| Long-term medium wave ultraviolet(UVB)radiation may cause skin photoaging,which is mainly reflected by dry,wrinkles,and pigmentation.Collagen peptides possess excellent physiological activity and are considered to exhibit potential applications in improving photoaging skin.The anti-skin photoaging activity of collagen peptide is related to its biological source,peptide sequence,average molecular weight ect.Studies have reported that fish skin and bone collagen are different in amino acid composition and the form and degree of hydroxylation,which may have diverse effects on enzymatic hydrolysates and their activities.The yield of tuna is huge,however,the by-products,such as skin and bone,which account for about50?70%of the total amount,can be produced during the processing,causing a great waste of resources.In this study,tuna skin and bone were selected as raw materials to explore the protective effect and mechanism of fish skin and bone collagen peptides on photoaging skin,and to clarify the differences and reasons between them.Firstly,tuna skin collagen peptides(TSCP)and tuna bone collagen peptides(TBCP)were prepared by different proteases and combinations.The differences in degree of hydrolysis,free amino group content,average molecular weight distribution,antioxidant activity,moisturizing and hygroscopicity were evaluated.On this basis,the anti-photoaging activity and molecular mechanism of TSCP and TBCP were explored by establishing UVB-induced photoaging NIH/3T3 cells and photoaging mice models,and to further analyze the differences between them.The main results and conclusions are as follows:(1)TSCP and TBCP were extracted by enzymatic hydrolysis of Neutrase(Neu),Alcalase(Alc),Flavorzyme(Fla),Alcalase+Neutrase(Alc+Neu),Alcalase+Neutrase+Flavorzyme(Alc+Neu+Fla),and their physicochemical properties,antioxidant activity and moisture absorption and retention were determined.The results showed that compared with a single enzyme,hydrolyzed by enzyme combination could promote the production of low-molecular-weight collagen peptides,and the obtained collagen peptides had a significant improvement in moisture absorption and retention properties.Under the same enzymatic hydrolysis conditions,TSCP had a higher proportion of low-molecular-weight peptides than TBCP.TSCP also had better moisture retention and absorption,and had better DPPH·scavenging activity.There was no significant difference in O2-·scavenging rate between them(P<0.05).While the scavenging effect of TBCP on·OH and ORAC antioxidant capacity were slightly better than TSCP.This indicated that both TSCP and TBCP had potential skin-protecting effects,but may be different in skin protection mechanisms.(2)NIH/3T3 cells were irradiated by UVB to establish the photoaging cell model.According to the free radical scavenging activity,moisture retention,moisture absorption and reactive oxygen species(ROS)scavenging ability of collagen peptides,Alc+Neu+Fla treated TSCP and TBCP had better moisture retention and absorption and could effectively scavenge free radicals.Therefore,TSCP and TBCP hydrolyzed by Alc+Neu+Fla were selected for the subsequent investigation of anti-photoaging activity.Cell experiment results showed that at the concentration of 0.1 mg/m L,TSCP and TBCP could enhance the viability of photoaging NIH/3T3 cells,had a significant scavenging effect on the excessive ROS(P<0.05),and could significantly inhibit the increase of mRNA and protein expression of MMP-1 and the decrease protein expression of TGF-?1 and type I collagen(P<0.05).TSCP and TBCP could also promote the mRNA expression of TGF-?1 and type I collagen.These results indicated that TSCP and TBCP had protective effects on UVB-induced photoaging NIH/3T3cells.Compared with TBCP,photoaging NIH/3T3 cells viability in TSCP group was significantly increased(P<0.05).TSCP could eliminate excessive ROS in photoaging cells to a normal level.The mRNA and protein expression of TGF-?1 in TSCP group was significantly higher than that in TBCP group(P<0.05).This suggested that TSCP had a more significant effect on improving cell viability and antioxidant capacity of photoaging cells and promoting the expression of TGF-?1.(3)ICR mice were irradiated by UVB to establish the photoaging mice model to study and compare the repairing effects of TSCP and TBCP on photoaging mice skin,and to explore action mechanism and differences.The results showed that at the oral dosage of 200 mg/kg,TSCP and TBCP could effectively suppress skin erythema and wrinkle in UVB-induced mice,attenuate hyperkeratosis and epidermal thickening,improve the hydration capacity and significantly increase hydroxyproline content and total antioxidant capacity(T-AOC)and significantly reduce the overexpression of MMP-1(P<0.05),besides increase the expression of TGF-?1 and type I collagen.In addition,TSCP and TBCP could inhibit the phosphorylation of ERK,JNK and p38.It is concluded that TSCP and TBCP could reduce UVB-induced skin damage in mice.It is speculated that TSCP and TBCP may alleviate the oxidative stress reaction of mice skin,inhibit the activation of MAPK signaling pathway,reduce the synthesis of MMP-1,promote TGF-?1 and type I collagen production,thereby protecting UVB-induced mice skin photoaging.Compared with TSCP group,the thickness of epidermis and stratum corneum of TBCP group was thinner,the structure of collagen fiber was more complete,the phosphorylation expression of ERK1 was significantly lower than that of TSCP group(P<0.05),and the phosphorylation expression of JNK2was no significant different from that in NC group(P>0.05);however,the skin water content of TSCP group was significantly higher than that of UVB group(P<0.05),and the expression of TGF-?1 was significantly higher than that of TBCP group(P<0.05),while the phosphorylation expression of p38 was significantly lower than that of TBCP group(P<0.05).It suggested that TBCP possessed a stronger ability to inhibit epidermal thickening and stratum corneous hyperkeratosis,maintain the integrity of collagen fiber structure,and reduce the phosphorylation expression of ERK and JNK protein and the expression of MMP-1 in mice skin.However,TSCP had a more excellent effect in improving the hydration ability of photoaging mice skin,promoting TGF-?1 protein expression and inhibiting the phosphorylation of p38.In conclusion,both tuna skin and bone collagen peptides could effectively attenuate UVB-induced skin photodamage in NIH/3T3 cells and ICR mice,however,there were differences in the molecular mechanisms of them.Tuna skin collagen peptides may possess more excellent antioxidant activity,scavenge excessive reactive oxygen species in photoaging body,inhibit the activation of p38 and could promote TGF-?1 expression and increase type I collagen synthesis,thereby exerting anti-photoaging activity.However,tuna bone collagen peptides may have a more excellent effect in inhibiting the MAPK pathway related proteins(ERK and JNK),reducing the expression of MMP-1 protein,thus inhibiting the degradation of type I collagen.This research not only provides a new theories basis for the study of collagen peptides in the field of photoaging research,but also provides new directions for the high-value utilization of tuna by-products. |
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