Study On The Protection Mechanism Of Antioxidant Peptides From Egg White On H₂O₂-induced Oxidative Injury In HEK293 Cells

Excessive free radicals lead to oxidative stress,which is manifested the imbalance between antioxidant defense systemsre and active oxygen species?ROS?or reactive nitrogen radicals?RNS?.Oxidative stress induces oxidative damage of enzyme and biological macromolecules,such as DNA,lipid,protein and carbohydrate.Moreover,oxidative stress will affect the expression of gene transcription and signal transduction,proliferation,differentiation and apoptosis of normal interference cells,eventually lead to the biological system function disorder,cause irreversible damage to the body finally.Cancer,aging,or many diseases are related to excessive free radicals.Egg is a source of high quality protein,it provides abundant nutrients for human.Furthermore,egg white protein and its hydrolysates have a variety of physiological activities which is drawing wide attention of the public,such as antioxidation,lowering blood pressure,bacteriostasis,immunoregulation,anti sticky and anticancer,etc.High-throughput RNA sequencing?RNA-seq?is a whole genome high-throughput transcriptome sequencing technology.It is widely used to analysis the expressions of genes on the level of genome and helps for understanding the function,metabolism and regulation mechanisms of some nutrients.The aim of this study is to determine the regulation of antioxidant capacity of egg white peptides in HEK293 cell.We firstly established an injury model of oxidative stress,and investigated protective effects of antioxidant peptides from egg white on H₂O₂-induced oxidative injury in HEK293 cell by using molecular biology and cytological methods.The survival rate,active oxygen content,antioxidant enzyme activity,and glutathione system effects of HEK293 cells were determined.Finally,RNA-seq was performed to explore the systemic transcriptome of the HEK293 cell before and after stimulation.Gene Ontology?GO?and Kyoto Encyclopedia of Genes and Genomes?KEGG?analysis were used to analysis of differential genes,signaling pathway in the cell and explore the mechanism of egg white peptides on H₂O₂ induced HEK293 cell.The main research contents are as follows:?1?Investigation the antioxidant activity of egg white-derived active peptides in vitroThe antioxidative activity of WNWAD and its modification peptides WNW,WAD,WN and AD in vitro were comprehensive evaluated.It was carried out by using the ABTS radical scavenging activity,DPPH radical scavenging activity,ORAC oxygen radical absorption capacity and hydroxyl radical scavenging activity.The results are as follows:The egg white-derived active peptides WNWAD,WNW,WAD and WN had strong ABTS radical scavenging activity in a concentration-dependent manner.However,the peptide AD had no ABTS radical scavenging activity.Egg white peptides WNWAD,WNW,WAD,WN,AD had low DPPH radical scavenging activity.WN showed the highest DPPH radical scavenging activity of 10.563%at concentration of 100?mol/L.The oxygen radical absorption capacity of WNWAD,WN,WAD,WNW were approximately 5.69,4.04,3.96,2.66 times higher than that of Trolox,respectively.In addition,WNWAD,WN,WAD and WNW showed low hydroxyl radical scavenging activities ranging from 10%to 15%.?2?Protective effect of egg white-derived active peptides on H₂O₂-induced oxidative damage in HEK293 cellsThe oxidative stress model of HEK293 cells was used to evaluate the antioxidant activity of peptides WNWAD,WNW,WAD,WN from egg white.The results are as follows:The egg white peptides showed no stimulatory or cytotoxic effects in HEK293cells.The optimal injury concentration of H₂O₂ was 400?mol/L,and the optimal time to treat the cells was 6 h.Results showed that WNWAD,WNW,WAD,WN exhibited the strongest anti-oxidative stress activity on H₂O₂-induced HEK293 cells.Within a certain concentration range,the peptides could increase the percentage of viable cell in a concentration-dependent manner.The level of intracellular ROS were monitored using the DCFH-DA fluorescent probe assay.The intracellular ROS levels of cells are induced by H₂O₂,and reduced by pretreatment with egg peptides.?3?Effect of egg white peptides on the antioxidant system of HEK293 cells injured by H₂O₂The oxidative stress model of HEK293 cells was used to evaluate the effects of egg white peptides WNWAD,WNW,WAD,WN on anti-oxidation enzyme activity and glutathione system.The results are as follows:The egg white peptides WNWAD,WNW,WAD,WN were able to improve the activity of the total superoxide dismutase?T-SOD?,hydrogenperoxidase?CAT?and glutathione peroxidase?GSH-PX?in HEK293 cells induced by H₂O₂.They could also increase the glutathione?GSH?content,reduce oxidized glutathione?GSSG?content,and maintain the stability of the GSH/GSSG ratio in a certain level.Egg white peptides WNWAD,WNW,WAD,WN could increase the relative expression of?-glutamylcysteine synthetase??-GCS?m RNA in HEK293 cells induced by H₂O₂,and protected cell from the injury caused by oxidative stress.?4?Effect of egg white-derived active peptides on the secretion of some cytokines in HEK293 cells injured by H₂O₂This study examined the effects of egg white peptides WNWAD,WNW,WAD,WN on the secretion of interleukin 8?IL-8?and tumor necrosis factor?TNF-??induced by H₂O₂ in HEK293 cells.The results showed that the content of IL-8 and TNF-?was significantly increased in the HEK293 cells treated with H₂O₂ and was decreased treated with the egg white peptides WNWAD,WNW,WAD,WN,indicating that egg white peptides could inhibit the release of interleukin-8?IL-8?and tumor necrosis factor?TNF-??.?5?RNA-seq analysis the effect of WNWAD on the genes expression and signaling pathways in HEK293 cells induced by H₂O₂RNA-seq was performed to explore the systemic transcriptome of the HEK293cells treated with H₂O₂ and egg white peptides WNWAD.The genes of the control group,oxidative stress injury group,and WNWAD antioxidative protection group were analyzed,and differentially expressed genes were screened.RNA-seq data showed that,866 genes were differentially expressed compared with the control group when HEK293 cells were injured by H₂O₂?P<0.05?.Of these genes,311 were significantly upregulated??2 fold?and 555 were significantly downregulated??-2fold?.In the presence of egg white peptides,a total of 226 genes were differentially expressed in the protected group compared to the injured group?P<0.05?.Among these genes,104 were significantly upregulated??2 fold?and 122 were significantly downregulated??-2fold?.GO and KEGG analysis showed that the signal transduction-related MAPKs signaling pathway was involved in the cellular antioxidnat activity of WNWAD.