【作者】 邵丹；

【导师】 魏道智；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 肝病已成为人类第9大杀手，肝病尤其是肝炎的治疗已成为医学界的重要研究课题，寻求安全有效的肝炎治疗药物是国内外医学界极为关注和研究的热点。我国历代用药实践和现代的大量研究成果表明：中医药治疗肝病，具有西药所不具备的多靶点、治调养结合和毒副作用小的特点，中医的治疗方法正被越来越多的中外医学专家和患者所接受和采用。中药悬钩子属植物锈毛莓具有抗菌消炎、活血化淤、去风湿、强筋骨之效，闽南民间广泛用于治疗各型肝炎，有良好的民间用药基础。本研究以福建上杭锈毛莓为材料，利用水提和有机溶剂浸提法对锈毛莓化学成分进行分离，一共获得6个组分，分别为锈毛莓水提物，锈毛莓醇提物，锈毛莓醇提石油醚部分，锈毛莓醇提氯仿部分，锈毛莓醇提乙酸乙酯部分，锈毛莓正丁醇部分。理化鉴定后发现这些组分含有黄酮类、皂苷类、多糖类、生物碱类和萜类物质。分别构建了小鼠耳肿胀模型和CCl₄肝损伤模型，对分离所得的6个组分分3个剂量梯度进行药效筛选，所得数据经Duncan’s Multiple-Range统计后发现最佳药效组分为锈毛莓醇提乙酸乙酯部分，其最佳用药剂量为3mg/kg。利用2-DE技术分离CCl₄肝损伤小鼠肝脏全蛋白，筛选出不同处理组差异表达蛋白点51个，经质谱鉴定含有39种蛋白质（表4.9）。这些蛋白大致可分为①小鼠CCl₄急性肝损伤胁迫相关的蛋白；②保肝抗氧化相关蛋白；③信号转导相关蛋白；④物质能量相关蛋白；⑤分子伴侣。利用生物信息学对以上蛋白质进行功能鉴定后，推测锈毛莓醇提乙酸乙酯组的药效机制可能为：诱使亚硫酸盐氧化酶、血清白蛋白上调表达，刺激谷胱甘肽的合成，增强机体内SOD的活性，祛除氧自由基，保护肝细胞膜不被氧化；调节赖氨酸、叶酸、鸟氨酸和S-腺苷-L-蛋氨酸的合成，提高机体的免疫力，在去除坏死肝细胞的同时刺激肝细胞的增生，加速肝组织的修复，保肝护肝，使ALT恢复正常水平，此外，酰基-CoA合成酶的上调减少了脂肪酸对肝细胞的伤害。磷脂酶和钙调素的发现说明磷脂酰肌醇信号途径参与了抗炎保肝作用。值得关注的是，实验中发现具有抑制细胞凋亡作用的葡萄糖调节蛋白78、线粒体热休克蛋白60上调表达，对导致肝纤维化具有重要作用的脯氨酰4-羟化酶下调表达。这些分子伴侣的差异表达说明锈毛莓醇提乙酸乙酯组分具有抗肝硬化的功效。更多还原

【Abstract】 The liver disease have become the ninth-largest killer of the human. How to treat liver d-iseases is an important research topic for the medical profession. To seek a safe and effectivetherapy of hepatitis is the hot spot with great concern and research in the medical professionat home and abroad. Ancient medicine and modern research results show that the treatment ofdiseases of the Chinese medicine having the characteristics of the multi-target, cure andaftercare binding and less side effects, which do not have in western medicine, are acceptedby more and more Chinese and foreign medical profession and patients. Rubus reflexus Kerbelong to Rubus, it is a kind of Chinese medicine which can antibacterial and anti-inflammat-ory, remove blood stasis, cure the rheumatism and strong your body. Local folk of Fujianwidely use it to treat various types of hepatitis. The basis of folk medicine is pretty fair.In this study, we use the Rubus reflexus Ker of Fujian Shanghang as the sample. thechemical composition of it were isolated by water extraction and organic solvent extraction.as a result, we get six components in all. They are water extraction, alcohol extraction,alcohol and petroleum extraction, alcohol and chloroform extraction, alcohol and ethylacetate extraction, alcohol and butanol extraction. Physical and chemical identification showthat these components contain flavonoids, saponins, polysaccharides, alkaloids andterpenoids.Mouse ear edema model and CCl₄liver injury model was constructed to screen the sixcomponents in three dose gradient. The date processing used the Duncan’s Multiple-Rangetest. The outcome reveal that the most efficacy composition is alcohol and ethyl acetateextraction,and its does is3mg/kg.2-DE separated and screened the liver protein of mouse injured by CCl₄. Finally, we get51protein spot of differentially expressed, which contains39kinds of proteins identified bymass spectrometry（Tab.4.9）. These proteins can be broadly divided into five groups:①CCl₄injure liver of mice acute inflammation-related protein,②Protect liver and anti-oxida-tion-related protein,③signal transduction-associated protein,④material and energy-rela-ted protein,⑤molecular chaperones. Using bioinformatics to identify the above proteinfunction,suggesting that alcohol and ethyl acetate extraction efficacy mechanism may be:ind-ucing sulfite oxidase, serum albumin upregulated and stimulating glutathione synthesisand toenhance the SOD activity, eliminate oxygen free radicals, keep the liver cell membrane awayfrom oxidized; regulate the synthesis of lysine, folic acid, ornithine and S-adenosyl-L-methi- onine, to improve the body’s immune system, remove the necrosis of liver cells whilestimulating the proliferation of liver cells, accelerated liver tissue repair, nourish and protectthe liver, returned ALT to normal levels, in addition,to the upregulated of acyl-CoA syntheta-se reduceing fatty acids reduce the damage to liver cells. The findings of Phospholipase andcalmodulin suggest that the phosphatidylinositol signaling pathway is involved in anti-infla-mmatory hepatoprotective effect. It is worth mentioning is that glucose-regulated protein78,mitochondrial heat shock protein60have been shown to inhibit cell apoptosisupregulated,prolyl4-hydroxylase plays an important role leading to liver fibrosis Cutexpress,these differentially expressed of molecular chaperones indicate alcohol and ethylacetate extraction component may have the effect of anti-cirrhosis.更多还原