、動物生存率降低；（2）ATZ抑制CAT后增強(qiáng)肝內(nèi)氧化應(yīng)激，表現(xiàn)為模型小鼠肝內(nèi)H2O2含量進(jìn)一步增高、MDA水平進(jìn)一步上升；（3）ATZ抑制CAT后促進(jìn)肝細(xì)胞凋亡，表現(xiàn)為模型小鼠肝內(nèi)TUNEL陽性細(xì)胞數(shù)目增加、激活型caspase-3含量進(jìn)一步增高、caspase-3，-8，-9活性進(jìn)一步提升。 第二部分實驗發(fā)現(xiàn)：（1）MET處理可提升正常小鼠和LPS/D-Gal模型小鼠肝內(nèi)CAT活性，ATZ可明顯解除MET對模型小鼠肝內(nèi)CAT活性的提升作用；（2）MET可顯著降低模型小鼠肝內(nèi)H2O2及MDA含量，ATZ僅能部分解除MET對模型小鼠肝內(nèi)H2O2含量的抑制作用，ATZ不能解除MET對模型小鼠肝內(nèi)MDA生成的抑制效應(yīng)；（3）ATZ不能消除MET對模型小鼠血漿ALT和AST水平的下調(diào)作用，也不能消除MET對模型小鼠肝組織損傷的改善效應(yīng)；（4）AICAR處理可顯著減低LPS/D-Gal模型小鼠血漿ALT和AST水平并減輕肝小葉結(jié)構(gòu)破壞及淤血。 本研究結(jié)果提示抗氧化酶CAT可能在LPS/D-Gal誘導(dǎo)的急性肝炎中發(fā)揮一定保護(hù)功效；MET雖可提升LPS/D-Gal模型小鼠肝組織內(nèi)CAT活性，但這一效應(yīng)可能在MET的保肝效應(yīng)中不占有主要地位；MET的保肝效應(yīng)可能與AMPK激活或其他抗炎、抗氧化機(jī)制有關(guān)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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