刺五加酸、红景天苷对小鼠急性肝衰竭的保护作用及干预机制研究

论文摘要

急性肝衰竭是一种递进的破坏性肝病,可在急性或慢性肝病、其他系统器官衰竭等过程中发生。急性肝衰竭是指发生于既往肝功能正常的患者,短期内出现肝脏功能急剧恶化,导致进行性神志改变直至昏迷和凝血功能障碍的症候群。主要病因是药物性肝脏损伤、病毒性肝炎、自身免疫性肝病、休克或低灌注,另外有近20%的急性肝衰竭无病因可寻。急性肝衰竭多发生于青年,死亡率极高。寻找有效的治疗药物及药物作用靶点已成为研究者们的重要课题。刺五加酸（Acanthoic acid）是由朝鲜刺五加（Acanthopanax koreanum Nakai）的根皮部提取的一种海松二烯二萜类物质。常用于滋补剂和镇静剂,还可以用于治疗风湿病和糖尿病。目前已发现刺五加酸可以抑制致炎细胞因子,如白细胞介素-1,肿瘤坏死因子、白细胞介素-8等,包括许多炎症介质和合成与释放。红景天苷是高山红景天（Rhodiola sachalinensis A.Bor.）主要成分之一。高山红景天具有中枢神经系统有较强的抑制和调节作用,有较好的抗疲劳、抗缺氧、抗衰老、抗辐射、防肿瘤及消炎、解毒、提高自身免疫力作用,是人体自身细胞的潜能激活剂,对人体神经系统、血循环系统、代谢系统、内分泌系统等具有极好的整体调理作用,在国内主要用于高山缺氧症的治疗。本研究采用对乙酰氨基酚、D-氨基半乳糖联合内毒素诱导的急性肝衰竭作为动物研究模型,探讨刺五加酸、红景天苷对急性肝衰竭的保护作用以及干预机制。分别从抗氧化作用、细胞凋亡途径以及小分子一氧化氮（NO）、缺氧诱导因子（HIF-1α,hypoxia-inducible factor-1α）进行研究。一氧化氮（nitric oxide,NO）在20世界80年代才被认识到是人体内的一种具有生物活性的分子,近十年来的研究已证实其在体内的多种病理生理过程中发挥着重要作用。缺氧诱导因子（HIF-1α,hypoxia-inducible factor-1α）是介导细胞对缺氧微环境进行适应性反应的关键性转录调控因子。目前的研究已经发现,在缺氧及肿瘤发生等病理条件下,机体通过增加HIF-1α的水平来调控促红细胞生成素（EPO）、血管内皮生长因子（VEGF）等基因的表达,从而在机体耐受低氧环境以及肿瘤的发生和转移中起重要的作用,但对HIF-1α是否参与急性炎症反应及其机制研究较少。本实验结果显示对乙酰氨基酚、D-氨基半乳糖联合内毒素诱导的急性肝衰竭时HIF-1α表达增加。同时探讨肿瘤坏死因子、一氧化氮以及一氧化氮合成酶与HIF-1α的相关性。HIF-1α表达增加,急性肝衰竭小鼠血清中TNF-α水平增加,NO和iNOS含量升高,从而显示这些因子的表达间存在一定的相关性,其确切关系尚待进一步探讨。HIF-1α与急性肝衰竭的发生、发展密切相关,抑制HIF-1α活性可能是治疗的良策。研究表明,刺五加酸、红景天苷显著降低急性肝衰竭模型中丙氨酸氨基转移酶（alanine aminotransferase）、门冬氨酸转氨酶（aspartate aminotransferase）、肿瘤坏死因子（tumor necrosis-alpha）、一氧化氮（nitric oxide）、丙二醛（malondialdehyde）,升高谷胱甘肽（glutathione）、超氧化物歧化酶（superoxidedismutase）、过氧化氢酶（catalase）以及谷胱甘肽过氧化物酶（glutathioneperoxidase）。通过免疫组化、蛋白印记和细胞凋亡进行组织病理学研究。综上所述,刺五加酸与红景天苷对由对乙酰氨基酚、D-氨基半乳糖联合内毒素诱导的急性肝衰竭均有显著的保护作用,干预机制的结果为将刺五加酸与红景天苷开发用于临床急性肝衰竭治疗提供了一定的理论基础。

论文目录

• List of Tables
• List of Figures
• Abstract in Chinese
• Abstract
• Chapter 1. Introduction
• 1.1 Liver
• 1.2 Acute liver failure
• 1.3 Animal models
• 1.3.1 Surgical models
• 1.3.2 Pharmacological models
• 1.3.3 Genetically modified animals
• 1.4 Drug-induced acute liver failure
• Chapter 2. Protective Effect and Molecular Mechanism of Acanthoic acid in Two Models of Acute Liver Failure Models in Mice
• 2.1 Protective Effect and Molecular Mechanism of Acanthoic acid on Acute Liver Failure Induced by Acetaminophen
• 2.1.1 Introduction
• 2.1.2 Materials and Methods
• 2.1.2.1 Isolation of AA
• 2.1.2.2 Materials
• 2.1.2.3 Animals
• 2.1.2.4 Experimental design
• 2.1.2.5 Serum Enzyme and TNF-α Assay
• 2.1.2.6 Glutathione and Malondialdehyde Determination
• 2.1.2.7 Histopathology and Immunohistochemistry
• 2.1.2.8 Statistical Analysis
• 2.1.3 Results
• 2.1.3.1 The Effect of AA in the APAP-induced Lethality in Mice
• 2.1.3.2. The Effect of AA on Serum AST and ALT Levels
• 2.1.3.3. The Effect of AA on Glutathione and Malondialdehyde Levels
• 2.1.3.4. The Effect of AA on TNF-α Level
• 2.1.3.5. The Effect of AA on Histopathology and Immunohistochemistry
• 2.1.4 Discussion and Conclusions
• 2.2 Protective Effect and Molecular Mechanism of Acanthoic acid on Acute Liver Failure Induced by D-Galactosamine and Lipopolysaccharide in Mice
• 2.2.1 Introduction
• 2.2.2. Materials and Methods
• 2.2.2.1. Animals
• 2.2.2.2 Experimental design
• 2.2.2.3. Determination of lethality
• 2.2.2.4. Blood biochemistry and serum TNF-alpha assay
• 2.2.2.5. Detection of apoptotic cells and histopathological analysis
• 2.2.2.6. DNA fragmentation analysis
• 2.2.2.7. Statistical analysis
• 2.2.3. Results
• 2.2.3.1. The acanthoic acid suppressed lethality in mice with D-GalN/LPS induced fulminant hepatitis
• 2.2.3.2. The effect of acanthoic acid on serum AST and ALT levels
• 2.2.3.3. The effect of acanthoic acid on serum TNF-α levels
• 2.2.3.4. The acanthoic acid reduced the histological changes of liver induced by D-GalN/LPS
• 2.2.3.5. The acanthoic acid reduced hepatocyte apoptosis in mice treated with D-GalN/LPS
• 2.2.4. Discussion and conclusions
• Chapter 3. Protective Effect and Molecular Mechanism of Salidroside in Two Models of Acute Liver Failure Models in Mice
• 3.1 Protective Effect and Molecular Mechanism of Salidroside on Acute Liver Injury Induced by Acetaminophen
• 3.1.1 Introduction
• 3.1.2 Materials and Methods
• 3.1.2.1 Materials
• 3.1.2.2 Animals
• 3.1.2.3 Experimental design
• 3.1.2.4 Serum Enzyme and TNF-α Assay
• 3.1.2.5. Glutathione and Malondialdehyde Determination
• 3.1.2.6. Histopathology and Immunohistochemistry
• 3.1.2.7. Statistical Analysis
• 3.1.3 Results
• 3.1.3.1 The Effect of SDS in the APAP-induced Lethality in Mice
• 3.1.3.2 The Effect of SDS on plasma AST and ALT Levels
• 3.1.3.3 The Effect of SDS on Glutathione and Malondialdehyde contents
• 3.1.3.4 The Effect of SDS on TNF-α Level
• 3.1.3.5 The Effect of SDS on Histopathology and Immunohistochemistry
• 3.1.4 Discussion and Conclusions
• 3.2 Protective Effect and Molecular Mechanism of Salidroside on Acute Liver Failure Induced by D-Galactosamine and Lipopolysaccharide in Mice
• 3.2.1 Introduction
• 3.2.2 Materials and Methods
• 3.2.2.1 Materials
• 3.2.2.2 Animals
• 3.2.2.3 Experimental design
• 3.2.2.4 Measurement of serum enzyme, TNF-α, NO, and NOS activities
• 3.2.2.5 Antioxidation System Determination
• 3.2.2.6 Histopathology and Immunohistochemistry
• 3.2.2.7 Western blotting analysis
• 3.2.2.8 Statistical Analysis
• 3.2.3 Results
• 3.2.3.1 The Effect of SDS on plasma AST and ALT Levels
• 3.2.3.2 The Antioxidation Effect of SDS
• 3.2.3.3 The Effect of SDS on TNF-α Production
• 3.2.3.4 The Effect of SDS on the production of NO, and the expressions of T-NOS and i-NOS
• 3.2.3.5 The Effect of SDS on Histopathology, Immunohistochemistry and western blotting
• 3.2.4 Discussion and conclusions
• Chapter 4. Overall Conclusion
• 4.1 Overall Conclusion
• Reference
• Acknowledgment
• Review
• Reference
• Appendix A