Purification,structure Identification Of Walnut Peptides And Mechanism Mediating Protective Effect Of Peptide On PC12 Cells Based On NF-?B/CASPASE Signaling Pathway

In this research,on the basis of the preparation of antioxidant walnut peptide with molecular weight<3 kDa,which has been proven to improve the memory impairment of mice,the walnut peptides were purified and identified by Sephadex G-25,Sephadex G-15,reverse high performance liquid chromatography?RP-HPLC?and HPLC-ESI-MS/MS.Peptides EVSGPGLSPN?Glu-Val-Ser-Gly-Pro-Gly-Leu-Ser-Pro-ASN?and LPLLR?Leu-Pro-Leu-Leu-Arg?with strong reactive oxygen species scavenging activity were obtained,and the protective mechanism of the two peptides from H2O2-induced oxidative damage in PC12 cells was investigated.The results are as follows:1.Walnut peptides?<3 kDa?were separated by Sephadex G-25 chromatography to obtain fraction A1,A2,A3 and A4.The fraction A3 with the highest activity was screened.When the maximum concentration was 2 mg/mL,the survival rate of PC12 cells was increased from 49.78%to 86.78%in the model group,and the ROS content in PC12 cells was 163.02 RFU;Then Sephadex G-15 chromatography was carried out to obtain fraction B1,B2,B3 and B4.It was verified that B3 had the highest activity.When the maximum concentration was 1 mg/mL,the survival rate of PC12 cells was increased from 51.36%to 81.36%in the model group,and the ROS content in PC12 cells was 114.31 RFU;Finally,fraction C1,C2,C3,C4,C5 and C6 were obtained by the purification of RP-HPLC.2.Fraction C1,C2,C3,C4,C5 and C6 were identified by HPLC-ESI-MS/MS.Combined with de novo sequence analysis and BIOPEP database screening,10 peptide sequences were obtained.The results showed that EVSGPGLSPN with molecular weight of 1148 Da and LPLLR with molecular weight of 610 Da had good protective effects on oxidative damaged PC12 cells.When the maximum concentration reached 100?M,the cell survival rate of the model group increased from 48.95%to 79.32%and 73.33%,respectively.The intracellular ROS content in model group decreased from 190.85 RFU and 216.35 RFU to 122.15 RFU and 146.22 RFU,respectively.Moreover,both peptides could significantly improve the activities of intracellular superoxide dismutase?SOD?,Catalase?CAT?and glutathione peroxidase?GSH-Px??P<0.01,P<0.05?.3.The effect of EVSGPGLSPN on the expression of key proteins in NF-?B signal pathway was investigated by immunofluorescence staining and western blot.Immunofluorescence staining results showed that EVSGPGLSPN could significantly reduce the expression of p65 protein?P<0.05?.Western blot results indicated that EVSGPGLSPN significantly reduced the expression of IKK?protein?P<0.05?,inhibited the activation of NF-?B signaling pathway,significantly reduced the expression of p65 protein?P<0.05?,and further inhibited the transcription of inflammatory factors IL-1?and TNF-?significantly?P<0.05?.4.The effect of EVSGPGLSPN on the expression of key proteins in Caspase signaling pathway was analyzed by western blot.The results showed that EVSGPGLSPN could reduce cell apoptosis by significantly down-regulating Cyt.C,Caspase-9,Cleaved caspase-3 and Cleaved PARP-1?P<0.05?,which could make PC12 cells resist ROS-induced damage.In addition,EVSGPGLSPN could significantly up-regulate the expression of CREB and synaptophysin protein?P<0.05?,which restored the function of PC12 cells to a certain extent.5.The effect of LPLLR on NF-?B/iNOS/NO signal pathway was analyzed by western blot.The results demonstrated that H₂O₂ could significantly up-regulate the expression of p65,iNOS and NO?P<0.01?.LPLLR could significantly down-regulate the expression of p65 and iNOS?P<0.05?,simultaneously reduce the secretion of NO?P<0.01?,which indicated that LPLLR may protect PC12 cells by inhibiting NF-?B/iNOS/NO signaling pathway from H₂O₂-induced oxidative damage.