【作者】 张征波；

【导师】 薛博瑜；

【作者基本信息】 南京中医药大学 ， 中医内科， 2012， 博士

【摘要】 背景非酒精性脂肪肝(Nonalcoholic Fatty Liver Disease, NAFLD)作为一种慢性肝脏损害性疾病在很多国家普遍存在。非酒精性脂肪肝的整体发生率在西方国家达到15%-40%,在亚洲发生率为9%-40%,并且在过去的十五年迅速地增长。本病包括了一系列的疾病过程,从单纯性脂肪肝,非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis, NASH)到肝纤维化,肝硬化,最终进展为肝细胞癌。非酒精性脂肪肝的存在与心包脂肪增加,颈动脉狭窄,心血管死亡率增加有关,这就使本病的预后更加严重。该病已成为严重影响我国人民身心健康和国民经济、社会发展和稳定的主要疾病之一。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,脂肪细胞分泌的细胞因子具有调节肝脏细胞脂质代谢的作用,其中脂联素成为研究热点,而存在于肝脏细胞中的脂联素受体2被认为NAFLD的发病机制有关。脂联素与其结合后通过调控与肝脏脂质氧化相关核转录因子PPAR-alpha及其控制的脂质氧化限速酶来增加肝脏细胞脂质氧化,从而减少肝脏脂质沉积。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脂联素受体2-PPAR-alpha信号途径,增加肝脏脂质氧化,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”“黄疽”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,而疲血内生。病理关键在“湿(痰)”“热”,“瘀”。大黄素为中药何首乌、大黄的主要活性成分之一,何首乌能补益肝肾,又能润肠通便而泄浊,大黄下瘀热祛浊瘀,于本病病机和主治最是合拍,自古为中医家治疗“肥胖”“胁痛”“黄疽”等病的要药,而大黄素在降脂、保护肝细胞等方面有一定效果,作用于NAFLD关键病理因素。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,为从中草药中开发治疗NAFLD药物作探索性研究。目的1.观察大黄素对非酒精性脂肪肝早期体内及体外模型的影响,包括对体质量、’肝质量、肝细胞脂肪变、肝功能、血脂代谢的影响,肝细胞株内TG、肝酶等影响。2.研究大黄素治疗非酒精性脂肪肝的可能作用机制,包括改善脂质沉积、保护肝细胞、减轻氧化应激以及对脂联素受体2、PPAR-alpha、CPT1、ACOX1的蛋白及基因表达的.影响方法C57BL6小鼠62只,正常喂养1周后随机分为分为2组,空白组对照(A,13只)和模型组(M,49只),组间大鼠体重差异无显著性(P>0.05)。空白对照组给以普通饲料,其余各组均给以高脂饲料。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4组：模型组、大黄素组、人参皂苷组、罗格列酮组。8周后,空白对照组灌胃给以DMSO,(5%DMSO20mg/kg体重)、人参皂苷组.灌胃给以人参皂苷(20mg/kg)、大黄素组灌胃给以大黄素(20mg/kg),罗格列酮组给予罗格列酮灌胃(10mg/kg)。治疗4周,取小鼠血清及肝脏组织标本。测体质量,称肝质量,计算肝指数,根据试剂盒说明测定血清谷丙转氨酶、谷草转氨酶、甘油三酷(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)；对肝组织进行病理学检查,HE染色观察其脂肪变程度;蛋白免疫印迹法(westeoblotting)检测脂联素受体2、PPAR-alpha、CPT1、 ACOX1蛋白表达;实时荧光定量(real-timePCR)检测脂联素受体2、PPAR-alpha、CPT1、 ACOX1基因表达。HepG2细胞,用1640培养基+10%FBS培养(37℃,5%CO2)。分组：设空白组、造模组、大黄素组、人参皂苷组、罗格列酮组。取对数生长期的HepG2细胞,每组设2个复孔,加入诱导液(油酸：棕榈酸2：1)诱导24h,分别加入大黄素、人参皂苷、罗格列酮,观察24h、48h、72h。油红染色观察细胞内脂质沉积,测定TG、肝酶含量,蛋白免疫印迹法(westeoblotting)检测脂联索受体2、PPAR-alpha、CPT1、ACOX1蛋白表达;实时荧光定量(real-timePCR)检测脂联素受体2、PPAR-alpha、CPT1、ACOX1基因表达。所有数值以均数士标准误表示,用SPSS11.5软件进行t检验或者方差分析(ANOVA),当P<0.05时,认为处理组和对照组之间有显著性差异。结果第一部分体内实验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小鼠早期单纯性肝脂肪变性阶段模型复制成功。2.与模型组相比,大黄素可降低小鼠体质量,但无明显差异(P>0.05)。降低肝脏质量,肝指数(P<0.05,P>0.05).肝脏病理显示,大黄素可明显改善肝脏脂肪沉积(P<0.01)。肝细胞形态、排列均明显好转。大黄素可明显降低血清ALT、AST (P<0.05),明显降低血清TG, FFA, CHO水平(P<0.05),降低血清LDL,升高HDL(P>0.05)。大黄素可降低MDA水平(P>0.05),明显升高SOD水平(P<0.01)。3.大黄素能上调脂联素受体2. PPAR-alpha、CPT1、ACOX1的蛋白及基因表达(P<0.01)。第二部分大黄素治疗非酒精性脂肪肝体内实验1.诱导液诱导HepG2细胞株24h后,油红染色显示细胞内有大小不等的脂滴存在,且细胞活力及形态较好,表明NAFLD单纯肝脂肪变性体外模型形成。2.大黄素可以改善细胞内脂质沉积、降低TG含量(P<0.05),降低ALT及AST含量,升高SOD含量(P<0.01)。3.大黄素能上调HepG2细胞株造模后脂联素受体2. PPAR-alpha、CPT1、ACOX1的蛋白及基因表达(P<0.01)。结论通过上述大黄素对NAFLD体内、体外药效及其可能的作用机制初步研究得出一下结论：1.大黄素能减轻NAFLD大鼠体质量,减轻肝脏肿大及肝脏湿质量,改善肝细胞内的脂质沉积。显示大黄素有一定的减肥作用,通过减轻内脏脂肪堆积达到治疗NAFLD。2.大黄素能降低小鼠血清肝酶及肝细胞释放的肝酶,改善NAFLD单纯性肝脂肪变性阶段由脂质沉积肝细胞导致的肝细胞受损。3.大黄素能减少血.清FFA,降低血清TG、TC、LDL-C,增加HDL-C,纠正血脂紊乱。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。4.大黄素能增强体内抗氧化能力,减少活性氧及自由基的产生,抑制脂质过氧化,减轻肝脏炎症及脂肪肝进展。其机制可能是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。5.大黄素能在体内及体外上调脂联素受体2及PPAR-alpha蛋白及基因的表达,并上调PPAR-alpha所调控的肝脏细胞内的脂肪酸氧化过程中的限速酶CPT1a及ACOX1的蛋白及基因表达,表明大黄素改善肝脏脂质沉积与增强肝脏脂质氧化有关,从而成为治疗NAFLD重要靶点。6.人参皂苷Rb1对于NAFLD单纯性肝脂肪变性与大黄素有相似的作用及机制。更多还原

【Abstract】 Non-alcoholic fatty liver (NAFLD) as a chronic liver disease is prevalent in many countries. the Mothidity of of Non-alcoholic fatty liver in Western countries is about15%-40%and9%-40%in Asia, and with rapid growth in the past15years. NAFLD consists of as pectrum of liver disease, ranging from simple steatosis to non-alcoholic steato hepatitis(NASH), fibrosis, cirrhosis and Hepatocellular carcinoma finally. The presence of non-alcoholic fatty liver disease is in connection with pericardial fat, carotid artery stenosis and increase in cardiovascular mortality, which makes the prognosis more serious. The disease has become one of the major diseases of serious impact on physical and mental health of our people, and the national economy, social development and stability in our country.Pathogenesis of NAFLD is no tvery clear until now,"two hits"theory is approved by most medical scientists."firsthit", is insulin resistance(IR). NAFLD almost exist insulin resistance. Insulin resistance lead liver cells to fatty degeneration through increasing lipolysis, in the mean time, sensitivity of damage factor inside and outside is rose."seeond 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 cytokines secreted by fat cells regulate lipid metabolism in liver cells. Signaling Pathways of adiponectin closely linked to two hits. Its regulation mechanism beeome the hot spot being researched by most medical scientists. Adiponectin receptor2existing in liver cells is suggested to relate to the pathogenesis of NAFLD. Adiponectin binding to its receptor in liver cells regulates the expression of nuclear transcription factor PPAR-alpha related to liver lipid oxidation, and increase expression of rate-limiting enzyme in lipid oxidation, thereby increases Liver lipid oxidation and reduces liver lipid deposition.There is no NAFLD in chinese tradional medicine, NAFLD is equivalent to Chinese "obesity""hypochondriac pain""jaundicet"..According toTCM theory, intake excessive energy caused dampness and heat stagnating in spleen, dampness-heat in middle jiao, then qiji is blocked by dampness-heat, with the passing of time blood stasis is indueed. To sum up, dampness, heat and blood stasis are the key for NAFLD. Emodin is one of the main active ingredient of Polygonum multiflorum and rhubarb. Polygonum multiflorum have the function of tonifing the liver and kidney, loosening bowel to relieve constipation. Rhubarb have the function of removing stagnated heat. Emodin has some effect in terms of lipid-lowering, protecting the liver cells, the role of the key pathological factor in NAFLD..The experiment aims to observe the systematic effect of Emodin on NAFLD, and make further reseach the mechanism. The conclusion of this experiment provid for exploratory research to develop the treatment of NAFLD drugs from Chinese herbal medicine.Objectives1. Observation of influence of emodin on nonalcoholic fatty liver early in vivo and in vitro models, including body weight, liver weight, liver steatosis, liver function, blood lipid metabolism and TG and liver enzymes in liver cell lines.2. Study on the possible mechanism of emodin in treating nonalcoholic fatty liver, including the improvement of lipid deposition, and protection on liver cells, reducing oxidative stress, and protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1.Methods62C57BL6mice were randomly divided into blank control group, model group and ginsenoside group and emodin group, the rosiglitazone group. The blank control group had been fed with normal diet, and other groups were given a high fat diet(88%ordinal forage+10%lard+2%cholesterin)for8w. After8W,two rats were randomly choosed from each group, liver tissue sections that stained with HE method is observed by light microscope to confirm NAFLD model mice replicated successfully. Then, the model group were randomly divided into4groups, namely blank model group, emodin group, ginsenoside group, rosiglitazone group. After eight weeks, the blank control group was given DMSO (5%DMSO20mg/kg)by intragastric administration, Ginsenoside group was given ginsenoside(20mg/kg),Emodin group was given emodin(20mg/kg), The rosiglitazone group was given given rosiglitazone(1Omg/kg). After treating for4w, all mice were fasted for12h,then were killed after being narcotized with chloraldurat to get serum and hepatic tissue rapidly according to routine, which is affixed by formalin, Packed with Paraffin, sectioned, stained with HE, evaluated the degree of hpatic steatosis and inflammation under the optical microscope. Then calculate liver index, to detect the content of serurn ALT, AST, TG,CHO, LDL, HDL. Hepatic tissue was leave over to make10%liver homogenate at4℃for detecting content of TG, FFA, SOD, MDA, detected the protein expression of AdipoR2, PPAR-alpha, CPT1a, ACOX1by wersten blotting.The mRNA expression levels of AdipoR2, PPAR-alpha, CPT1a, ACOX1by real time quantitative RT-PCR.HepG2cells were cultured in1640medium supplemented with7%newborn calf serum.50Upenicillin/mL and50μg streptomycin/mL. For subculturing purposes, cells were detached by treatment with0.25%trypsin/0.02%EDTA at37℃. Cultures were used at75%confluency. Cultures were divided into5groups. To induce fat-overloading of cells, HepG2cells at75%confluency were exposed to a long-chain mixture of FFAs(oleate and palmitate) for24h. Emodin, ginsenoside and rosiglitazone were added to each group for24h、48h、72h. Cells were stained with Oil-Red-O to examine the amount of fat accumulation in the cells. TG,ALT, AST, SOD, MDA in homogenates from cells were measured using a commercial kit. The mRNA and protein expression levels of AdipoR2, PPAR-alpha, CPT1a, ACOX1were detected by real time quantitative RT-PCR and wersten blotting respectively. Data are expressed as mean±S.D. The Student’s t-test was used to determine the statistical significance of the experimental data.ResultsExperiment1Emodin treatment of nonalcoholic fatty liver in vivo experiments1. According to the hepatic tissue pathology and the state of hepatic function of mice, we succeeded in replicating the models of rats with NAFLD.2. Compared with model group, the body weight and liver index of emodin group decrease, but has no considered significant (P>0.05). the liver weight of emodin group decrease significantly (P<0.05). Emodin can significantly improve liver fat deposition according to liver pathology (P<0.01). Emodin can improve Liver cell morph and arrangement significantly. Emodin can significantly reduce the serum ALT, AST, TG, FFA and CHO (P<0.05), reduce serum LDL and increase HDL, but has no considered significant(P>0.05). Emodin can reduce content of FFA and increase SOD in hepatic tissue significantly (P<0.05),can reduce content of TG and MDA in hepatic tissue, but has no considered significant (P>0.05)3. Compared with model group, protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1in emodin group increase significantly (P<0.01) Experiment2Emodin treatment of nonalcoholic fatty liver in vivo experiments1. After induced by FFAs for24h, Intracellular lipid vacuoles in HepG2cell line were seen by phase-contrast microscopy and confirmed by Oil Red O staining with fine cell viability and morph, indicating that the formation of the NAFLD lipid deposition in vitro model.2. Emodin can reduce intracellular lipid deposition reduce the TG, ALT and AST content significantly (P<0.05), and increase SOD content significantly (P<0.05)3. Emodin can increase protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1of the HepG2cell line after induced by FFAs (P<0.01)Conclusions1. Emodin can decrease significantly the body weight, improve hepatomegaly, reduce liver wet weight and the liver index. It is suggested that to reduce the abdomen fat may be one of the mechanism of emodin in the treatment of NAFLD.2. Emodin can decrease liver enzymes in serum and released from liver cell, improve damage on liver cells caused by lipid deposition in the simple fatty liver stage of NAFLD.3. Emodin can decrease the serum of FFA, TG, TC and LDL-C and increase HDL-C to correct the dyslipidemia. The possible mechanism for emodin to reverse liver fat deposition, improve liver function and protect the liver cells is reducing the entrance of free fatty acids into the liver cells, reducing triglyceride synthesis and reducing the toxic effects of excess FFA.4. Emodin can enhance the anti-oxidization ability, reduce lipid peroxidation. Emodin reduce liver cell free fatty acids flowing into the liver cells, reduce oxidative load of mitoehondrial, thereby improve (3-oxidation, reduce Ros and free radicals.5. Emodin can increase protein and gene expression of adiponectin receptor2and PPAR-alpha in vitro and in vivo, and raise protein and gene expression of CPTla and ACOX1, the rate-limiting enzyme in the fatty acid oxidation process regulated by PPAR-alpha in liver cells, indicating that the emodin can alleviate the NAFLD lipid deposition through this way. It is concluded that emodin can be an important target for the treatment of NAFLD.6. Ginsenoside Rbl has the same effect and mechanism on NAFLD as emodin.更多还原