| Wilson disease(WD)is an autosomal recessive inherited copper metabolic disorder that mainly occurs in the adolescence.ATB7 B gene mutation gives rise to the metabolic disturbance of copper in the body,which results in the abnormal deposition of copper in the tissues and organs,such as livers,brains and corneas.Under the circumstances,patients with WD might have symptoms of liver damage,neurological/psychiatric symptoms,and clinical manifestations of the K-F ring of the cornea.In more severe cases,it can even be life-threatening.The etiology and pathogenesis study of WD disease shows that the liver is not only the original defect site of WD,but also the most important affected organ.Many clinical data studies have shown that almost all patients with WD might be subject to various degrees of liver damage,including acute and chronic hepatitis,cirrhosis and even acute(sub-acute)hepatic necrosis,the latter of which is one of the leading causes of death among WD patients.Therefore,the treatment of various liver damages is one of the main tasks in the clinical diagnosis and treatment of WD.Since the 1970 s,a favorable curative effect has been achieved for treating WD patients by adopting the Traditional Chinese Medical Gandou Decoctionon(GDD)by the project researchers.Previous studies have shown that Gandou Decoction can increase the utilization and facilitate the excretion of coppers in the livers through promoting the expression of copper-metabolism-related proteins.From the perspective of regulation of copper metabolism,preliminary elucidation of the therapeutic mechanism for Gandou Decoctionon for treatment of WD patients has been made but further studies are still required for its therapeutic mechanism.Recent studies have shown that the Wnt/?-catenin signaling pathway plays an important role in the growth,development and metabolism of hepatocytes,participates in the progression of various liver diseases and suggests that this pathway is one of the key factors controlling and influencing hepatocyte function.Abnormal activation of signaling pathway is closely related to various pathological changes of the livers.Previous studies by this research group have found that high copper can suppress Wnt/?-catenin activity in hepatocytes and aggravate oxidative stress injury induced by high copper.Based on this,the study regards the WD model of copper-loaded rats as the reaearch subject.Further discussion for the therapeutic mechanism of Gandou Decoctionon on WD liver injury will be made by observing the changes of Wnt/?-catenin pathway-related protein expression in hepatic before and after copper-removing treatment,so as to provide a theoretical basis for finding molecular targets for the treatment of WD liver injury.Research Objectives: To explore the effect of Gandou Decoctionon on the Wnt/?-catenin signaling pathway in the liver of copper-loaded rats and its regulatory mechanism,and thereby to provide a theoretical basis for finding molecular targets for the treatment of WD liver injury.Research method: Male SD rats were fed a 1g/kg/d Cu SO4 feed and 0.185% Cu SO4 water of 0.2ml/10g/d for 12 weeks to make a copper-loaded rat model.After the successful establishment of the model,0.09g/kg/d penicillamine and 0.2ml/10g/d Chinese medicine Gandou Decoctionon were administered intragastrically for 4consecutive weeks.ICP-AES was used to detect the contents of Cu,Fe,and Zn in rat livers.HE staining was used to detect the pathological changes of rat liver.Immunohistochemistry was used to detect expression of Nitrotyrosine,8-Hydroxyguanosine proteins and Wnt/?-catenin pathway-related proteins ?-catenin,p-?-catenin,GSK3?,p-GSK3?,DKK1,Dishevelled3,c-Myc.Western blotting(WB)was used to detect expression of ?-catenin,p-?-catenin,GSK3?,p-GSK3?,DKK1,Dishevelled3,c-Myc in the Wnt/?-catenin of rat livers.Research result:(1)Results that ICP-AES was used to detect trace elements of Cu,Fe and Zn in rat liver: compared with normal group,model group significantly increased the content of liver Cu,Fe elements(P<0.01),zinc content(P>0.05)no difference;compared with model group,contents of Cu,Fe element in the rat livers of GDD group reduced(P< 0.05),zinc element content of no statistical significance(P>0.05);PCA group compared with model group rats liver Cu element content decreased significantly(P<0.01),Fe element were decreased(P<0.05),zinc element content of no statistical significance(P>0.05).(2)HE staining results showed that compared with normal group,the liver cells of model group were of different sizes,disordered arrangement,partial loss of hepatic cord,extensive hepatic vesicular degeneration,and cytoplasmic vacuolization;compared with model group,GDD and PCA groups of rat liver tissue damage degree is decreased obviously.(3)Immunohistochemical detection results show that compared with normal group,model group rats liver tissue in ?-catenin,p-GSK3?,c-Myc protein positive expression rate significantly reduced(P<0.01),p-?-catenin,GSK3?,DKK1,8-Hydroxyguanosine,Nitrotyrosine protein positive expression rate increased significantly(P<0.01),Dishevelled3 protein positive expression rate of increase(P<0.05);GDD and PCA groups compared with model group,?-catenin,p-GSK3?,c-Myc protein positive expression rate increased significantly(P<0.01),GSK3?,p-?-catenin,DKK1,8-Hydroxyguanosine,Nitrotyrosine protein positive expression rate significantly reduced(P<0.01),Dishevelled3 protein positive expression rate of decrease(P<0.05).(4)Western blotting method,according to the results of model group compared with normal group of ?-catenin,p-GSK3?,c-Myc expression quantity significantly reduced(P<0.01),p-?-catenin,GSK3?,DKK1,Dishevelled3 expression quantity increased significantly(P<0.01);GDD and PCA group compared with model group rats,?-catenin in the liver,p-GSK3?,c-Myc expression quantity increased significantly(P<0.01),p-?-catenin,,Dishevelled3 protein expression significantly reduced(P<0.01),GSK3?,DKK1 expression decreased(P< 0.05).Conclusions:1.Excessive copper deposition in the copper-loaded rat livers might induce oxidative stress injury of liver mitochondria,causing inactivation of Wnt/?-catenin pathway and inducing liver cell injury.2.Gandou Decoctionon can reduce copper content in copper-loaded rats,alleviate copper-induced oxidative stress injury,activate Wnt/?-catenin signaling pathway,and lessen liver damage of the copper-loaded rats.Therefore,Wnt/?-catenin pathway is a potential molecular therapeutic target for WD liver injury. |
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