苦参碱对NASH大鼠氧化应激的影响以及NASH患者氧化应激相关指标的检测及意义

【作者】 唐彬；

【导师】 姚树坤；

【作者基本信息】 北京协和医学院 ， 内科学， 2013， 博士

【摘要】 非酒精性脂肪性肝病(NAFLD)是一种与胰岛素抵抗(IR)和遗传易感密切相关的代谢应激性肝脏损伤,疾病谱包括非酒精性单纯性脂肪肝(NAFL)、非酒精性脂肪性肝炎(NASH)及其相关肝硬化和肝细胞癌。NAFLD发病机制复杂,目前多数学者认同“二次打击”学说：胰岛素抵抗(IR)作为初次打击造成游离脂肪酸的增加,肝内脂肪蓄积形成脂肪肝;氧化应激及脂质过氧化损伤作为第二次打击导致脂肪变的肝脏发生炎症、坏死和纤维化。氧化应激在NASH的发生发展中起了关键作用。因此,抑制氧化应激、脂质过氧化可阻止NASH进程,对于NAFLD的预防和治疗具有重要意义。目前对NAFLD的治疗多采用多烯磷脂酰胆碱、还原型谷胱甘肽、维生素E、熊去氧胆酸、水飞蓟素等细胞膜稳定剂和抗氧化剂,但治疗效果并不十分理想。因此,选择新型的抗氧化应激药物治疗对NAFLD有着非常重要的临床价值。日前,中医中药在抗炎抗氧化方面的疗效日益受到重视。目前研究发现生物碱,黄酮类,酚类等中药成分具有较明确的抗氧化作用,中药单体具有疗效确实,多靶点多环节起效,毒副作用小等独特优势。其中的佼佼者苦参碱(MT),是生物碱类的中药活性成分,其抗炎、抗氧化、抗纤维化等作用显著。环氧合酶(COX)是一种膜结合蛋白,为前列腺素(PG)合成过程中的一个重要的限速酶,在慢性炎症刺激时可促进TNF-α等炎症介质大量释放,诱导肝脏炎症细胞浸润。诱导型一氧化氮合酶(iNOS)在炎症刺激下,可大量表达和激活,持续催化产生大量一氧化氮(N0),对肝细胞产生毒性作用。已有研究表明,肝脏发生炎症和坏死的同时,COX-2、 iNOS均表达增高,且与肝脏炎症呈正相关。本研究旨在通过建立动物脂肪肝模型观察苦参碱对脂肪肝大鼠的疗效,并探讨其对肝脏环氧化物酶(COX-2)、诱导型一氧化氮合酶(iNOS)表达的影响。方法：选用4周龄Wistar大鼠30只,体重为113-138g,平均125.6±7.0g。随机分为3组：正常对照组(C组,n=10)、高脂模型组(M组,n=10)、苦参碱治疗组(Ma组,n=10),雌雄各半。正常对照组投喂普通颗粒饲料,其它组以高脂饲料替代普通饲料投喂。动物造模3周后,C组和M组给生理盐水,Ma组苦参碱36mg/kg/d灌胃治疗,共30天。所有实验动物末次给药后麻醉,腹主动脉采血,分离血清,取肝脏。测定肝指数,肝中甘油三酯(TG)、总胆固醇(TC)的含量,血清TG、TC、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量,HE组织病理学分析,荧光定量PCR和免疫组化检测肝脏COX-2、iNOS mRNA转录和蛋白表达水平。结果：高脂饮食能够引起大鼠脂肪代谢紊乱,肝指数升高,肝组织中TG、TC和血清中TG、TC、HDL-C、LDL-C升高(P<0.05),肝细胞变性坏死和肝组织炎症损伤(P<0.05),肝脏COX-2、iNOS mRNA转录和蛋白表达量增高(P<0.01)；与模型组比较,苦参碱能够降低肝指数,肝组织中TG、TC和血清中TG、TC、HDL-C、LDL-C含量(P<0.05),阻止肝细胞变性坏死,抑制炎症损伤(P<0.01),降低肝脏COX-2、iNOS mRNA转录和蛋白表达水平(P<0.01)。结论：苦参碱对高脂-脂肪肝大鼠疗效明显,其作用机制可能与抑制COX-2、iNOS mRNA转录和蛋白表达,阻止炎症发生和抗氧化有关。非酒精性脂肪性肝炎(NASH)为非酒精性脂肪性肝病较严重的病理类型,目前理论认为“二次打击”学说是其重要发病机制之一。“二次打击”主要是多种原因引起的氧化应激或脂质过氧化损伤。肿瘤坏死因子-α(TNF-α)是导致胰岛素抵抗的主要炎症因子之一,肝型脂肪酸结合蛋白(liver type fatty acid binding protein, L-FABP)是一种脂肪酸转运蛋白,其作为有效的内源性抗氧化物,有减轻氧化应激损伤的功能,本研究通过检测NASH患者血清中L-FABP、NF-α的水平,探讨二者在NASH发生发展中的相关性及意义。方法：收集肝功能异常(ALT在40-200IU/L)的NASH患者50例,对照组健康人群32例。所有受试者经B超检查符合弥漫性脂肪肝的诊断标准,测量身高、体重、腰围及臀围,并计算BMI和WHR,晨起空腹取血检测L-FABP和TNF-α及相关临床指标。结果：NASH组患者的ALT、AST、CH0、LDL-C、TG、BMI、WHR、hs-CRP、L-FABP和TNF-α水平均显著高于对照组人群(P<0.05)。Spearman相关分析提示,NASH组血清L-FABP水平与TNF-α、CHO、LDL-C、ALT、UA、hs-CRP显著正相关(P<0.05), TNF-α水平与ALT、UA、hs-CRP显著正相关(P<0.05)。结论：L-FABP和TNF-α可能在NASH患者肝功能损伤与炎症反应中发挥重要作用。更多还原

【Abstract】 Non-alcoholic fatty liver disease (NAFLD), which is considered to be the hepatic injury related to insulin resisitance and hereditary susceptibility. It encompasses a spectrum of disorders ranging from simple steatosis to inflammatory steatohepatitis (NASH) and cirrhosis. Although the mechanisms are complicated, a large amount of information on the key mechanism is "Two Hits Theory". The first hit of steatosis, giving rise to the first lesions is caused by excess free fatty acids (FFA) in the liver, which are sterified to triglycerides (TG). These initial lesions make the liver vulnerable to aggressive factors of the second hit, which is caused by the oxidative stress and lipid peroxidation. This leads to the occurrence of lesions in the hepatocytes, inflammation and fibrosis, and consequently the evolution of hepatic steatosis to steatohepatitits. Oxidative stress, along with the insulin resistance, plays one of the essential roles in NAFLD pathogenesis. Therefore, treatment with different antioxidants (vitamin E, vitamin C, betaine, etc) has been used in prevent the process of NASH. However, there are no optimal drugs for NASH treatment so far. Some monomers extracted from Chinese herbs have the property of antioxidative stress, such as alkaloid, flavonoid, phenols. Matrine (MT), is an active ingredients of alkaloids, shows an remarkable effect in anti-inflammatory, antioxidant, anti fibrosis aspects.Cyclo-oxgenase2(COX-2) is a kind of membrane-binding proteins involved in prostaglandin synthesis, it can promote the inflammatory mediator release such as TNF-awith a chronic inflammatory stimulate, induce the liver inflammation cell infiltration. iNos also has high expression and activation with inflammatory stimulate, continuous catalyze and generate a lot of NO, produce toxic effects on liver cells. This study aim to observe the therapeutic effects of Matrine on the expression of COX-2and iNOS in rats with non-alcoholic fatty liver diseaseMETHODS:30Wistar rats (4-weeks,113～138g weight),were randomly divided into3groups, including the control (C, n=10), high fatty diet model (M, n=10), and matrine treatment groups (Ma, n=10). The groups of M and Ma were fed with a high-lipid diet for3weeks to induce non-alcoholic fatty liver model. After3weeks, the Ma group was administrated with Matrine36mg/kg/d for30days, and the control and model groups were administrated with normal saline. At the last day, rats were sacrificed. Then, the liver index, content of TC, TG in the liver, and the concentrations of TC, TG, HDL-C, LDL-C in serum were examined. In addition, histopathology analysis and NAS Score were performed. Furthermore, the expression of COX-2and iNOS were measured by real-time fluorescent PCR and the immunohistochemistry methods.RESULTS:High-lipid diet increased the content of TC, TG in liver, the concentrations of TC, TG, HDL-C and LDL-C in serum (P<0.05) and increased the expression of the COX-2and iNOS (P<0.01). Matrine decreased the liver index and the content of TC, TG in liver and the concentration of TC, TG, HDL-C and LDL-C in serum (P<0.05), arrest hepatic cell adipose degeneration, inhibit inflammation and hepatic cell ballooning degeneration (P<0.01), and decreased COX-2and iNOS significantly (P<0.01).CONCLUTION:Matrine is an effective therapeutic in treating rat non-alcoholic fatty liver disease through synergistical inhibition of COX-2and iNOS, resulting in preventing inflammation and oxidation. Objective:To investigate the serum level of liver-type fatty acid binding protein (L-FABP) and tumor necrosis factor-α (TNF-α) and their relationship with the function and inflammation of the liver in non-alcoholic steatohepatitis(NASH).Methods:50cases of NASH patients and32cases of healthy person as control group were included. B ultrasonic inspection has been performed to confirm the diagnosis of diffuse fatty liver. The height, weight, waist, and hipline of all subjects were measured, BMI and WHR were calculated. The level of serum L-FABP, TNF-a and related biochemical indexes were measured.Results:Compared with the control group, the serum level of ALT, AST, CHO, LDL-C, TG, BMI, WHR, hs-CRP, L-FABP and TNF-α in the NASH group was significantly increased (P<0.05). Spearman correlation analysis revealed that the level of L-FABP is positively correlated with TNF-α, CHO, LDL-C, ALT, UA, and hs-CRP (P<0.05), and the level of TNF-α is positively correlated with ALT, UA, and hs-CRP. Conclusion:L-FABP and TNF-α may play important role in the impaired function and inflammation in the liver of NASH.更多还原