【作者】 张霖；

【导师】 吕志平；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 背景非酒精性脂肪肝(nonalcoholic fatty liver disease, NAFLD)是指除外酒精和其他明确的损肝因素所致的,以弥漫性肝细胞大泡性脂肪变为主要特征的临床病理综合征,包括单纯性脂肪肝以及由其演变的脂肪性肝炎(NASH)、肝纤维化和肝硬化。各种原因使肝内脂肪堆积超过5%或肝活检中发现单位肝细胞有1／3以上有脂肪滴被称为脂肪肝。在脂肪肝中,非酒精性脂肪肝占80%。NAFLD发病率在不同国家也各有不同,其发病率在不同国家也各有不同,目前西方国家约为20-30%,亚太地区约为12%～24%；儿童发病率约2.6%,肥胖儿童可达52.8%。随着我国经济条件的改善,人民生活水平不断提高,饮食结构、工作的环境及方式的改变,NAFLD发病率明显增加,而且有年轻化趋势。资料显示在我国上海市和广东省NAFLD患病率分别已达到15.4%、11.7%。有资料显示在排除其他肝病后,NAFLD通常是无症状转氨酶升高的最主要原因,约占90%。单纯性脂肪肝(NAFL)有12-40%患者在8-13年内进展为NASH,后可发展为肝硬化,与其他肝病引起的肝硬化患肝癌危险性无明显差异。此外,还与肥胖、2型糖尿病、高动脉压症、高脂血症等代谢综合征密切相关。NAFLD已经成为病毒性肝炎之后第二大慢性肝病,实行肝移植的第三大适应症。NAFLD的总死亡率比普通人显著增高。防治本病已成为全球普遍关注的医学和社会问题。NAFLD病机不甚明了,大多数学者较为认可的有“二次打击”学说,“初次打击”主要为胰岛素抵抗,各种病因所致NAFLD几乎普遍存在胰岛素抵抗现象。胰岛素抵抗通过促使外周脂解增加和高胰岛素血症引起肝细胞脂肪储积,并诱致对内外性损害因子敏感性增高；二次打击主要为反应性氧化代谢产物增多,导致脂质过氧化及其异常细胞因子的作用,进而引起坏死、甚至进展性肝纤维化。最近研究显示,调节肝细胞调节脂质代谢平衡的主要是核转录因子PPAR-α和SREBP-1c,分别促进脂肪的氧化和合成,其信号途径与两次次打击各个环节均有密切的联系,关于其调控的机制成为众医家研究的热点。目前世界范围内尚无疗效确切,安全稳定的治疗NAFLD的药物,研发通过化学合成或从中草药中寻找干预PPAR-α和SREBP-1c信号途径,降低脂质合成,达到治疗目的药物成为热点。非酒精性脂肪肝属中医“肥胖”“胁痛”“黄疸”等病范畴。多由过食肥甘厚味,湿热蕴藉中焦,湿热阻滞,气机受阻,气血运行不畅,而瘀血内生。病理关键在“湿(痰)”“热”“瘀”。虎杖功能清热、利湿、破瘀,作用于NAFLD关键病理因素,自古为中医家治疗“肥胖”“胁痛”“黄疸”等病的要药,虎杖苷为中药虎杖的主要活性成分之一,资料显示在降脂、保护肝细胞等方面有一定效果,本实验旨在用现代分子生物学手段系统观察其疗效,并深入探讨其可能的作用靶点,为临床更确切广泛使用虎杖提供科学依据,为从中草药中开发治疗NAFLD药物作探索性研究。目的1观察虎杖苷对非酒精性脂肪肝早期大鼠的影响,包括在体重、肝重、肝细胞脂肪变、肝功能、血脂代谢的影响。2、研究虎杖苷治疗非酒精性脂肪肝的可能作用机制,包括氧化应激、胰岛素抵抗、肿瘤坏死因子-α(TNF-α)以及过氧化物酶体增殖激活物受体-α(PPAR-a),固醇调节元件结合蛋白-1c (SREBP-1c)、肝X受体-α(LXR-α)、蛋白及基因表达的影响。方法：雄性SPF级SD大鼠44只,体重200±10g,正常喂养1周后,完全随机法分为2组,正常组对照(A,8只)和模型组(M,36只),组间大鼠体重差异无显著性(P>0.05)。正常组给予标准饲料,模型组给予高脂饲料(88%标准饲料+10%猪油+2%胆固醇)喂养,自由饮水和进食,动物房保持安静,自然采光,温度25℃左右,喂养持续8周,分别从正常对照组和空白模型组随机挑选一只,取肝脏做病理切片,证实非酒精性脂肪肝形成。再将模型组完全随机法分为5组：空白模型组(B)、虎杖苷高剂量组(C1)、虎杖苷中剂量组(C2)、虎杖苷低剂量组(C3)、非诺贝特组(D),每组7只。8周后,A组、B组予以生理盐水灌胃,1ml/100kg.d, C1、C2、C3分别以160mg/kg.d、80mg/kg.d、40mg/kg.d虎杖苷混悬液灌胃,D组以100mg/kg.d非诺贝特混悬液灌胃。持续4周。第12周末取大鼠血清及肝组织样本,称体重,肝湿重,计算肝指数,全自动生化仪测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C),游离脂肪酸(FFA)；酶法测定空腹血糖(FBG)；放免法检测血清空腹胰岛素(FIN),计算胰岛素抵抗指数(IRI),胰岛素敏感指数(ISI)；对肝组织进行病理学检查,HE染色观察其脂肪变程度；酶联免疫法(ELISA)检测血清肿瘤坏死因子α(TNF-α);蛋白免疫印迹法(western blotting)检测PPAR-α、LXR-α、SREBP-1c蛋白表达；实时荧光定量(real-time PCR)检测PPAR-α、LXR-α、SREBP-1c基因表达。计量资料结果以x±s表示,两组间比较采用t-test。多组间比较采用One way ANOVA,首先对数据进行方差齐性检验,方差齐则用LSD法进间两两比较,如果数据方差不齐,则采用Welch法进行方差分析,应用Dunnett T3法进行两两比较。等级资料资料采用Kruskal-Wallis H非参数检验进行分析,P<0.05为有显著性差异。所有数据采用SPSS 13.0软件包进行统计分析。结果第一部分：虎杖苷对非酒精性脂肪肝药效研究1.根据肝脏病理状态,可见弥漫性肝细胞大泡样脂肪变,无明显炎症、坏死及纤维化,NAFLD大鼠早期单纯性脂肪肝阶段模型复制成功。2.与空白模型组相比,虎杖苷高剂量组体重明显减低(P<0.01),增长速度较慢,肝脏体减小,肝湿重减轻,肝指数明显降低(P<0.01)。肝脏病理显示,虎杖苷高、中剂量组大鼠肝细胞脂肪沉积明显减少(P<0.01),肝细胞形态、排列均明显好转。3.与空白模型组相比,虎杖苷高、中剂量组大鼠血清ALT明显降低(P<0.01,P<0.05),虎杖苷高剂量组明显优于非诺贝特组(P<0.05)；虎杖苷高、中剂量组大鼠血清TG降低(P<0.01, P<0.05);虎杖苷高剂量组大鼠血清TC、LDL-C降低(P<0.05, P<0.01), HDL-C升高(P<0.01)。虎杖苷各剂量组均能降低FFA(P<0.01)。第二部分：虎杖苷治疗NAFLD部分机制研究1.与正常对照组比较,空白模型组大鼠总抗氧化能力(T-AOC)、超氧化物岐化酶(SOD)、谷胱甘肽-S转移酶(GST)明显减低(P<0.01),与空白模型组比较,虎杖苷高、中剂量组均能增高肝组织T-AOC、SOD、GST,同时降低肝组织MDA(P<0.01),其中虎杖苷高剂量组提高肝脏抗氧化能力效果均优于非诺贝特组(P<0.01)。2.与正常对照组比较,空白模型组大鼠空腹血糖(FBG)、空腹胰岛素(FIN)明显增高(P<0.01),IRI增高,ISI明显降低(P<0.01, P<0.05)。与空白模型组比较,虎杖苷高、中剂量组及非诺贝特组能降低FBG、FIN(P<0.01, P<0.05),减低胰岛素抵抗指数(IRI),增高胰岛素敏感指数(ISI) (P<0.01, P<0.05),其中虎杖苷高剂量效果均明显优于优于非诺贝特组(P<0.01, P<0.05)。虎杖苷低剂量各指标与空白模型组无差异(P>0.05)。虎杖苷高、中剂量组在降低IRI,增加ISI效果优于虎杖苷低剂量组(P<0.01)。3.与正常对照组比较,空白模型组大鼠血清TNF-α明显高于(P<0.01),与空白模型组相比,虎杖苷高、中剂量组及非诺贝特组均能明显降低血清TNF-α含量(P<0.01),虎杖苷高剂量组效果明显优于虎杖苷中、低剂量组(P<0.01, P<0.01)。但与非诺贝特组无显著差异(P>0.05)。虎杖苷低剂量组与不能显著降低血清TNF-α含量,与空白模型组无显著差异(P>0.05)。4.与正常组比较,空白模型组大鼠PPAR-α蛋白及基因表达明显降低(P<0.01),SREBP-1c、LXR-α蛋白及基因表达升高(P<0.01),与空白模型组比较,虎杖苷高、中剂量组均能明显降低大鼠肝脏SREBP-1c、LXR-α蛋白及基因表达,明显优于非诺贝特组(P<0.01)；虎杖苷各组PPAR-α蛋白及基因表达与空白模型组无显著差异(P>0.05),非诺贝特能显著增加PPAR-α蛋白及基因表达,与空白模型组及虎杖苷各剂量组存在显著差异(P<0.01)。结论1.虎杖苷能减轻NAFLD大鼠体重,减轻肝脏肿大及肝脏湿重。显示虎杖苷有一定的减肥作用,通过减轻内脏脂肪堆积达到治疗NAFLD。2.虎杖苷能减少血清FFA,降低血清TG、TC、LDL-C,增加HDL-C,纠正血脂紊乱。同时虎杖苷能减少NAFLD单纯性脂肪肝阶段肝细胞脂质沉积,从而减轻脂肪对肝细胞造成的水肿、缺氧以及炎症。可能机制是通过减少进入肝细胞游离脂肪酸,从而降低肝脏甘油三酯合成,减轻过量FFA的毒性作用,逆转肝脏脂肪沉积,改善肝功能,保护肝细胞。3.虎杖苷能增强体内抗氧化能力,减少脂质过氧化,减轻肝脏炎症及脂肪肝进展。虎杖苷可通过减少循环中血清FFA,减少肝细胞FFA的摄入,从而减轻线粒体氧化负荷,改善线粒体β氧化,减少活性氧及自由基的产生。4.虎杖苷降低循环中TNF-α的含量,改善胰岛素抵抗,减少脂肪组织分解及循环中FFA的释放,减轻肝细胞脂质沉积。5.虎杖苷能下调脂质合成因子SREBP-1c、LXR-α蛋白及基因的表达,减少脂质合成。更多还原

【Abstract】 BackgroundNonalcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by excessive fat storing in liver and hepatic cellular degeneration,without alcohol over ingestion. NAFLD consists of a spectrum of liver disease, ranging from simple steatosis to non-alcoholic steatohepatitis(NASH),fibrosis,eirrhosis.When weight of fat in liver exceed 5% of the liver or 1/3 above of liver cells have steatosis in liver biopsy,the liver is called fatty liver. Non-alcoholic fatty liver disease account for 80% of all the fatty liver.Morbidity of NAFLD is different in different countries, about 10%～24% in western country,but in Asian and Pacific regions the number is 12%-24%. In obesity incidence in patients the morbidity is 57.5%～74%. Incidence of children is about 2.6 %, while in those overweight the numbe can reach 52.8%.With the improvement of the standard of living, the change of the eating habits,more and more people are suffering nonalcoholic fatty liver(NAFLD) in china. Data shows in China, NAFLD prevalence has reached 15.4%,11.7% respectively in Shanghai and guangdong provinces. It appears that obesity is concerned with the cause of NAFLD. And there is a tendency of young. NAFLD is the most medium cause of liver dysfunction, accounting for 90%.The spectrum of NAFLD ranges from simple fatty liver (hepatic steatosis), with benign prognosis, to a potentially progressive form, nonalcoholic steatohepatitis (NASH), which may lead to liver fibrosis and cirrhosis, resulting in increased morbidity and mortality. WHO estimated that at least two million patients will develop cirrhosis due to hepatic steatosis in the years to come. Longitudinal cohort studies have demonstrated that those patients with cirrhosis have a similar risk to develop hepatocellular carcinoma as those with other causes of cirrhosis. All features of the metabolic syndrome, including obesity, type 2 diabetes, arterial hypertension, and hyperlipidemia (in the form of elevated triglyceride [TG] levels) are associated with NAFLD/NASH. NAFLD has become the third most important indication for liver transplantation. Total mortality of NAFLD significantly higher than normal. Prevention NAFLD has become a global attention as a medical and social problems.Pathogenesis of NAFLD is not very clear until now, "two hits" theory is approved by most medical scientists."first hit"is insulin resistance(IR).NAFLD almost exist insulin resistance. Insulin resistance lead liver cells to fatty degeneration through increasing lipolysis, in the meantime, sensitiity of damage factor inside and outside is rose."second hit"is lipid peroxidation caused by exceed lipid peroxide(LPO) and some cytokine. the "second hit"leads liver cells to inflammation,necrosis, fibrosis. Recent research suggests that PPAR-αand SREBP-1c is the main regulating factor to balance lipid synthesis and decomposition. Signaling pathways of PPAR-αand SREBP-1c closely linked to two hits. their regulation mechanisms become the hot spot being researched by most medical scientists. At the present time,there is no special drug to cure NAFLD, physicians focus on developing drug can decreace liver lipidosages by regulating signaling pathways of PPAR-αor/and SREBP-1c, especially from Chinese herbal medicine. There is no NAFLD in chinse tradional medicine, NAFLD is equivalent to Chinese "obesity""hypochondriac pain""jaundicet".According to TCM theory, intake excessive energy caused dampness-heat stagnating in spleen, dampness-heat in middle jiao, then qiji is blocked by dampness and heat, with the passing of time blood stasis is induced. To sum up,dampness, heat and blood stasis are the key for NAFLD.Giant Knotweed Rhizome,as a common herb,have the function of clearing heat, promoting diuresis,removing blood stasis. It can deal with most key Pathological factors,for that, Giant Knotweed Rhizome is used to treat obesity,hypochondriac pain or jaundicet.Polydatin is one of the main active ingredients Giant Knotweed Rhizome.Data shows that polydatin can decreace fat accumulation in liver, protective liver cell, etc. The experiment aims to observe the systematic effect of polydatin on NAFLD,and make further reseach the mechanism. The conclusion of this experiment provide more scientific basis for using Giant Knotweed Rhizome in clinic,and the author expect to explore a drug for treating NAFLD from Chinese herbal medicineObjectivesUsing the model of NAFLD recognized by the world and linking to the evolution of modern research of NAFLD,we observed the body weight,liver wet weight,the liver index,hepatic tissue pathology,Liver function,blood lipid,FFA,oxidative stress,TNF-αand hepatic tissue expression of PPAR-a, SREBP-lc and LXR-a protein and mRNA by the technique of pathology, biochemistry and molecular biology in the research.The aim of this investigation was to evaluate the efficacy and the poteintial mechanisms of the polydatin on the experiment nonalcoholic fatty liver disease(NAFLD) induced by high fat forage. MethodsForty-four clean graded male SD(Sprague Dawley) rats,weight 200±10g,were fed 1W normally,then were randomly divided into 2 groups,namely normal control group(A,n=8),model group(M,n=36), The normal control group had being fed with ordinary forage,while the rest with high fat forage(88% ordinary forage+10% lard+ 2% cholesterin) for 8w. After 8W,two rats were randomly choosed from each group,we observed samples of liver tissue sections that stained with HE method by light microscope to confirm NAFLD model rats replicated successfully.Then,the model group were randomly divided into 5 groups,namely blank model group(B,n=7),PD groups:low dosage,medium dosage,high dosage (C1,C2,C3, n=7, respectively) and Fenofibrate Tablets group(D,n=7). The forage of the normal control group don’t change, the rest group were fed with high fat forage unceasingly. Since then, we poured PD Suspension liquid into stomach of rats of C1、C2、C3 groups,160mg/kg/d、80mg/kg/d、40mg/kg/d qd separately;the rats of D group are poured suspension of Fenofibrate tablet into stomach, 100mg/kg/d,qd;the rats of A and B group are poured isotonic nachloride into stomach,1 ml/100g.d,qd. At the end of twelve week of the experiment,all rats were fasted for 12h,then were killed after being narcotized with chloraldurat to leave over serum and hepatic tissue rapidly according to routine, which is fasted by formalin,packed with paraffin, sectioed,stained with HE,evaluated the degree of hepatic steatosis and inflammation under the optical microscope. Then reckoned liver index,to detect the content of serum ALT,AST,TC,TG,LDL,HDL,FPG、FIN、TNF-α. We leave over hepatic tissue to make 10% liver homogenate at 4℃for detecting content of T-AOC、SOD、GST、MDA, detected the protein expression of PPAR-α、LXR-α、SREBP-1c by Western blotting. The expression levels of PPAR-amRNA、LXR-amRNA、SREBP-1cmRNA were determined by real time quantitative RT-PCR. The data were analyzed with SPSS 13.0:Measurement data is expressed as the mean±SD(x±s). Differences between two group were analysed using unpaired Student’d-test,and on-way ANOVA for multiple comparisons. During ANOVA, data were tested via homogeneity test for variance first, if variance is homogenous, data were analyzed by on-way ANOVA, and groups were compared by LSD; if not, data would be analyzed by Dunnett T3 method after data tested via welch for vatiance.Rank data were analyzed by Kruskal-Wallis non-Parametric test. P<0.05. was considered significant.ResultsThe experiment I is to study the effect of PD on NAFLD.1.According to the hepatic tissue pathology and the state of hepatic function of rats,we succeeded in replicating the models of rats with NAFLD.2.Compared with the blank model group,the body weight and the liver index of PD high dosage (C1) group decrease significantly(P<0.01).During the experiment, weight of PD high dosage group grows slower, liver volume decreases.Liver wet weight and liver index decreased significantly (P<0.01). Hepatic pathology revealed, high polygoni cuspidati glycosides in rats, the fat deposition in liver cells reduced significantly (P< 0.01). Liver cell morphology, arrangement are improved significantly.3. Compared with the blank model group,the serum ALT of the PD high-dosage group and PD medium dosage group decreased significantly (P<0.01, P<0.05), and the effect of improving liver function is superior than fenofibrate group (P<0.05). High, medium polygoni cuspidati nucleoside dosage group of serum TG reduce rat (P< 0.01, P<0.05). Serum TC,LDL-c of the PD high dosage group and PD medium dosage group reduced significantly (P<0.05, P<0.01), at the same time HDL-C increased (P<0.01). FFA of Each PD group reduced markedly (P<0.01). The second part:Research of partly mechanisms of polydatin on nonalcoholic fatty liver disease1.Compared with the normal control group, T-AOC, SOD, GST of the blank model rats significantly reduced (P<0.01), the liver tissue T-AOC, SOD, GST of PD high and medium dosage group all are heighten(P<0.01), while reducing liver tissue MDA compared with blank model group(P<0.01). Liver anti-oxydation ability of PD high dosage group is more effective than fenofibrate group (P<0.01).2.Compared with the normal control group, FBG FIN of the blank model rats are notably higher (P<0.01), the IRI increased, and ISI are lower (P<0.01, P<0.05). Compared with blank model group, FBG、FIN、IRI of PD high dosage group,medium dosage group and fenofibrate reduced (P<0.01, P<0.05), at the same time there ISI heighten (P<0.01, P<0.05), including effect of PD high dosage is better than fenofibrate group (P<0.01, P<0.05). While each index of PD low dosage is no differences with blank model group (P>0.05). PD High and medium is more effective than PD low dosage group (P<0.01).3.Compared with the normal control group, serum TNF-αof the blank model rats increased significantly (P<0.01).Compared with blank model group, PD high and medium dosage group and fenofibrate group significantly reduced (P<0.01), PD high dosage group is obviously superior to medium and low dosage groups (P<0.01, P<0.01),but compared with fenofibrate group,there are no difference(P>0.05). PD low dosage group can not reduce serum TNF-αcontent, there are no significant differences from the blank model group (P>0.05).4.Compared with the normal control group, The expression of SREBP-1c,LXR-αprotein of the blank model rats increased significantly (P<0.01). while the expression of PPAR-a protein decresced. Compared with blank model group, the expression of SREBP-1c、LXR-αprotein PD high and medium dosage group significantly reduced (P<0.01), the two group are more effective than fenofibrate and PD low group(P<0.01). But the expression of PPAR-a protein was higher than the blank model group(P<0.01), The expression of PPAR-a protein of PD groups are no differences from the blank model group (P>0.05).Conclusions1.Polydatin can decrease significantly the body weight, improve hepatomegaly, reduce liver wet weight and the liver index. The experiment hints:to reduce the abdomen fat may be one of the mechanism of polydatin in the treatment of nonalcoholic fatty liver.2.Polydatin can reduce serum TG, TC,LDL-C and ALT,while increase HDL-C, and it shows that polydatin can correct blood disorder and keep the liver cells intact. According to the pathological polydatin can reduce the fat deposition in the hepatocyte of NAFLD rats,when there are in the stage of simple fatty liver (hepatic steatosis), thus reduce fat cause Swelling of liver cells and hypoxia and inflammation.The possible mechanism for polydatin to reverse liver fat deposits, improve liver function and protect liver cells is reducing free fatty acids flowing into the liver cells, to reduce excess synthesis of liver triglycerides and its toxicity.3.Polydatin can enhance the anti-oxidization ability, reduce lipid peroxidation. Polydatin reduce liver cell free fatty acids flowing into the liver cells, reduce oxidatie load of mitochondrial, thereby improve (3-oxidation, reduce Ros and free radicals.4.Polydatin can reduce TNF-a in blood, and improve insulin resistance. Polydatin reduces lipoclasis, and FFA released from fat tissue, through which Polydatin reduces the at deposits of liver cells.5.Polydatin can cut lipid synthesis factor SREBP-lc, LXR-a protein and gene expression,through decreacing expression of SREBP-lc, polydatin reduce lipid synthesis, which may be an key mechanism for polydatin to treat NAFLD.更多还原