Protective Effect Of Food-derived Peptides WY、WJ On Human Skin Fibroblasts’ Resistance To Injury

Food-derived peptides WY and WJ are are digestion products of fish proteins and poultry proteins.5-FU, as a thymidylate synthase inhibitor, interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate into thymidine monophosphate.5-FU causes a scarcity in dTMP, so rapidly dividing cancerous cells undergo cell death via thymineless death. H2O2is an important reactive oxygen species, which can easily through cell membranes and act on iron in cells by Fenton reaction to form high active oxygen free radicals. High active oxygen free radicals act on biological macromolecules and destroy the cellular structure, which eventually led to atherosclerosis, diabetes, inflammation, cancer.The present study use HSF and H22as modal cells, A series of experiments (MTT assay, single cell gel electrophoresis (SCGE)and flow cytometry) were designed to investigate the effect of WY and WJ on HSF against5-FU-induced DNA damage, H2O2-induced oxidative damage and antituvmorigenesis. The main conclusions are as follows:1. WY and WJ were produced by enzymatic hydrolysis of papain。WY was divided into two parts by70%ethanol:WY-1was the alcohol-soluble part and precipitation was WY-2; WJ was divided into two parts by70%ethanol:WJ-1was the alcohol-soluble part and precipitation was WJ-2; The molecular weight degradation products of WY,WY-1,WY-2,WJ,WJ-1,WJ-2were determinated through HPGPC assay. We found that WY and WY-2were large size peptides with the molecular weight of2000Da.However, WY-1, WJ and WJ-1were small size peptides with the molecular weight of240Da,WJ-2were also small size peptides with the molecular weight of400Da.2. Amino acid analyzer was used to determinate for the determination the amino acid composition of WY, WY-1, WY-2, WJ, WJ-1, WJ-2.The result showd that WY, WY-1, WY-2are were rich in glutamate and glycine. WJ mainly contains glutamate, and glycine; WJ-1mainly contains aspartic acid, glycine. WJ-2mainly contains leucine, glycine.3. MTT experiment was used to study the effect of WY, WY-1, WY-2on HSF proliferation after72h.We found that WY-2could significantly promot HSF proliferation, Wich is higher than that of WY, WY-1and carnosine. Compared with the comparison group, WY and WY-2could not have the obvious difference. We also study the effect of WY-2on the growth curve of HSF. The result showed than WY-2can promote HSF cell proliferation, and the value-added effect is most obvious in its logarithmic phase, unconspicuous in stable period, the most important is WY-2couldn’t change the natural growth law of HSF.4. MTT experiment was used to study the effect of WJ, WJ-1, WJ-2on HSF proliferation after72h.We found that WJ-1could significantly promot HSF proliferation, Wich is higher than that of WY-1and carnosine. Compared with the comparison group, WJ-2could not have the obvious difference. We also study the effect of WJ-1on the growth curve of HSF. The result showed than WJ-1can promote HSF cell proliferation, and the value-added effect is most obvious in its logarithmic phase, unconspicuous in stable period, the most important is WJ-1couldn’t change the natural growth law of HSF.5. MTT experiment was used to study the effect of WY, WY-1, WY-2, WJ, WJ-1, WJ-2on5-FU-induced the inhibition of HSF proliferation. From the results, we concluded that the products (WY, WY-1, WY-2, WJ, WJ-1, WJ-2) had no cytotoxicity the growth of HSF cell. They can protect DNA damage induced by5-FU in HSF cell, however, these samples showed different degree of protection under different condintions, including different sample concentrations, different doses of5-FU and different action time. On the whole, WY-2and WJ-1showed strong and wide protection.6. MTT experiment and SCGE were used to study the effect of WY, WY-1, WY-2, WJ, WJ-1and WJ-2on5-FU-induced DNA damage of HSF. From the results, in5-FU group, the survival rate is more than80%, the comet rate of HSF is98.09%, which provided the platform of DNA damage model in HSF. Based on this platform, we investigated that compared with5-FU group, WY and WJ attenuated5-FU-induced DNA damage, in which the tail length, the tail moment, the Olive tail moment and the tail DNA content significantly decreased, wherease the content of hydroxyproline increased dramatically. This suggested that WY and WJ could protect HSF against5-FU-induced DNA damage in a dose-dependent manner.7. The morphology of HSF was studied. Cell viability was observed by MTT experiment, the growth curve of HSF cell was drawn. In addition, we also explored the damage on HSF by different concentrations of H2O2. The death rate of HSF cell can be up to half induced by500μmol/L H2O2, which provided the platform of oxidative stress damage model in HSF cell. Based on this platform, studied the effects of different products(WY, WY-1, WY-2, WJ, WJ-1, WJ-2) at2-250μg/mL concentration to HSF induced by H2O2at high dose for a short time. From the results, we concluded that they can protect oxidative stress damage induced by H2O2in HSF, however, WY-2has a better protective effect on HSF than WY and WY-1. WJ-1has a better protective effect on HSF than WJ and WJ-1.8. MTT experiment was used to study the effects of different products(WY-2, WJ-1, WY-2+WJ-1) at2-250μg/mL concentration to HSF induced by H2O2at low dose for a long time. From the results, we concluded that they can dose-dependent protect oxidative stress damage induced by H2O2in HSF. When the studies use WY-2with WJ-1, the effect is better than each one of them, so we can find that WY-2and WJ-2may act synergistically.9. MTT experiment was used to study the effects of WY, WY-1, WY-2, WJ, WJ-1, WJ-2on the inhibition of H22proliferation. From the results, we concluded that they can significantly inhibit H22proliferation after48h. At250μg/mL, the inhibition rate on H22of WY, WY-1and WY-2is21.6%,27.9%and34.2%. At50μg/mL, the inhibition rate on H22of WJ, WJ-2and carnosine is24.6%,23%�'�13.1%, which is higher than sample concentrations. Both WY-2and WJ-1can act synergistically with5-FU. WY-2+WJ-1and5-FU can’t act synergistically, but WY-2+WJ-1has no effect on anti-tumor of5-FU. 10. Flow cytometry was used to detect the effect of WY-2and WJ-1on the cell cycle distribution of H22. From the results, we concluded that WY-2at different concentration, H22cell were accumulated at differently degree in G0/G1phase; WJ-1at250μg/mL, WY-2+WJ-1at50-250μg/mL and carnosine at10-250μg/mL, H22cell were accumulated at differently degree in S phase, and decreased in G0/G1phase. So WJ-1, WY-2+WJ-1can induce H22cell to be accumulated in S phase.