OXIDATIVE DAMAGE OF SOYBEAN AQUEOUS EXTRACT AND SOYBEAN MEAL AQUEOUS EXTRACT TO GRASS CARP (CTENOPHARYNGODON IDELLUS) PRIMARY HEPATOCYTES

In order to investigate whether soybean meal and soybean have a damaging effect on hepatocytes and its mechanism, Soybean aqueous extract (SAE) and Soybean meal aqueous extract (SMAE) were used as the experimental material for culturing primary hepatocytes of grass carp for 24 hours. The final concentrations of SMAE and SAE were 0, 0.5, 5.0 and 10.0 mg/mL. The results showed that with the increase of the concentration of aqueous extract, the activity of hepatocytes decreased gradually, with a significant dose-response relationship (P<0.05). No significant difference in relative cell activity was observed among the low SAE and SMAE concentration (0.5 and 2.5 mg/mL) groups and the control group. In the 5.0 and 10.0 mg/mL SAE dose groups, the relative cell viability was negatively correlated with the concentration. The cell viability was (78.10±8.57)% and (65.97±7.35)% respectively, which was significantly different from the control group (P<0.01). The relative cell viability in the SMAE groups (5.0 and 10.0 mg/mL) was (86.35±7.17)% and (80.26±7.08)%, respectively, which was significantly lower than that of the control group (P<0.01). At the same concentration, the relative activity of cells in SAE group was lower than that in SMAE group. The ultrastructure of hepatocytes showed that the annular condensation of chromatin along the nuclear membrane, sparse light density, swelling of mitochondria, decrease of number and accumulation of lipid droplets. Hoechst 33285 staining showed that the fluorescence intensity of SAE and SMAE groups (10.0 mg/mL) was significantly weakened, the staining was uneven (chromatin agglutination), the nucleus was broken, and some apoptotic bodies appeared. Compared with the control group, the contents of Lactate dehydrogenase (LDH) and Malondialdehyde (MDA) in the cell culture medium were significantly increased, and the activities of Superoxide dismutase (SOD) and Glutathione (GSH) were significantly different (P<0.05). SAE and SMAE decreased MMP and increased ROS. With the increase of water extract concentration, the early apoptosis rate of cells increased. There was no significant difference in the apoptotic ratio between the low dose group (0.5, 2.5 mg/mL) and the control group. The apoptotic ratio of the SAE group with 5.0 and 10.0 mg/mL concentrations was (22.55±4.35)%, (55.03±2.76)%, and the SMAE group was (32.67±5.79)%, (37.11±8.57)%, respectively, which was significantly different from the control group (P<0.01). Transcriptome results showed that the enriched pathways were mostly related to inflammation, protein metabolism, amino acid metabolism and lipid metabolism. The above results show that the degree of damage to hepatocytes by SAE and SMAE is positively correlated with the amount of addition, and SAE and SMAE have the same damage effect. SAE and SMAE can cause cell ultrastructure changes, increase ROS levels, abnormal cell antioxidant systems and abnormal mitochondrial structure and function, which ultimately affect cell viability and trigger cell death pathways.