METHODS: Hepatic injury was induced in a copper-loaded rat model using the intragastric administration of copper(II) sulfate pentahydrate (CuSO4·5H2O). The water extract of GDD (0.4 g/kg/d) was administered twice a day for 4 weeks. Copper content and alanine aminotransferase (ALT) level, structural observation under the microscope, and immunohistochemical analysis of liver tissue were performed after the final administration. Moreover, the expression of β-catenin, GSK3β, Dishevelled-3, c-Myc, and p-GSK3β of liver tissue were detected to explore the relationship between the hepatic protection of GDD and the Wnt/ β-catenin signal pathway of GDD. We also stimulated the rat hepatic cell line BRL-3A with CuSO4·5H2O to establish a hepatic injury cytomodel. GDD serum at a concentration of 20% was administered into the model cell for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry were performed to detect cell viability, mitochondrial membrane potential (MMP), and the expression of reactive oxygen species (ROS). Meanwhile, the expression of the Wnt/β-catenin signal pathway-related proteins was evaluated.
RESULTS: GDD reduced copper and ALT while inhibiting oxidative stress and the degeneration and necrosis of liver tissue and hepatocytes. Treatment with GDD improved cell viability and MMP while suppressing the ROS level. Furthermore, GDD rectified the expression of Wnt/β-catenin signal pathway-related proteins in both livers of the copper-loaded and copper-stimulated BRL-3A cell lines.
CONCLUSIONS: GDD had apparent therapeutic effects on the hepatic injury of copper-loaded rats and copper-stimulated BRL-3A cells. Its mechanism is related to its regulatory effect on the Wnt/β-catenin pathway rectification and oxidative stress antagonism.