Establishment Of A Rat Model For Wilson Disease With Excessive Copper And A Study On The Mechanism Of Its Hepatic Injury

【BACKGROUND&OBJECTIVE】Wilson disease is an autosomal recessive disorder of copper homeostasis, the incidence of WD is 30/1000, 000. WD typically occurs in young people, the etiological factor of WD is the mutations in ATP7B gene, which is important for transport of copper, lead to defect of the function of ATP7B. Then copper accumulate in the liver and other tissues caused by dysfunction of removing excessive copper. The clinical manifestations are hepatitis, renal and neurological abnormalities. However, metabolism disorder of copper first occur in the liver, and liver is the most severe tissue. So more than 50% patients characterized by liver dysfunction, hepatocytes necrosis, prtal and the surrounding inflammation and fibrosis.At present, the specific pathogenesis of liver injury caused by excessive copper accumulation in the liver is unclear. The research indicated that apoptosis played an important role in the process of liver injure. However, the specific pathogenesis is still unclear. The imbalance of the ratio of Bcl-2 family proteins is the central component in the regulation process of hepatocyte apoptosis caused by many agents. The animal models of WD are varied, such as LEC rats, TX mice and ATP7B gene knock out mice. But the pathology and physiology changes of the animal models are not completely consistent with WD.In this experiment, our purpose is to establish a convenient and reliability rat model to investigate the pathogenesis of liver injure caused by accumulation of excessive copper, and to offer new ideas for clinical therapy.【METHODS】1. Rats are divided into 4 groups randomly: control group, copper loading 4 weeks group, copper loading 8 weeks, copper loading 12 weeks. All the rats are kept for 12 weeks, group A was fed by normal food and drink, group B, group C, group D were given food containing copper sulfate at the dose of 1g/kg and water that the copper sulfate concentration of 0.185% from the beginning of the ninth, fifth, first week respectively. At the end of the twelfth week, liver tissues and serum of all the rats were harvested for the detection.2. Copper concentrations of the rats serum and liver tissues were detected by flameless atomic absorption spectrometry. Serum levers of ALT were detected by biochemistry analysator.3. The TUNEL method was used to detect hepatocytes apoptosis and then AI (apoptotic index) was evaluated.4. Bcl-2 and Bax mRNA expression levers of rats liver tissue were detected by RT-PCR method, and then use gel imaging and analysis system to analyze.5. Bcl-2 and Bax protein expression levels were detected by immunohistochemistry staining, and then use graphic analysis system to analyze.【RESULTS】1. With the increase of copper accumulation time, the liver tissue and the serum copper contents increased gradually, with the highest level in the twelfth week (serum copper content is 16.43±4.51 mg/kg, liver tissue copper content is 4.53±0.44 mg/kg).2. With the increase of copper accumulation time, the serum ALT level increased gradually, with the highest level in the twelfth week (200.50±16.15U/L).3. With the increase of copper accumulation time, hepatocyte apoptosis index increased gradually, with the highest level in the twelfth week (AI=19.8±1.2%).4. The experiment groups Bax mRNA expression levels of liver tissue were significantly higher than the control group, and with the increase of copper accumulation the level was increasing. Meanwhile, the experiment groups Bcl-2 mRNA expression levels of liver tissue were also higher than the control group, and with the increase of copper accumulation the level was increasing. But the increase degree is lower than Bax, so the mRNA expression of Bcl/Bax ratio decreased gradually. The changes of Bax and Bcl-2 protein expression were consist with mRNA, so with the increase of copper accumulation the protein expression of Bcl/Bax ratio decreased gradually.【CONCLUSION】1. In this study we have successfully established a excessive copper accumulation rat model, which can be used to investigate the mechanism of liver injury caused by excessive copper accumulation.2. In this study, we observed that excessive copper accumulation induced hepatocytes apoptosis, meanwhile, with the increase of copper accumulation, Bax/Bcl-2 ratio increased gradually. It can be concluded that, excessive copper accumulation can lead to liver injury, and the mechanism may be the relative up-regulation Bax induced hepatocytes apoptosis.