Study On Protective Effect Of Bovine Elastin Peptides Against Skin Photoaging And Its Action Mechanism

Elastin is an essential protein in extracellular matrix to bestow skin with unique and indispensable structure and mechanic.However,over-expression of matrix metalloproteinase?such as elastase?induced by UV usually causes a wide set of physiological and pathological processes including the degradation of elastin,which can finally lead to photoaging of skin.Therefore,elastase inhibitors are considered to be an effective method to protect skin from photoaging.In the current study,we studied the hydrolysis of elastin by protease Alcalase^?2.4.Besides,the protective effects of elastin hydrolysates?EH?with high elastase inhibitory activity on skin photoaging were investigated in a UV-induced photoaging model in mice and fibroblasts.Additionally,four elastase inhibitory peptides were purified and identified by UPLC-Q-TOF-MS/MS,and then these peptides were synthesized and analyzed for their potential elastase inhibitory mechanisms.Furthermore,the mechanisms of anti-photoaging on UV damaged fibroblasts and bioavailability of elastase inhibitory peptides were further studied.The main research results are as follows:?1?Bovine elastin is difficult for deep hydrolysis,and low hydrolysis degree and free amino acids content were observed?maximum at 2.16%and 33.23?g/mL,respectively?.A large number of amino acids including Gly,Ala,Val,Leu and Pro were found in the hydrolysates,while Cys,Met and Trp were nearly absent.This was mainly due to the special amino acid composition of elastin.Peptide fingerprints,generated from stop-flow size-exclusion chromatography?SEC?×reversed phase liquid chromatography?RPLC?,revealed that peptides of higher molecular weight and/or weaker polarity were hydrolyzed to peptides of lower molecular weight and/or stronger polarity as the extending hydrolysis time,and similar peptide fingerprints were found in EH after hydrolyzing for 10 h.Additionally,405 peptides were identified from EH,and most fragments derived from elastin's hydrophobic domains which contained repeated hydrophobic units,while few peptides were found from the terminal region,especially C-terminal region of elastin.?2?The ingestion of elastin peptides could obviously ameliorate the epidermis hyperplasia and fibroblast apoptosis,and increase the content of hydroxyproline and water in photoaging skin in vivo?p<0.05?.Besides,EH could effectively prevent fibroblasts from apoptosis,and it could also help to inhibit the degradation of collagen and elastin by decreasing MMP-1and elastase in UV-damaged fibroblasts.?3?The most active fraction was obtained from bovine elastin hydrolysates through separation by ultrafiltration,macroporous resin,C18 chromatography and HPLC.Four peptides were identified by UPLC-Q-TOF-MS/MS and these four peptides were sequenced as PY,GLPY,GLGPGVG and GPGGVGAL.Among them,GLPY exhibited the highest elastase inhibitory activity with 58.77%,followed by GPGGVGAL and GLGPGVG,while the dipeptide PY displayed the weakest elastase inhibitory activity.The competitive inhibition of GLGPGVG and mixed inhibition of PY,GLPY,GPGGVGAL on elastase were illustrated by Lineweaver-Burk.Furthermore,GLPY,which displayed the highest affinity to elastase?6.4 kcal/mol?,could strongly interact with elastase.Obviously,many hydrogen bonds were observed between PY,GLPY,GLGPGVG,GPGGVGAL and elastase,which implied that these peptides could enter into the hydrophobic channel of elastase and obstructed hydrolysis of elastin by elastase.?4?PY,GLPY,GLGPGVG and GPGGVGAL could suppress the increase of ROS induced by ultraviolet radiation and alleviated the UV-irradiation-induced oxidative stress.All of four peptides could inhibit Ca²⁺influx while only PY showed effects on mitochondrial membrane potential,and finally inhibit the apoptosis of fibroblast.In addition,PY,GLPY,GLGPGVG could reduce the degradation of elastin by decreasing the content of elastase while GLGPGVG could reduce the degradation of type I collagen by decreasing the content of MMP-1.Furthermore,PY,GLPY,GLGPGVG and GPGGVGAL could contribute to the increase of type I collagen by up-regulating mRNA expression of type I collagen and could also regulate the abnormal expression of elastin mRNA in UV-induced fibroblasts.?5?The further hydrolysis of PY,GLPY,GLGPGVG and GPGGVGAL by gastrointestinal enzymes was performed in simulated in vitro digestion,which led to the decrease in elastase inhibitory activity,especially PY and GPGGVGAL exhibiting no inhibition effect after in vitro digestion.The elastase inhibitory activity of GL and PY was much lower than GLPY,as well as LGPGVG and G,which might be the reason for the decrease in elastase inhibitory activity of GLPY and GLGPGVG after in vitro digestion.Moreover,the resistance of the peptides to proteolytic degradation by Caco-2 cell enzymes was also investigated.Although 6.50%of GLPY and 56.51%of GLGPGVG was cleaved during the transport study,both GLPY and GLGPGVG could be transepithelial transported intact across Caco-2 cell monolayers,and the apparent permeability coefficient was 1.02×10^-66 cm/s and 0.47×10^-6 cm/s for GLPY and GLGPGVG,respectively.Particularly,GLPY are mainly transported by the tight junctions-mediated transcytosis pathway and partly transported by PepT1.