Study On Protective And Mechanism Of Duck Embryo Antioxidant Peptide TD12 On Oxidative Stress Injury In HepG2 Cells

In this study,the antioxidant activity of duck embryo antioxidant peptide TD12 was examined in vitro.The model of oxidative damage induced by H₂O₂ in human hepatoma cells（HepG2）was established.The effects of duck embryo antioxidant peptide TD12 on the survival rate,ROS content,antioxidant enzyme activity,protein expression level,secretion of some cytokines and gene expression of HepG2 cells injured by H₂O₂ were studied by molecular biological methods.The Gene expression profiling of HepG2 cells induced by H₂O₂ before and after TD12 treatment was determined by the RNA-Seq technique,and the genes of difference expression were screened.The function of Gene Ontology and the signal pathway of Kyoto Encyclopedia of Genome were analyzed in order to explore the protective mechanism of duck embryo antioxidant peptide TD12 on oxidative damage of HepG2 cells induced by H₂O₂ at cell and gene levels.The main results are as follows:1.The results of antioxidant activity of duck embryo-derived antioxidant peptide TD12 in vitro showed that TD12 has strong radical scavenging activity,and its radical scavenging ability and total antioxidant activity increased gradually with increasing concentration,and the scavenging rates of TD12 on ABTS,DPPH and hydroxyl radicals was enhanced in a concentration-dependent manner.2.The results of protective effect of duck embryo-derived antioxidant peptide TD12 on H₂O₂-induced oxidative damage in HepG2 cells showed that TD12 had neither growth-promoting,toxic or growth-inhibiting effects on HepG2 cells;The concentration of H₂O₂with 400μmol/L was the appropriate concentration for the establishment of the induced damage model,and the intervention time was 1 h.The TD12 had a protective effect on H₂O₂-induced damage in HepG2 cells,and HepG2 cells were protected by TD12 in a concentration-dependent manner within a certain concentration range,and the level of intracellular ROS was significantly reduced after TD12 pretreatment.3.The results of the effect of duck embryo-derived antioxidant peptide TD12 on the antioxidant system of HepG2 cells induced by H₂O₂ showed that the activities of SOD,GSH-Px,CAT and LDH were increased and malondialdehyde（MDA）content was reduced within a certain concentration of TD12 in HepG2cells damaged by H₂O₂-induced.The expression levels of SOD1,CAT and LDHA proteins in HepG2 cells were increased to restore the stability of enzymes in the antioxidant system.and the release and m RNA expression of cytokines such as TNF-ɑ,IL-8,IL-4 and IL-10 in HepG2 cells has been inhibited by TD12 to alleviate oxidative stress.4.The results of RNA-seq analysis showed that a total of 2843 genes were differentially expressed in the injury group compared with the control group,of which 1664 were significantly up-regulated and 1179 were significantly down-regulated when HepG2 cells were damaged by H₂O₂-induced injury,.and a total of 282 genes were differentially expressed in the protected group compared to the injured group,of which 89 were significantly up-regulated and 193 were significantly down-regulated.GO function and KEGG pathway enrichment analysis of these differentially expressed genes revealed that many important genes were significantly regulated,such as MAPK,NF-k B,FOXO,HIF-1,cancer and other signaling pathways,which have a strong correlation with oxidative stress and apoptosis.The expression levels of proteins such as Nrf2,NQO-1 and HO-1were increased,and the expression of proteins and m RNAs such as Keap1 were decreased by TD12 in HepG2 cells damaged by H₂O₂.In conclusion,the duck embryo-derived antioxidant peptide TD12 is beneficial to the survival of HepG2cells through the transcriptional regulation of genes related to MAPKs,cancer signaling pathway and Nrf2-Keap1 signaling pathway.