Abstract:Fatty liver was a common disease in aquaculture. In recent years, the occurrence of fatty liver in fish has significantly increased, but the mechanism of this disease remains unclear. Most researches have focused on the pathogenesis of the disease, while little is known at the cellular level. In this study, we used primary hepatocyte culture of Jian Carp (Cyprinus carpio var. Jian) as the experimental material, and induced the hepatocyte injury by optimal exposure (dose and time) to oleic acid. The hepatocytes of Jian Carp were isolated and purified with trypsin digestion and then were cultured in a medium. To identify the proper concentration of oleic acid, the hepatocytes were treated with a gradient of concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8 mmol/L) for 24–48 h, and the supernatants and hepatocytes were then collected at different time points. We measured the contents of multiple biochemical parameters in the collected samples, including triglyceride (TG) and the total cholesterol (TC), as well as the activities of enzymes such as alanine aminotransferase (GPT), aspartate aminotransferase (GOT), γ-glutamyltranspeptidase (γ-GT), lactate dehydrogenase (LDH), and superoxide dismutase (SOD). We also determined the survival rate of hepatocytes using the MTT method. Lipid droplets in the cells were observed using the oil red staining method. Our results suggested that the cultured hepatocytes grew properly with a normal morphology, and that the hepatocytes could be used for the study of lipid metabolism. After the oleic acid treatment at different concentrations of oleic acid for 24–48 h, we found that the exposure to 0.4 mmol/L oleic acid for 48 h significantly elevated the levels of GOT, GPT, LDH, TG,TC, and γ-GT, but reduced the activity of SOD (P﹤0.05 or P﹤0.01). Lipid droplets were detected in the hepatocytes under light microscope after the treating with 0.4 mmol/L oleic acid for 24–48 h. The biochemical indices described above indicated that 0.4 mmol/L oleic acid and 48 hours could be the optimal conditions to induce hepatocyte steatosis in cultured hepatocytes. Therefore, in this study we successfully established a hepatocyte steatosis model that will be a useful tool in the future study of fatty liver disease in fish.
