【作者】 胡巢凤；

【导师】 陆大祥；

【作者基本信息】 暨南大学 ， 病理生理学， 2009， 博士

【摘要】 近年来脂肪肝已成为一种严重危害人类身体健康的常见疾病,为寻找有效治疗脂肪肝的药物,根据中药理论,我们自拟了中药复方GST。本研究将对此中药复方进行筛选,探讨其防治脂肪肝的作用和机制。研究分为5个部分进行:第1部分:采用高脂饲料喂养小鼠,分别于2周、3周、4周观察小鼠血脂、肝脂、肝指数和肝组织病理改变(HE及苏丹Ⅲ染色),建立小鼠高脂性脂肪肝模型。结果发现高脂组小鼠饲养2周后,血清TC、LDL-C和HDL-C含量、TC/HDL-TC比值、肝指数、肝组织TC、TG含量显著升高,第3-4周继续维持在高水平:肝组织病理切片观察到高脂组小鼠2周时脂肪肝已经形成,在实验过程中肝脂肪变程度随时间的持续而逐渐加重。实验表明高脂饲料喂养的小鼠2-4周内可形成不同程度脂变的脂肪肝,为抗脂肪肝的药物研究提供了良好的动物模型。第2部分:采用小鼠高脂性脂肪肝模型(高脂饲养2周),运用正交设计方法,观察肝指数和肝组织甘油三酯(TG)含量变化,对中药复方GST进行筛选。通过正交分析和药效学研究,筛选出了人参、三七和天麻为防治脂肪肝的最佳组方,此组方即为中药复方GST;并确定了药物的最佳用药剂量(国家知识产权局专利申请号:200910038735.2)。第3部分:建立人肝细胞株(L02细胞)脂肪变性模型(50%小牛血清+2%乙醇),油红O染色证实建模成功后,应用正交实验对中药GST主要成分进行筛选。研究证实GST主要成分的最佳组方为:人参茎叶皂苷、三七总皂苷和天麻素,并确定了体外培养的最佳用药浓度,此主要成分组方具有明显改善L02细胞脂肪变性的作用(国家知识产权局专利申请号:200910038736.7)。第4部分:在第3部分研究的基础上,探讨中药GST主要成分组方防治肝细胞脂肪变性的机制。应用流式细胞术检测肝细胞活性氧和线粒体膜电位;采用反相高效液相色谱分析法测定肝细胞ATP含量;应用RT-PCR方法检测肝细胞CYP2E1及PPARαmRNA表达。实验证实中药GST主要成分组方改善肝细胞脂肪变性的作用可能与增加肝细胞PPARαmRNA表达,减少肝细胞TG产生;降低CYP2E1 mRNA表达,抑制活性氧(ROS)产生、抗脂质过氧化反应,改善线粒体膜电位,增加ATP合成有关(国家知识产权局专利申请号:200910038736.7)。第5部分:采用小鼠高脂性脂肪肝模型(高脂饲养12周),肝组织病理切片(HE染色)证实建模成功后,分别用中药GST、GST主要成分组方和降血脂药物辛伐他汀给小鼠灌胃2周。观察这些药物对血清TC、TG,HDL-C、LDL-C、ALT及肝组织TC、TG含量、肝指数、肝组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、PPARαmRNA和CYP2E1 mRNA表达、肝组织病理变化的影响。研究结果表明中药GST及其主要成分组方具有有效治疗脂肪肝的作用,其机制可能与增加肝组织PPARαmRNA表达,降低血清和肝组织TG含量,改善肝细胞脂肪变性,抑制CYP2E1 mRNA表达,抗脂质过氧化反应有关。而辛伐他汀具有明显降血脂作用,但却增加肝脂含量,促进脂质过氧化反应,加剧肝脏脂肪沉积。(国家知识产权局专利申请号:200910038736.7.200910038735.2)。更多还原

【Abstract】 Fatty liver is a common disease,which is seriously harmful to human health in recent years.To explore the valid drugs of curing hepatic steatosis,new compound GST of traditional Chinese medicine is composed under the direction of Chinese medicine theries.Then we are decided to screen GST and investigate its effects of preventing and treating fatty liver and underlying mechanisms.The studies were divided into five parts:Part 1:To establish the model of hyperlipidemic fatty liver,the mice were fed with high-fat diet(HF group).The serum TC,TG,HDL-C,LDL-C and hepatic TC, TG levels were detectd from two to four weeks.At the same time,liver index was observed and hepatic steatosis was analyzed by HE and SudanⅢstaining.The results showed that serum TC,TG,HDL-C,LDL-C,TC/HDL-C and hepatic TC,TG contents were elevatd significantly in HF group two weeks after experiment.In the following three-four weeks,the serum lipid,hepatic lipid and liver index kept rising. Hepatic steatosis was induced in HF group two weeks later and the degree of steatosis was aggravated with time going on.The results suggest that feeding with high -fat diet,different phases of fatty liver in mice have been developed from two weeks to four weeks,which establishes favourable animal model for further study on drugs of anti-fatty liver.Part 2:The compound GST of traditional Chinese medicine was sieved by liver index and hepatic TG level in orthogonal design,and pharmacodynamics was verified in mice with fatty liver induced by high-fat diet for two weeks.The results indicated that the confirmed compound of improving fatty liver was composed of ginseng, panax notoginseng and gastrodia elata,which just was GST.The optimized dosage was also determined by the experiments.(Chinese patent applied No. 200910038735.2). Part 3:Hepatocyte steatosis of human liver cell line L02 was established induced by 2%alcohol and 50%calf serum,and was demonstrated by oil red staining.Then orthogonal design was used to screen main components of GST.The results showed that the optimized compound of main components was made up of ginsenosides of stem and leaf,panax notoginseng saponins and gastrodin,which obviously improved hepatocyte steatosis.Meanwhile,its optimal concentration was also confirmed. (Chinese patent applied No.200910038736.7 ).Patr 4.On the basis of part 3,the underlying mechanisms of main components of GST for ameliorating hepatocyte steatosis were investigated.The level of ROS and mitochondrial membrane potential in cells were tested by flow cytometry.Reversedphase high-performance liquid chromatography(RP-HPLC) was applied to assay cellular ATP content.Expressions of PPARαand CYP2E1 mRNA in liver were examined by RT-PCR.The results demonstrated that the effects of main components of GST on hepatocyte steatosis were associated with increasing PPARαmRNA expression in liver cells,then decreasing cellular TG level,and inhibiting CYP2EI mRNA,thus suppressing ROS level and resisting lipid peroxidation,further improving mitochondrial function and raising ATP level.(Chinese patent applied No.200910038736.7).Part 5:Mice was fed with high-fat diet for twelve weeks.When the experimental model of fatty liver was confirmed by HE staining,mice were treated intragastrically with GST,main components of GST and simvastatin respectively for two weeks. Then serum TC,TG,HDL-C,LDL-C,ALT contents,hepatic TC,TG,MDA levels and SOD activity were detected.Moreover,liver index and pathology were also observed.Expressions of PPARαand CYP2E1 mRNA in liver were examined by RT-PCR.The results indicated that GST and its main components were effective in the treatment of hepatic adipose infiltration of mice,which may be related to up-regulating PPARαmRNA,then reducing serum and hepatic TG levels,and down-regulating CYP2E1 mRNA expression in the liver,thus inhibiting lipid peroxidation.We also found that simvastatin significantly decreasd serum lipid contents,but increased hepatic lipid levels and lipid peroxidation,then aggravating fatty liver(Chinese patent applied No.200910038736.7,200910038735.2).更多还原