Copper Overloading Induced Hepatic Injury And The Protective Effect Of Curcumin

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.