Transcriptomics Analyze Hepatocytes Oxidative Damage Of Grouper(Epinephelus Coioides) Induced By Hydrogen Peroxide

1.Construction of hepatic cell oxidative damage model in grouper?Epinephelus Coioides?.To set up a dependable and reliable model of hepatocytes oxidative damage in grouper?Epinephelus Coioides?,various H₂O₂ concentration?0,100,200,40,600,800 and 1000?mol/L?and reation time points?2,4,6,8,12 and 24 h?were included to optimize the concentration and time point,then detect their effects to the number and activity of hepatocytes.The cell viability and number were detected by trypan blue and MTT assay was used to test the cell rate.The results show that only 61.98% of hepatocytes survive under 800?mol/L H₂O₂ for 8 h.?0,100,200,600,800,1000 ?mol / L?and the time of action?2,4,6,8,12,24 h?were used to explore the effects of different concentrations of hydrogen peroxide cell oxidative damage concentration and time in order to establish a stable and reliable model of hepatocyte oxidative damage in grouper,and to detect the changes in the number and activity of hepatocytes during culture.The cell viability and number were determined by using the blood cell counting plate according to trypan blue for dead cell rejection.The cell rate was measured by MTT assay.The results showed that the survival rate of the hepatocytes of the grouper was reduced to 61.98% at800 ?mol/L H₂O₂ for 8h.2.Effects of hydrogen peroxide on the activity and function of primary hepatocytes in grouper?Epinephelus Coioides?.To investigate the effects of hydrogen peroxide on the activity and function of primary hepatocytes in grouper?Epinephelus Coioides?,the hepatocytes were cultured in vitro.Malondialdehyde?MDA?,superoxide dismutase?SOD?,catalase?CAT?,and glutathione peroxidase?GSH-PX?,lipid peroxides?LPO?in the supernatant were determinated under different H₂O₂ concentration and time points,what's more we analyzed the cell growth and metabolism status.The data reveal that in both groups?800 ?mol/L and 1000 ?mol/L H₂O₂?,the SOD,GSH-PX and CAT were significantly decreased?P<0.05?,and the MDA and LPO were significantly increased?P<0.05?compared with other group.However,there is no significant difference between these two group?P>0.05?.The results present that the 800?mol/L H₂O₂ for8 h results in obvious oxidative stress to the primary hepatocytes in Epinephelus coioides,whicn is the optimum condition to form the oxidative damage model.In this study,hepatocytes were cultured in vitro to investigate the effects of hydrogen peroxide on the activity and function of primary hepatocytes of grouper?Epinephelus coioides?.Malondialdehyde?MDA?,superoxide dismutase?SOD?,catalase?CAT?,and glutathione peroxidase?GSH-PX?,lipid peroxides?LPO?,were measured in the supernatants of hepatocellular culture cells under different culture times and to analyze the cell growth and metabolism status.The results showed that the activities of SOD,GSH-PX and CAT were significantly decreased in the 800 ?mol/L and 1000 ?mol/L groups after treatment with hydrogen peroxide,and the contents of MDA and LPO were significantly increased?P<0.05?And 1000?mol/L group,the difference was not significant?P>0.05?.The results showed that the concentration of H₂O₂ was 800 ?mol/ L and the action time was 8h,which resulted in obvious oxidative stress in the primary hepatocytes of grouper?Epinephelus coioides?.This condition could be used as a model to establish the oxidative damage model Suitable condition.3.To study the effects of hydrogen peroxide on hepatocytes in grouper?Epinephelus coioides?using transcriptomics techniques.The cell gene expression is not exactly the same under different growth periods and growth environment.Currently the sequencing technology is the most commonly mean to reveal this differences.Based on the established oxidative damage model,we used the RNA-Seq technique to detect the hepatocytes in grouper?Epinephelus Coioides?under different treatment of H₂O₂,and analyzed the intracellular genes transcription regulation and changes in cell phenotype and function after hydrogen peroxide treatment.The transcriptome data displayed that 112,618 Unigenes were obtained by screening and exclusion of 83,802,103 original reads.And the Unigenes' s average length is 957.45 bp and N50 is 1,687 bp.110,560 functional annotation genes are obtained by the analysis of Blast and other related softwares.The different expression genes were mainly in the pathway of cell growth,metabolism and biological regulation according to the GO enrichment,and the metabolic pathway involved the most genes?170?from the KEGG pathway enrichment.2,570 genes were differentially expressed between hydrogen peroxide treatment group and control group,1,520 up-regulated and 1,020 genes down-regulated genes.The results indicated that 800 ?mol/L H₂O₂ had influence to metabolism,biological regulation,etc.related gene expression of hepatocytes in grouper?Epinephelus Coioides?.In conclusion,the prediction and analysis of differentially expressed genes has great significance to the growth,metabolism and disease prevention in grouper?Epinephelus Coioides?.