Research On Mechanism Of Vibrio Anguillarum Metalloproteinase Inducing Apoptosis Of Head Kidney Cell Of Turbot(Scophthalmus Maximus)

In order to clarify the toxic mechanism of Vibrio anguillarum on the head kidney cells of Scophthalmus maximus.In this experiment,metalloproteinase used to treat head kidney cells,which were divided into control group(0μg/mL),low concentration group(1.6 μg/mL),middle concentration group(8 pg/mL)and high concentration group(40μg/mL).LDH and MTT methods were used to determine the integrity of cell membranes and cell survival rate.Hoechst 33342 fluorescence staining was used to detect apoptotic morphology.Flow cytometry was used to detect the changes of cell apoptosis,reactive oxygen species and mitochondrial membrane potential.RT-qPCR and ELISA were used to detect the expression of apoptotic-related genes and the changes of enzyme activity.The results showed that compared with the control group,the intracellular LDH release rate of the experimental group increased with the prolongation of stress time,and was significantly higher than that of the control group(P<0.05).The highest release rate of LDH was 82.61±9.48%in the high concentration metalloproteinase group for 24 hours.MTT results showed that the cytotoxicity of low concentration of metalloproteinase for 3h was small,and there was no significant difference between the two groups(P<0.05).Other time points were significantly lower than those in the control group(P<0.05),the cell survival rate was 76.19±0.04%after 24 hours.but the survival rate of cells in the high concentration group of metalloproteinase was below 50%after 6 hours of being stressed.Hoechst 33342 fluorescence staining and Annexin V-FITC/PI double staining showed that the apoptosis rate was highest in the high concentration metalloproteinase group at 24 hours.Mitochondrial membrane potential results showed that the mitochondrial membrane potential of low concentration group,middle concentration group and high concentration group showed a downward trend,and significantly lower than the control group from 6 to 24 hours(P<0.05).The fluorescence signal of DCF in high concentration group of metalloproteinase at 24 hours of reactive oxygen species(ROS)was the largest and significantly higher than that of control group(P<0.05).In addition,the expression levels of Caspase 3,Caspase 9 and Cyt C genes displays the tendency declining at the begining and rising up in late.Cyt C and Caspase 9 gene expression in high concentration group of metalloproteinase reached the maximum at 12 h and the expression of Caspase 3 reached the highest level at 6 hours,which was significantly different from that of the control group(P<0.05).The activity of Caspase 3 in high concentration group presented a rising trend within 3-24 hours.Except for 3h,the other time points were significantly higher than those in the control group(P<0.05).In summary,metalloproteinases has cytotoxic effect on head kidney cells of turbot,and high concentration of metalloproteinase can induce apoptosis of head kidney cells in a short time,and there is a relationship between time and dose.