Protective Effect Of Selenium-enriched Peptide From Cardamine Violifolia On Ethanol-Induced Hepatocyte Injury

Cardamine violifolia is a selenium-rich plant,which is approved as a food raw material in 2021.The organic selenium content of Cardamine violifolia is high and the organic selenium is mainly concentrated in the protein.At present,the preparation process of selenium-enriched protein and selenium-enriched peptide of Cardamine violifolia is relatively perfect,but the influence law and mechanism of selenium-enriched peptide of Cardamine violifolia on regulating oxidative stress are not clear enough.Therefore,this study takes the seleniumenriched peptide of Cardamine cordifolia(SPE)as the research object.Vitro chemical method was used to determine the free radical scavenging rate of SPE.An oxidative stress model was constructed via the alcohol-induced injury of hepatocytes to evaluate the cell protection and antioxidant capacity of SPE.The mechanism was preliminarily discussed by transcriptome analysis.The main conclusions of this study are as follows:The protein of Cardamine violifolia(CPR),peptide of Cardamine violifolia(CPE),selenium-enriched protein of Cardamine violifolia(SPR)and SPE were prepared and their antioxidant activities were studied by in vitro chemical methods(DPPH·scavenging ability,ABTS+·scavenging ability,OH·scavenging ability and O2-·scavenging ability).The results showed that the ability of SPE to scavenge four free radicals was stronger than that of SPR,CPR and CPE.The IC50 values of SPE scavenging DPPH·,ABTS+·,OH·,and O2-·were 1.40 mg/mL,1.17 mg/mL,0.42 mg/mL and 0.37 mg/mL,respectively.Two hepatocyte models of ethanol injury were constructed.In the model constructed with L-02 cells,the action time of ethanol was 6 h,and the concentration of ethanol was 600 mmol/L;in the model constructed by THLE-2 cells,the action time of ethanol was 12 h and the concentration of ethanol was 1000 mmol/L.The effects of three incubation methods(SPE preincubation,ethanol pre-incubation and co-incubation)on cell viability were investigated.The survival rates of L-02 and THLE-2 cells were increased to 82.44%and 78.01%respectively by SPE pre-incubation.Therefore,SPE pre-incubation was determined as a suitable incubation method for intervening ethanol-induced liver cell injury model.The cell protection and oxidative stress regulation ability of SPE were evaluated by studying cell damage and oxidative stress related indicators.The ethanol-induced L-02 and THLE-2 hepatocytes were treated with SPE at different selenium concentrations,the results showed that the SPE group with 12 μmol/L selenium concentration could improve the cell status of the two hepatocytes,reduce the release of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase,and reduce the early apoptosis rate and total apoptosis rate.The results of oxidative stress-related indicators showed that SPE pretreatment at 12 μmol/L selenium concentration could effectively reduce intracellular reactive oxygen species content,increase mitochondrial membrane potential,and increase the content of intracellular antioxidant enzymes(superoxide dismutase,catalase and glutathione peroxidase)and glutathione,thereby inhibiting ethanol-induced oxidative damage.Among them,glutathione peroxidase was highly expressed.After SPE intervention,the content of GSH-Px in L-02 cells and THLE-2 cells was 3.5 times and 5.6 times that of the injury group,respectively.L-02 cells were selected for transcriptome analysis.The results showed that compared with the injury group,SPE intervention changed the expression of 630 genes,of which 331 genes were up-regulated and 299 genes were down-regulated.The results of GO functional annotation showed that the biological functions of differential genes were mainly related to cellular components.KEGG functional annotation showed that differential genes were mainly enriched in signal transduction metabolic pathways.Enrichment analysis of metabolic pathways showed that glutathione metabolism and apoptosis pathways played a significant role in SPE protecting ethanol-induced L-02 cell injury.The key genes involved in these two metabolic pathways were verified by qT-PCR.After SPE intervention,the expression of GPX1 and NGFR genes in L-02 cells increased,which was consistent with the results of transcriptome analysis.