【作者】 万小华；

【导师】 罗小平；

【作者基本信息】 华中科技大学 ， 儿科学， 2007， 博士

【摘要】 肝豆状核变性又称Wilson病(Wilson’s disease,WD),是一种因铜代谢障碍所致的常染色体隐性遗传病,主要表现为肝脏损害和中枢神经系统病变,发病率约为3/10万[1,2]。1993年WD致病基因被克隆并定位于13q14. 3,编码一种P型铜转运ATP酶(copper transporting P type ATPase,ATP7B),参与铜跨膜转运的代谢过程[3]。WD基因突变呈现遗传异质性,至今已发现了300多种突变形式,是导致其发病人种和地区明显差异的分子病理学基础及WD基因诊断与治疗的分子基础。由于WD的分子病理学的复杂性,对于WD的治疗至今还仅限于对症治疗而无法进行病因治疗。目前主要是应用螯合剂(如青霉胺)和锌盐等,螯合剂具有一定副反应,一般仅能用于维持治疗;肝脏移植被选择性用于具有急、慢性肝损伤的某些病例;WD的基因治疗可能尚需较长时日。因此,探讨铜损伤肝脏的机制对于WD疾病的治疗可提供新的方向和依据。以往对于铜损伤的研究表明体内过多的铜诱导自由基反应和脂质过氧化反应,损伤肝细胞,引起脂肪变性和炎症,导致肝细胞死亡。但对于铜诱导肝细胞凋亡及其凋亡信号途径的研究报道不多。姜黄素(Curcumin;diferuloylmethane)是从姜黄中提取的一种黄色酸性酚类物质,具有抗肿瘤、抗氧化、抗炎、清除氧自由基等药理作用,且毒性相对较低[4],临床应用潜能较大。姜黄素可以抑制激活蛋白1 (AP1)和核因子(nuclear factor kappa B, NF-κB)等转录因子以及依赖这些转录因子的基因表达[5],目前对姜黄素的药理及临床应用的研究涉及的内容越来越多,尤其是对肝脏的保护作用研究已成热点。近年来研究表明,许多疾病都与细胞凋亡正、负向精确调控失误有关。资料显示,肝缺血再灌注损伤、炎症、肿瘤等疾病都与细胞凋亡存在着密切联系。研究表明氧化应激与凋亡有着密切的关系[5],氧化应激诱导凋亡的机制与活性氧激活NF-κB、凋亡相关基因Bcl-2、c-Myc、Fas/FasL等有关[7,8]。参与凋亡的信号通路很多,JNK/SAPK通路研究较多,JNK通路的激活与细胞凋亡有密切关系[9],介导紫外线照射、氧化应激、细胞因子等引起的细胞凋亡。活化的JNK通过对转录因子c-Jun,ATF2,ELK1等磷酸化促基因的表达及新蛋白质的合成,新生蛋白质可能作用于细胞凋亡途径的某一环节而促进或引起细胞凋亡,对于不同的刺激因素和不同的细胞类型,作用的环节可能不同。Pan等[10,11]发现姜黄素能够通过抑制IκB激酶活性而抑制NF-κB活化,防止细胞因子、趋化因子等的诱生。姜黄素对凋亡有双重作用,对不同的细胞其作用有一定的选择性。在白介素1β诱导的大鼠肝细胞损伤实验中,有人发现,姜黄素可以抑制JNK被细胞应激和细胞因子的激活,从而抑制JNK途径的激活[12]。铜是人体必需的微量元素,但过多的铜对机体会产生毒性作用,金属催化生成的羟自由基是细胞氧化损伤的有效介质。在铜过多沉积的损伤过程中凋亡和凋亡信号通路改变以及其上下游调节机制有哪些?姜黄素对铜损伤肝脏是否有保护作用及其可能的机制如何?本实验拟通过建立大鼠的铜负荷模型,体外高浓度铜孵育培养BRL细胞(大鼠肝细胞),探讨铜负荷对肝脏的损伤,观察其凋亡情况和凋亡通路的变化、一些细胞因子以及姜黄素在铜负荷肝损伤中的作用,从细胞内信号转导和转录因子等角度研究凋亡在铜损伤肝脏过程中的意义,为进一步阐明WD疾病的发病机制提供理论依据,为寻求更有效的治疗方法和药物提供实验依据。研究内容:1.建立铜负荷大鼠模型和应用不同剂量的姜黄素干预:检测脂质过氧化水平,病理图像分析大鼠肝细胞损伤和凋亡状况,TUNEL检测大鼠肝脏凋亡指数,免疫组化法检测Fas和FasL在肝脏的表达,逆转录-聚合酶链反应(RT-PCR)检测TNF-αmRNA、IL-8 mRNA和ICE(IL-1β转化酶)mRNA的表达,ELISA法检测肝匀浆TNF-α和IL-8的含量变化,免疫印迹法(Western blot)检测肝组织JNK磷酸化水平的变化,EMSA法检测肝组织NF-κB的活性,从而探讨凋亡和凋亡信号通路以及特定的一些细胞因子是否在铜负荷大鼠肝损伤过程起作用,同时研究姜黄素对铜负荷大鼠肝损伤有无保护作用及其可能的机制。2.体外高浓度铜孵育培养BRL细胞,建立铜诱导肝细胞损伤的模型:采用流式细胞仪、四甲基偶氮唑盐(MTT)法、Hoechst33258荧光染色、Annexi V-FITC/PI染色法观察BRL细胞的凋亡,免疫荧光染色激光共聚焦显微镜分析Fas和FasL的表达,RT-PCR检测TNF-αmRNA、IL-8 mRNA和ICE mRNA的表达,免疫印迹法检测JNK磷酸化水平的变化,EMSA法检测NF-κB的活性,从而探讨高浓度铜孵育培养BRL细胞时凋亡、凋亡相关基因蛋白以及通路的变化,同时建立铜诱导损伤肝细胞的模型。3.应用铜诱导肝细胞损伤的模型,采用不同浓度的姜黄素或YVAD-cmk (四肽重氯甲酮或ICE抑制剂)(100μmol/L)进行干预,应用DCFH-DA法流式细胞仪测定细胞活性氧(ROS)水平,Annexin-V FITC/ PI双染色法观察BRL细胞的凋亡,免疫荧光染色激光共聚焦显微镜分析Fas和FasL的表达,RT-PCR检测TNF-αmRNA、IL-8 mRNA和ICE mRNA的表达,Western免疫印迹检测p-JNK蛋白的表达,EMSA法检测NF-κB的活性,从而探讨姜黄素对铜诱导损伤肝细胞BRL凋亡的影响及其可能的机制。结果1.铜负荷大鼠脂质过氧化水平明显升高;电镜结果显示铜负荷4周大鼠肝脏可见细胞早期凋亡改变,核染色质边集浓缩,呈半月形;随着铜负荷时间延长,线粒体肿胀,内质网扩张,溶酶体颗粒明显增多;TUNEL结果显示肝脏凋亡指数上升,免疫组化法检测显示Fas和FasL在肝脏的表达增强;TNF-αmRNA、IL-8 mRNA和ICE mRNA的表达以及肝匀浆TNF-α、IL-8的含量都增多;肝组织JNK磷酸化水平升高;肝组织NF-κB的活性增强。2.高浓度铜孵育培养的BRL细胞凋亡百分率和早期凋亡百分率明显升高;激光共聚焦显微镜显示Fas和FasL在肝细胞表达增强;TNF-αmRNA、IL-8 mRNA和ICE mRNA的表达增强;BRL细胞的JNK磷酸化水平升高;NF-κB的活性增强;选择100μmol/L铜孵育BRL细胞6h可以作为铜诱导损伤肝细胞的模型。3.与模型组比较,姜黄素组BRL细胞内的活性氧水平明显减低;与模型组比较,姜黄素或ICE抑制剂干预组的凋亡率下降;与模型组比较,姜黄素组TNF-αmRNA、IL-8 mRNA和ICE mRNA的表达和JNK磷酸化率以及NF-κB的活性明显降低。结论:1.铜负荷大鼠血清和肝组织的铜含量以及血清ALT升高,电镜显示肝脏组织病理改变包括线粒体肿胀,内质网扩张,溶酶体颗粒增多等。实验结果与文献报道基本一致[13],说明该模型建立成功。2. 100μmol/L铜孵育BRL细胞6h可以作为铜诱导损伤肝细胞的模型。3.过多的铜诱导肝细胞的凋亡,其机制与Fas和FasL家族和ICE(caspase-1)有关。JNK可能是介导铜损伤肝脏的重要信号之一。4.铜损伤时活性氧增多,NF-κB的活性被激活,NF-κB促进TNF-α、IL-8和ICE因子释放从而造成肝损伤。5.YVAD-cmk可以抑制铜损伤时BRL细胞的凋亡,间接地说明Caspase家族是其下游调节机制。6.姜黄素在过多的铜对肝损伤实验中表现出抗脂质过氧化,抗炎,抗凋亡作用,可能与其抑制NF-κB的活化、抑制JNK通路有关。本实验为其临床应用提供了一定的理论依据。由此可见,凋亡和凋亡信号通路在铜损伤肝脏中起重要的作用,可能通过NF-κB的活化、Fas和FasL系统及TNF-α、IL-8和ICE(caspase-1)起调控作用。姜黄素对铜损伤肝脏有保护作用,可能与其抑制NF-κB的活化、抑制JNK通路有关,这为研究WD疾病的发病机理提供了一定的理论依据,也为将来治疗WD疾病提供了新的思路。更多还原

【Abstract】 Hepatolenticular degeneration (Wilson’s disease, WD) is an autosomal recessive disorder of copper metabolism .The clinical manifestations are heterogeneous as well as their presentation, dominated by the neuropsychiatric and hepatic symptoms. The incidence of WD is 3 of one hundred thousand [1, 2]. Gene of WD was cloned and located on chromosome 13q14.3 in 1993, that encodes a hepatic copper transport protein (ATP7B) [3]. A principal characteristic of this disease is its wide phenotypic and genotypic variability. More than 300 mutations of this gene was molecular machinery involved in the pathogenesis of genetic hemochromatosis, which was the molecular fundament of diagnosis and therapy of WD.Because of complexity of molecular pathology in WD, therapy of WD is staying in thsymptomatic treatment, not etilogical treatment. The traditional treatment for WD is based on copper chelation with agents such as D-penicillamine, but use of this drug has been questioned because of reported side effects. Liver transplantation is indicated in cases with fulminant hepatitis, end-stage liver cirrhosis.Genetic therapy is still in research. So investigation of mechanism of copper injury should be helpful for elucidating the pathogenesis and treatment in WD. Previous researches demonstrated that excessive copper deposition resulted in liver injury and necrosis by reducing free radical and lipid peroxidation reaction. But little is known about apoptosis and its signaling transduction pathway involved in liver injury of copper deposition.Curcumin (diferuloylmethane), a plant-derived polyphenol, exhibits the propetities of anti-tumor, anti-oxidative, anti-inflammation, removing oxygen radical et al, and has no evident side effects [4] and has potentiality in clinic apply. Curcumin was found to inhibit the transcriptional activity of NF-kB and activator protein-1 (AP-1) [5]. Today curcumin is studied by more and more researchers in pharmacologic action and clinic application especially for its protective effect in liver diseases. Studies demonstrated that many diseases were related with abnormal apoptosis. Hepatic ischemic reperfusion, drug metabolism and metal toxic can generate lots of free radical, which unbalanced oxidation and antioxidation system in body, and result in oxidative stress on hepatocyte. Oxidative stress had tight correction with apoptosis [6],which related with activation of NF-κB,Bcl-2,c-Myc and Fas/FasL [7,8]. JNK is one of signaling transduction pathway which taking participate in apoptosis induced by ultraviolet irradiation, oxidative stress and cytokines [9]. Activating JNK maybe induce apoptosis by promoting phosphorylation of c-Jun,ATF2,ELK1 and expression of some proteins that different in kind of cell and stimulation. Pan et al found that curcumin could inhabit activation of NF-κB by inhabiting IκB kinase[10,11]. Curcumin had double effect in apoptosis, which different from cell category. Curcumin was found it could inhabit activation of JNK induced by oxidative stress and cytokines in rats injuried by IL-1β[12].Copper is one of essential trace elements in body, excess coppers are toxic to organs,but the mechanisms how coppers injury liver are not well understood.Little is known about the regulation of JNK and apoptosis in copper deposition in liver.Whether curcumin protect liver injury and the mechanism is still unknown. In this study, we focused on liver injury and apoptosis and regulation of apoptosis signaling pathway and effect of curcumin in copper overloading rats model and BRL cell in vitro cultured with high concentration of copper.The results should be helpful to eluminate the mechanism of WD and provide with experiment proof for effective therapy and drug for WD.Methods1. Copper-overloading rat model was established and various concentrations of curcumin were applied to the rats for different time period. Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in liver homogenates were measured to reflect the cupper induced lipid peroxidation. Histological changes induced by copper in liver were detected by using image analysis system. The apoptosis index in liver was detected by TUNEL assay. The expressions of Fas and FasL were detected by immunocytochemical staining. The expressions of TNF-αmRNA, IL-8 mRNAand ICE(IL-1βconverse enzyme)mRNA were observed by RT-PCR . Contents of TNF-αand IL-8 in liver homogenates were measured by ELISA. Levels of phosphorylation of SAPK/JNK were measured by Western blotting. Activity of NF-κB in liver was detected by EMSA. Thus this study was designed to investigate apoptosis and pathway of apoptosis and other accommodate factors in rat model of copper overloading and whether curcumin could attenuate liver injury.2. BRL cells were cultured with high concentration of copper in vitro and BRL cell model injuried by copper was established.Apoptosis of BRL was detected by methyl thiazolyl tetrazolium (MTT) colorimetric assay and Hoechst33258 fluorescence stains and Annexi V-FITC/PI stains through flow cytometry analysis.Expression of Fas and FasL were observed by immunofluorescence staining with confocal microscopy. Expressions of TNF-αmRNA, IL-8 mRNA and ICE mRNA were detected by RT-PCR. Levels of phosphorylation of JNK/SAPK were measured by Western blotting. Activity of NF-κB was detected by EMSA. Thus we investigated relations of apoptosis and apoptosis signaling pathway and proteins in BRL cell and established BRL cell model injuried by copper.3. Various concentrations of curcumin or 100μmol/L YVAD-cmk were applied to BRL cell model for different time period.Levels of reactive oxygen species (ROS) were detected with DCFH-DA by FCM.Apoptosis of BRL were observed by Annexin-V FITC/ PI staining. Expression of Fas and FasL were observed by immunofluorescence staining with confocal microscopy.Expressions of TNF-αmRNA, IL-8 mRNA and ICE mRNA were detected by RT-PCR. Levels of phosphorylation of JNK were measured by Western blotting. Activity of NF-κB was detected by EMSA.Thus we investigated effect of curcumin on BRL cell injuried with copper.Results1. Levels of lipid peroxidation in cupper-overloading rats increased significantly. Early apoptosis of liver, such as nuclei chromatin fringly condensing like half moon, existed in copper-overloading rats for 4w.Chondrosome swelled and reticulum distended and lysosome grains increased significantly in model rats.Apoptosis index (AI) was increasing by TUNEL, The enhanced expressions of Fas and FasL appeared obviously increased in liver tissue and the expressions of mRNA of TNF-α,IL-8 and ICE or protein of TNF-αand IL-8 were significantly increased in model rats. Levels of phosphorylation of JNK and activity of NF-κB were enhanced in liver of model rats.2. Percentage of apoptosis and early apoptosis were significant increasing in BRL cell cultured with high concentration of copper. The enhanced expressions of Fas and FasL appeared obviously increased and the expressions of mRNA of TNF-α,IL-8 and ICE were significantly increased in BRL cell cultured with high concentration of copper, same as levels of phosphorylation of JNK and activity of NF-κB. BRL cell cultured with 100μmol/L copper for 6h was suitable for cell model of copper injury.3.Levels of ROS were decreased significantly in curcumin groups.Percentage of apoptosis and early apoptosis were significant decreased in treatment groups with curcumin or YVAD-cmk. Curcumin down regulated expression of TNF-αmRNA, IL-8 mRNA and ICE mRNA and levels of phosphorylation of JNK and activity of NF-κB.Conclusions1. Levels of copper content in the serum and liver of copper overload rats were gradually increased along with copper overload time, the same as the level of liver alanine aminotransferase (ALT) in the serum of copper overload rats. Transmission election microscopy (TEM) revealed many of changes of liver ultrastructure:mitochondria were swollen, lysosomes grains were increased and endoplasmic reticulum were distend. The administration of copper, characterized by copper deposition and liver damage which consistent with used reports, was a useful model in vivo for studies of copper-induced damage events in the liver [13].2. BRL cell cultured with 100μmol/L copper for 6h was suitable for cell model of copper injury.3. Apoptosis induced by excess copper had relation with Fas/FasL and ICE (caspase-1). JNK pathway possible was one of important signaling pathways which mediated liver injury with copper.4. Excess copper promoted generation of ROS and activated NF-κB. NF-κB promoted the release of TNF-α, IL-8 and ICE and enhanced liver injury.5. YVAD-cmk inhabited apoptosis of BRL cell cultured with copper, which indirectly demonstrated involving of Caspase family in apoptosis induced by copper.6. Curcumin exhibited the propetities of anti anti-lipid peroxidation, anti-inflammation and anti-apoptosis in copper overloading models. The effect of curcumin may be related with inhibition of activation of NF-κB and downregulation of activation of JNK.So, our results suggested that apoptosis and apoptosis signaling pathway were crucial for liver injury by copper. Excess copper played a pivotal role on activation of NF-κB and enhanced expressions of Fas and FasL and release of TNF-α,IL-8 and ICE. Curcumin attenuated liver injury,which may be related with inhibition of activation of NF-κB and downregulation of activation of JNK. The results should be helpful for providing the novel clue for further elucidating the mechanism and treatment in WD.更多还原