| Alcoholic liver disease(ALD)is a chronic liver disease in the world,which may lead to a series of liver diseases and damage of internal organs of the body.The pathogenesis of the disease is complicated,which is related to oxidative stress,glutathione depletion,abnormal metabolism of methionine,malnutrition and endotoxin induced apoptosis.The most common treatment for alcoholic liver disease is through no alcohol,nutritional supplements,and medication.These treatments have difficulties to carry out and have sideeffects.It is necessary to develop a new kind of therapeutic product instead of medicine.Bioactive peptides are a kind of natural therapeutic products,including a varity of functional peptides,and antioxidant peptides are a kind of bioactive peptides.Antioxidant peptides have the function of scavenging free radical,antioxidation,antibacterial and inhibiting lipid peroxidation.Casein derived antioxidant peptides have been shown to have antioxidant activity.Therefore,this paper selected casein as raw materials,preparation of antioxidant peptides,verification of its antioxidant activity and protective effect of antioxidant peptide on ethanol-induced liver cells,and explored the protective effect of antioxidant peptide and some componts in the blood in the microenvironment by ethanol-induced hepatocytes HHL-5.In this study,casein hydrolysate was used to obtain casein hydrolysate,and three kinds of plastein(phenylalanine,tyrosine and tryptophan)were modified.Objective to evaluate the effects of four kinds of antioxidant peptides on the cell survival rate,cell content,intracellular ROS content,cell apoptosis and cell cycle of ethanol-induced HHL-5 cells.The results of CCK-8analysis showed that 4 kinds of antioxidant peptides had obvious protective effects on ethanol-induced injury of liver cells,cell survival rate increased from 74.02% to 95.57%,97.28%,90.22% and 99.25%.As time increased,the greater the concentration,the protective effect was more obvious.The intracellular ROS content showed that the fluorescence intensity was 33.34 in normal cells,and the fluorescence intensity of liver cells after injury was about 76.42,and the fluorescence intensity of the four protection groups were about 52.34,50.52,50.27 and 48.48,respectively.The results of flow cytometry showed that the percentage of apoptosis was decreased from 24.00% to 6.00%.In the experimental group,the proportion of apoptosis decreased from24.00% to 10.00%.4 kinds of antioxidant peptides showed that the cells of ethanol injury showed anti apoptotic cells,and the effect was better than that of rescue.Further studies in the prevention period,4 kinds of antioxidant peptides have different effects on the cell cycle in S phase,and theintroduction of antioxidant peptide tryptophan on liver cells in S phase was 24.98%,the results showed that the antioxidant peptide changeed in cell cycle of liver cell HHL-5,and changed the value of efficiency the cell,thereby preventing apoptosis group protection effect was stronger than the rescued apoptosis group.On the whole,the 4 kinds of antioxidant peptides had obvious protective effects on HHL-5 cells damaged by ethanol.Using CCK-8 method,determined the content of intracellular ROS,intracellular MAD and LDH culture medium,that proofed antioxidant peptide(CH-try)with vitamins(vitamin A,vitamin C and vitamin E)and ion(potassium ion,zinc ion,magnesium ion and calcium ion)affected the interaction of liver cells in ethanol the oxidative damage.CCK-8 and LDH were detected.The results showed that the content of cultured antioxidant peptide(CH-try)and vitamins on ethanol damage liver cells showed a synergistic protective effect,with the increase of time,synergistically enhanced,the cell survival rate changed from 66.35% to 96.22%,the effect was obvious;antioxidant peptide(CH-try)and ions of ethanol damage liver cells,indicated that CH-try and potassium ions had no synergistic and antagonistic effect,synergistic protective effect of zinc ion,magnesium ion and calcium ion concentration showed low concentration effect was stronger,while high concentration had antagonistic effect.The content of intracellular ROS and MDA determination results showed that the antioxidant peptide 2 mg·mL-1(CH-try)and vitamins on ethanol damage liver cells were again showed synergistic protective effect;in the 1 mg·mL-1(CH-try)and ion of ethanol damage liver cells were once again showed a synergistic protective effect,with the the growth of the time,enhanced synergy.In short,antioxidant peptide of casein sources,had a significant protective effect on ethanol induced oxidative damage in human liver cells,and the physiological range of antioxidant peptide can improve the protective effect on liver cells. |
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