【作者】 贾彦红；

【导师】 南月敏；

【作者基本信息】 河北医科大学 ， 内科学， 2012， 博士

【摘要】 非酒精性脂肪性肝病（Non-alcoholic fatty liver disease，NAFLD)为与肥胖、糖尿病等疾病密切相关的一大慢性肝病，其在发达国家的发病率可达20%，近年在国内也大幅增长，发病年龄趋于低龄化，病情可进一步发展为肝纤维化、肝硬化甚至肝癌，严重威胁着人类健康。纤维化为机体对慢性损伤的修复反应，是一种可逆性改变。因此，寻找一种有效的抗纤维化治疗措施，将会对临床治疗NAFLD具有重要意义。扶正化瘀复方为针对慢性肝炎肝纤维化、肝硬化的“瘀血内结、正气虚弱”病机，在反复药理实验研究基础上配伍组方研制的具有抗纤维化作用的传统中药复方，由桃仁、丹参、绞股蓝、虫草菌丝、松花粉、五味子六味中草药组成，可以显著改善慢性乙型病毒性肝炎或肝炎后肝硬化患者的临床症状与肝功能，逆转肝纤维化，降低门静脉压力，但其对非酒精性脂肪性肝纤维化是否具有保护作用及作用机制尚未见报道。本课题采用高脂、胆碱-蛋氨酸缺乏（high fat, methionine-cholinedeficient diet, MCD）饮食建立非酒精性脂肪性肝纤维化动物模型，观察扶正化瘀复方对非酒精性脂肪性肝纤维化小鼠的抗纤维化作用并探讨其作用机制，为非酒精性脂肪性肝纤维化的临床治疗及传统中药的开发提供理论依据。第一部分扶正化瘀复方对MCD饮食小鼠一般情况、血清生化学及肝组织病理学的影响目的:探明扶正化瘀复方对MCD饮食所诱导肝纤维化的预防及治疗作用，明确扶正化瘀复方与血红素氧合酶-1（heme oxygenase-1，HO-1）激动剂联合是否具有协同拮抗非酒精性脂肪性肝纤维化的作用，为非酒精性脂肪性肝纤维化的防治奠定基础。方法:采用MCD饮食喂养健康雄性C57BL/6J小鼠8周，建立非酒精性脂肪性肝纤维化模型，同时应用扶正化瘀复方或联合应用HO-1激动剂血晶素进行干预实验，以胆碱-蛋氨酸充足饮食设为对照组。酶法测定血清丙氨酸氨基转移酶（alanine aminotransferase, ALT）及天门冬氨酸氨基转移酶（aspartate aminotransferase，AST）水平；HE和Masson染色观察肝脂肪变、炎症活动和纤维化程度，并依照2010年中华医学会脂肪肝学组《非酒精性脂肪性肝病诊疗指南》及美国NASH临床研究协会病理工作组2005年制定的NAFLD组织学评分（NAFLD activity score，NAS）系统对肝脂肪变、炎症及纤维化程度进行评估。结果:1实验小鼠行为学改变各组实验小鼠均无死亡，与对照组相比，模型组小鼠毛发紊乱、无光泽，体重明显减轻，活动减弱、少动，精神萎靡。与模型组相比，扶正化瘀复方组及扶正化瘀复方与HO-1激动剂联合应用组小鼠一般状态尚可，毛发略显紊乱，较黯淡，摄食减少、体重减轻，活动较少，精神较为呆滞。2实验小鼠肝脏解剖学及组织病理学改变2.1大体观察对照组小鼠肝脏为暗红色，被膜光滑，明亮有光泽，质地柔软有弹性。模型组小鼠肉眼观可见肝脏体积明显减小，边缘圆钝，颜色变黄，无光泽，有明显油腻感,且与周围组织有粘连，质地较硬，弹性差。血晶素组与扶正化瘀复方组小鼠无明显差别，毛发略显紊乱，较黯淡，摄食减少、体重减轻，活动减弱，精神呆滞；扶正化瘀复方与血晶素联合组小鼠毛发较整齐、略有光泽，摄食较少，体重稍有下降，活动尚可，反应较灵敏，精神较饱满。2.2光镜下观察对照组小鼠肝组织中肝小叶结构均基本正常，肝板走行整齐，肝细胞索排列整齐清晰，围绕中央静脉呈放射状排列，肝细胞形状规则，排列致密、较均一，细胞核呈圆形；模型组小鼠呈非酒精性脂肪性肝纤维化表现，肝细胞呈大泡性脂肪变，小叶内可见灶状肝细胞坏死，伴有淋巴细胞和中性粒细胞浸润，肝窦间隙变窄，窦周细胞增生活跃，汇管区扩大，纤维组织增生，可见纤维间隔形成，NAS评分7~8S2~3；与模型组比较，血晶素组、扶正化瘀复方组、扶正化瘀复方与血晶素联合组小鼠肝脏损伤明显减轻，肝细胞呈轻度水样变，可见少数肝细胞呈脂肪变，个别呈气球样变，未见明显的肝细胞坏死及炎细胞浸润，偶见轻度纤维组织增生，汇管区扩大不明显（NAS评分5~6S1~2），尤以血晶素联合扶正化瘀复方组病变为轻（NAS评分1~2S0~1）。3实验小鼠血清ALT、AST水平对照组小鼠血清ALT、AST水平分别为：30.60±5.86U/L和32.00±5.57U/L，模型组小鼠血清ALT、AST水平分别为222.00±6.96U/L和316.00±14.2U/L，显著高于对照组（P<0.001；P<0.001）；血晶素组血清ALT、AST分别为146.80±9.20U/L和134.00±11.03U/L；扶正化瘀复方组血清ALT和AST分别为106.20±8.44U/L和108.60±10.48U/L，两组血清转氨酶水平均显著低于模型组（P<0.01；P<0.01）；扶正化瘀复方与血晶素联合应用组血清ALT、AST分别为71.25±12.76U/L和55.25±8.50U/L，与扶正化瘀复方组（P<0.001；P<0.001）、血晶素组（P<0.01；P<0.01）小鼠相比均显著降低。结论：1扶正化瘀复方可以显著改善MCD饮食小鼠一般情况，降低血清ALT、AST水平，减轻肝脂肪变、炎症及肝纤维化；2扶正化瘀复方与血晶素具有协同拮抗非酒精性脂肪性肝纤维化发病及进展的作用。第二部分扶正化瘀复方对非酒精性脂肪性肝纤维化小鼠肝脏脂质过氧化与炎症相关因子表达的影响目的：观察扶正化瘀复方对MCD饮食小鼠肝脏脂质过氧化与炎症相关因子表达的影响，初步探讨扶正化瘀复方拮抗非酒精性脂肪性肝炎及肝纤维化的机制。方法：采用MCD饮食喂养C57BL/6J小鼠8周，建立非酒精性脂肪性肝纤维化动物模型，分别应用扶正化瘀复方、HO-1激动剂血晶素（hemin）进行干预实验，以胆碱-蛋氨酸充足饮食设为对照组。采用硫代巴比妥酸法测定肝脏组织丙二醛（malondialdehyde，MDA）含量；采用实时定量PCR和Western blot方法检测脂质过氧化相关基因（HO-1）、氧化应激相关基因（CYP2E1）的表达；采用实时定量PCR方法检测炎症相关因子（TNF-α，IL-6）mRNA的表达。结果：1实验小鼠肝组织中MDA含量的改变对照组小鼠肝脏组织MDA含量为1.72±0.42nmol/mg，MCD模型组小鼠为5.67±1.25nmol/mg，明显高于对照组（P<0.001）；与模型组比较，血晶素组、扶正化瘀复方组小鼠肝脏组织MDA含量均明显降低，分别为3.58±0.71nmol/mg与3.88±0.79nmol/mg（P<0.05；P<0.05），而扶正化瘀复方与血晶素联合应用组MDA含量为3.17±0.77nmol/mg，分别与扶正化瘀复方组（P<0.01）、血晶素组（P<0.05）小鼠相比显著降低。2扶正化瘀复方对实验小鼠肝组织中CYP2E1表达的影响实时定量PCR和Western blot方法检测结果一致，MCD模型组小鼠肝组织CYP2E1mRNA及蛋白表达水平与对照组比较明显升高；与模型组相比，血晶素组、扶正化瘀复方组小鼠肝组织CYP2E1表达水平均显著下降；而血晶素与扶正化瘀复方联合应用组分别与扶正化瘀复方组、血晶素组小鼠相比CYP2E1表达水平亦显著下降。3扶正化瘀复方对实验小鼠肝组织中HO-1表达的影响与对照组比较，MCD模型组小鼠肝组织HO-1mRNA及蛋白表达水平明显升高；血晶素组小鼠肝组织HO-1表达水平与模型组相比显著升高；而扶正化瘀复方组小鼠肝组织HO-1表达水平较模型组显著下降，扶正化瘀复方与血晶素联合应用组小鼠肝组织HO-1表达水平高于扶正化瘀复方组，低于血晶素组。4扶正化瘀复方对实验小鼠肝组织炎症因子TNF-α、IL-6mRNA表达水平的影响MCD模型组小鼠肝组织TNF-α与IL-6mRNA表达水平与对照组比较明显增高；与模型组比较，血晶素组、扶正化瘀复方组小鼠肝组织TNF-α、IL-6mRNA表达水平均显著下降；而血晶素与扶正化瘀复方联合应用组分别与扶正化瘀复方组、血晶素组小鼠相比TNF-α、IL-6mRNA表达亦显著下降，提示血晶素、扶正化瘀复方均可从mRNA水平抑制TNF-α与IL-6的表达，减轻MCD饮食诱导的炎症反应，尤其两者联合应用其作用更为显著。结论：1在MCD饮食诱导的非酒精性脂肪性肝纤维化小鼠模型中，存在脂质过氧化与氧化应激反应，此可能为非酒精性脂肪性肝纤维化发病的重要致病机制之一；2扶正化瘀复方可以下调CYP2E1、HO-1基因的表达，阻止或减轻脂质过氧化与氧化应激导致的肝损伤；3扶正化瘀复方可以抑制炎症相关因子TNF-α与IL-6的表达，进一步阻止非酒精性脂肪性肝炎的发生与进展；4扶正化瘀复方与血晶素具有协同拮抗MCD饮食诱导的肝脏脂质过氧化、氧化应激反应、调节肝脏脂质代谢及炎症因子表达的作用，进一步减轻或阻止肝脏炎症向肝纤维化的进展。第三部分扶正化瘀复方对非酒精性脂肪性肝纤维化小鼠纤维化相关因子表达的影响目的：探明扶正化瘀复方阻止非酒精性脂肪性肝纤维化的主要作用靶点和分子生物学机制。方法：采用MCD饮食喂养C57BL/6J小鼠8周，建立非酒精性脂肪性肝纤维化模型，实验分组及干预方式同第二部分。采用实时荧光定量PCR和Western blot方法检测α-平滑肌肌动蛋白（alpha-smooth muscleactin，a-SMA）、转化生长因子β1（transforming growth factor beta1，TGF-β1）、Col-1、Col-3mRNA和蛋白表达。结果:1扶正化瘀复方对各组实验小鼠肝组织α-SMA、TGF-β1mRNA与蛋白表达的影响与对照组比较，MCD模型组小鼠肝组织α-SMA、TGF-β1mRNA与蛋白表达水平明显升高；与模型组比较，血晶素组、扶正化瘀复方组小鼠肝组织α-SMA、TGF-β1mRNA与蛋白表达水平显著降低，而血晶素组与扶正化瘀复方联合应用组分别与血晶素组、扶正化瘀复方组比较，α-SMA、TGF-β1mRNA、蛋白表达水平亦显著降低，证实扶正化瘀复方与血晶素均可以抑制α-SMA、TGF-β1mRNA的表达，尤其扶正化瘀复方与血晶素联合应用其抑制作用更为显著。2扶正化瘀复方对实验小鼠肝组织Col-1、Col-3mRNA与蛋白表达的影响与对照组比较，MCD模型组小鼠肝组织Col-1、Col-3mRNA与蛋白表达水平显著升高；与模型组比较，血晶素组、扶正化瘀复方组小鼠肝组织Col-1和Col-3mRNA、蛋白表达水平显著降低，而血晶素组与扶正化瘀复方联合应用组分别与血晶素组、扶正化瘀复方组比较，Col-1、Col-3mRNA、蛋白表达水平亦显著降低，证实扶正化瘀复方与血晶素均可以抑制Col-1、Col-3mRNA与蛋白的表达，尤其扶正化瘀复方与血晶素联合应用其抑制作用更为显著。结论：1扶正化瘀复方可显著抑制促纤维化相关基因α-SMA、TGF-β1、Col-1、Col-3的表达，尤其以扶正化瘀复方与HO-1激动剂联合其抑制作用更为显著。2扶正化瘀复方为有效防治非酒精性脂肪性肝纤维化的传统中药。更多还原

【Abstract】 Non-alcoholic fatty liver disease (NAFLD) is one of the chronic liverdiseases which is strongly linked with metabolic syndrome including obesity,dyslipidemia, type2diabetes, hypertension and so on. The prevalence ofNAFLD is estimated to be about20%in the western world, while it isincreasing rapidly in China and assumes the younger age tendency. Someindividuals can progress to hepatocacinoma eventually after reaching thefibrotic stage and subsequently cirrhosis threatening human health seriously.Fibrosis is a reversible healing response to chronic injuries, hence, searchingfor the effective antifibrotic methods is of great importance to the treatment ofNAFLD.Fuzheng Huayu recipe (FZHY), a compound of Chinese herbal medicinefor hepatic fibrosis, consists of six Chinese medicinal herbs, namely SemenPersicae, Radix Salvia Miltiorrhizae, Gynostemma Pentaphyllammak,Cordyceps, Pollen Pini, Fructus Schisandrae Chinensis. Based onpharmacological experiment repeatedly，it was produced in the light ofChinese medicine theory that deficiency of healthy energy and blood stasis arethe basic pathological changes of hepatic fibrosis. The previous clinical trialsshowed that FZHY could significantly improve clinical symptoms, liverfunction, reverse hepatic fibrosis and decrease portal pressure in patients withchronic hepatitis B and liver cirrhosis. However, the effect and mechnism ofFZHY on fibrosing steatohepatitis remains unknown.In this study, we investigated the protective role and molecular basis ofFZHY in the evolution of nutritional fibrosing steatohepatitis induced by highfat, methionine and choline deficient (MCD) diet in mice, which will providetheoretical evidence for the therapy of nutritional fibrosing steatohepatitis andthe development of traditional Chinese medicine. Part1Effect of FZHY recipe on general condition, serum ALT, AST,histopathologal changes in mice fed with MCD dietObjective: To explore the protective role of FZHY recipe in anti-fibrosis,and whether the co-administration of FZHY and hemin showed a synergeticeffect on anti-fibrosis, which could lay the foundations for antifibrotictreatment.Methods: C57BL/6J mice were fed with methionine-choline deficient(MCD) diet for8weeks to induce fibrosing steatohepatitis. Control animalswere fed with choline-methionine supplemented diet (control group). FZHYand/or heme oxygenase-1(HO-1) chemical inducer (hemin) wereadministered to mice, respectively. The effect of FZHY was assessed bycomparing the severity of hepatic injury, levels of hepatic lipid peroxides, andthe expression of oxidative stress, and inflammatory related genes. Serumalanine aminotransferase (ALT) and aspartate aminotransferase (AST) weremeasured by enzymic kinetic method using an automatic biochemical analyzer.Hematoxylin and eosin (HE) stained and Masson trichromatism stainedparaffin-embedded liver sections were scored for hepatic steatosis,inflammation and fibrosis according to the guidelines for diagnosis andtreatment of non-alcoholic fatty liver diseases and histological scoring systemfor NAFLD.Results:1Behavior alterationsAll the experimental mice survived. Mice in control group lookedenergetic, swift with shiny hair and without weight loss, while mice fed withMCD diet lost weight significantly, looked exhausted. There was nosignificance in comparing the general condition of mice between hemin andFZHY group with unbalanced hair, weight loss, less ingestion intake. Whilemice administered with hemin and FZHY presented worse general conditioncompared to mice fed the control diet.2Histopathology examinationThe liver sections from mice fed an MCD diet alone exhibited disordered lobule structure, severe macrosteatosis, spot or focal hepatocyte necrosis andinflammatory infiltration, portal fibrosis and fibrous septum (NAS7~8S2~3).However, mice treated with FZHY in the presence or absence of hemin couldnotably ameliorate hepatic steatosis, necrotic inflammation and improved liverfibrosis (NAS5~6S1~2). Co-administration of FZHY and hemin had a furtherimproved effect on hepatic inflammation and fibrosis (NAS1~2S0~1).3Serum levels of ALT and AST in experimental miceSerum ALT (222.00±6.96U/L) and AST (316.00±14.2U/L) of mice fedwith MCD diet significantly hegher than that of mice fed the control diet(30.60±5.86U/L,32.00±5.57U/L)(P<0.001, P<0.001). A significant decreaseof ALT and AST levels was noted after hemin (146.80±9.20U/L,134.00±11.03U/L) or FZHY (106.20±8.44U/L,108.60±10.48U/L) treatmentcompared to mice fed with MCD diet (P<0.01, P<0.01). An additive effectwas observed in the mice treated with FZHY and hemin, the ALT and ASTlevels in which (71.25±12.76U/L,55.25±8.50U/L) were markedly decreasedthan those of mice treated with FZHY (P<0.001, P<0.001) or hemin (P<0.01,P<0.01) respectively.Conclusions:1FZHY treatment could improve the general condition, reduce the serum ALT,AST levels, ameliorate hepatic steatosis, necrotic inflammation and improvedliver fibrosis.2Co-administration of FZHY and hemin showed a synergetic effect onanti-fibrosis in mice with nutritional fibrosing steatohepatitis induced by MCDdiet.Part2Effect of FZHY recipe on hepatic expression of lipid peroxidationmediator, oxidative stress related genes and inflammatory genes innutritional fibrosing steatohepatitis in miceObjective: To explore the effect and mechnism of FZHY recipe onhepatic expression of lipid peroxidation mediator, oxidative stress relatedgenes and inflammatory genes in nutritional fibrosing steatohepatitis in mice.Methods: Grouping and intervention methods in accordance with part1. Malondialdehyde (MDA) content was measured by Thiobarbituric acidreactive substances. The hepatic mRNA and protein expressions ofanti-oxidative gene HO-1and pro-oxidative stress gene cytochrome P4502E1(CYP2E1) were deteimined by quantitative real-time PCR. The mRNAexpression of inflammatory related genes TNF-α and IL-6was detected byquantitative real-time PCR.Results:1Effect of FZHY on hepatic oxidative stressThe levels of MDA in mice fed with MCD and control diet were in linewith part1. FZHY (3.88±0.79nmol/mg) or hemin (3.58±0.71nmol/mg)lowered hepatic oxidative stress as demonstrated by TBARS assay comparedwith MCD fed mice (P<0.05, P<0.05), while the combination of FZHY andhemin (3.17±0.77nmol/mg) showed a better effect on suppressing MDAconcentrations than FZHY (P<0.01) or hemin (P<0.05) treatment.2Effect of FZHY on the expression of lipid peroxidation mediator CYP2E1The mRNA and protein expressions of CYP2E1elevated significantly inMCD feeding mice. After FZHY or hemin treatment for8weeks, the mRNAand protein expressions of CYP2E1were down-regulated by compared withthat of mice fed MCD diet. However, Co-administration of hemin and FZHYfurther reduced the hepatic expression of CYP2E1.3Effect of FZHY on the expression of anti-oxidative stress factor HO-1The mRNA and protein expressions of HO-1in MCD feeding miceincreased significantly compared with mice fed with control diet. After hemintreatment the mRNA and protein expressions of HO-1were higher than that ofMCD fed mice. While FZHY treatment could down-regulated the HO-1expression. Co-administration of FZHY and hemin showed higher HO-1expression than FZHY and lower than hemin treatment.4Effect of FZHY on the mRNA expression of inflammatory ralated genesTNF-α and IL-6Relative to control mice, hepatic TNF-α and IL-6were up-regulated inMCD diet-fed mice, which was significantly blunted by treatment with FZHY or hemin. The combination of FZHY and hemin led to a further effect onpreventing the TNF-α and IL-6mRNA expression compared withMCD+hemin groupConclusions：1Oxidative stress and lipid peroxidation existed in the liver of nutritionalsteatohepatitis induced by MCD diet, which might lead to fibrogenesis.2FZHY treatment significantly reduce expression of HO-1and CYP2E1, thusprevent or ameliorate the hepatic injury triggered by excessive lipidperoxidation products.3Administration of FZHY and/or hemin significantely reduced the hepaticexpression of TNF-α and IL-6, consequently, alleviated or prevented fibrosingsteatohepatitis by decreasing liver inflammation.4Co-administration of FZHY and hemin showed a synergetic effect on liverinjury through inhibiting oxidative stress by mediating key oxidative stressrelated factors CYP2E1and HO-1, inflammatory ralated genes TNF-α andIL-6, which is beneficial for the treatment of fibrosing steatohepatitis.Part3Effect of FZHY recipe on hepatic expression of pro-fibrosis genesin nutritional fibrosing steatohepatitis in miceObjective: To explore the role and the molecular mechanism of FZHYrecipe in nutritional fibrosing steatohepatitis in mice.Methods：Grouping and intervention methods in accordance with part1.Hepatic mRNA and protein expressions of alpha-smooth muscle actin(α-SMA), transforming growth factor-β1(TGF-β1), collagen type I (Col-1)and Col-3were assayed by quantitative real-time PCR and Western blotrespectively.Results:1Effect of FZHY recipe on hepatic expression of α-SMA, TGF-β1In MCD feeding mice, mRNA and pritein expressions of α-SMA,TGF-β1had a marked elevation. However, administration of FZHY and/orhemin could reduce hepatic expressions of the two genes. The combinationof hemin and FZHY showed an additive effect on suppressing the above genes’ expressions.2Effect of FZHY recipe on hepatic expression of Col-1and Col-3The mRNA and protein expressions of Col-1and Col-3were induced byMCD diet. After hemin or FZHY treatment the expression of Col-1andCol-3was significantly down-regulated in the livers of mice, especially inthe livers of mice treated with the combination of hemin and FZHY.Conclusions:1FZHY recipe supressed hepatic expression of pro-fibrosis genes such asα-SMA, TGF-β1, Col-1, Col-3in MCD diet induced fibrosing steatohepatitis,especially the combination of FZHY and hemin showed a synergetic effecton inhibiting hepatic expression of pro-fibrosis genes.2Compound of Chinese herb medicine FZHY plays a protective role inameliorating nutritional fibrosing steatohepatitis.更多还原