Purification And Identification Of Anti-skin Photoaging Peptides From Oyster(Crassostrea Hongkongensis) Protein And Their Antiphotoaging Mechanisms

Although numerous studies showed that oyster and oyster-derived peptides possess multiple activities,few studies reported its antiphotoaging effect.This study aimed to purify and identify anti-skin photoaging peptides from Oyster(Crassostrea hongkongensis)protein enzymatic hydrolysates(OPEH),and investigate the possible mechanism underlying its skin photoaging effect.Thus,Multiple methods(including ultrafiltration,G25 Chromatography,RP-HPLC,UPLC/MS/MS)had been employed for this purpose and several peptides were identified and artificially synthesized in the last step.The protective effects of samples on UVB-irradiated Ha Ca T cells viability and MMP-1 inhibition were used for the screening of its antiphotoaging effect.The possible mechanism of the most effective peptide was further studied by measuring the activities of the MAPK/NF-?B signaling pathway,and the protein expression MMP-1 and apoptosis related bax,bcl-2)in UVB irradiation exposed Ha Ca T cells.An UVB-irradiated skin photoaging mice model was used to confirm anti-photoaging activityof samples,various molecular biological methods were used to explore its possible mechanism underlying its anti-skin photoaging effects.Several findings were observed including:(1)Compared with OPEH and its other three ultrafiltration fractions,OPEH-1(molecular weight less than 3k Da)has the lowest IC50 value(156.083?g/m L)for free radical scavenging.Meanwhile,OPEH-1 significantly increased cell viability of UVB-irradiated Ha Ca T cells starting the concentration of 50?g/m L in a dose-depended pattern.In addition,OPEH-1 reduced the cellular ROS accumulation,promoted intracellular type I collagen precursors secretion,and improved cell morphology at the concentration of 100?g/m L.(2)The topical application of OPEH-1 alleviated UVB irradiation-induced photoaging symptoms including skin moisture loss,epidermal hyperplasia,collagen and elastic fiber degradation.Meanwhile,OPEH-1 promoted the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),reduced the content of malondialdehyde(MDA),and increased the expression of collagen in the dorsal skin of UVB irradiated mice.Inadition,topical application of OPEH-1inhibited the expression of inflammatory cytokines(IL-1?,IL-6,TNF-?)and matrix metalloproteinase 1(MMP-1).The molecular mechanism of underlying OPEH-1anti-photoaging effect was associated to down-regulating the MAPK/NF-?B signaling pathway,as well as activating the skin TGF-?signaling pathway.(3)Four peptides of Tyr-Thr-Val-Thr--Phe(YTVTF),Arg-Lys-Asn-Glu-Val-Leu-Gly-Lys(RKNEVLGK),Val-Thr-Tyr(VTY),Trp-Asn-Leu-Asn-Pro(WNLNP)were identified after separation and purification.RKNEVLGK and WNLNP were showed anti-skin photoaging activity after artificial synthesis.The anti-skin photoaging activity of WNLNP was higher than that of RKNEVLGK.(4)In an UVB irradiation-photoaged Ha Ca T cell model,WNLNP exerted its antiphotoaging effect by reducing the production of intracellular ROS while promoting the production of type 1 collagen precursors.In addition,WNLNP inhibited the phosphorylation of the MAKP(p38,JNK,ERK)signaling pathway and nuclear transcription factor NF-?B(p65),the overexpression of MMP-1,and the occurrence of apoptosis.The molecular docking results showed that WNLNP could form 4 and 7 hydrogen bonds with NF-?B(p65)and MMP-1 respectively.(5)Mathematical simulations showed that WNLNP has poor skin passive penetration ability,with the Kp value 2.68*10^-4 cm/h.To improve its skin passive penetration ability and bioavailability,WNLNP was performed N-terminal acetylation and palmitoylation,as well as C-terminal amidation.N-terminal palmitoylation WNLNP(Palm-WNLNP,Pep)not only improved its skin penetration activity but also improved its anti-photoaging effect.(6)In the UVB irradiation-induced skin photoaging model of Kunming mouse,Pep attenuated UVB irradiation-induced photoaging symptoms including wrinkles,decreased water content,thickened skin,decreased elasticity,loss of collagen fibers.In addition,Pep promoted the activities of antioxidant enzymes(SOD,GPH-px)and the expression of anti-inflammatory cytokines(IL-10),while reduced the expression of pro-inflammatory cytokines(IL-1?),Furthermore,Pep decreased MMP-1,MMP-9and bax,but increased bcl-2 expression by inhibiting the the phosphorylation of the MAKP(p38,JNK)signaling pathway and nuclear transcription factor NF-?B(p65).The results from this study may provide a strong foundation for the application of peptides derived from Hongkong Oyster(Crassostrea hongkongensis)as therapeutic or cosmetic products.