| Pinctada martensii,known for its high-quality pearls,is the main shellfish that cultivates pearls in the coastal areas of southern China.Pearls are mainly carried out by artificial "nucleation operation" via wrapping the pearl nucleus with the mantle.The whole cultivation process will be accompanied by the inflammation and immune response of the shellfish.Therefore,the mantle of Pinctada martensii plays an important role in pearl formation.At present,pearl shellfish are often discarded after harvesting pearls,and mantle is rich in protein and is a valuable source of protein for human nutrition,so it is very necessary to fully develop and utilize the functional value of the mantle.Herein,enzymatic method was used to prepare enzymatic hydrolysates of Pinctada martensii mantle,and the function of skin wound healing was studied through in vitro and in vivo experiments,which provided theoretical basis and direction for further research.The main research contents and results of this study are as follows:(1)The enzymatic hydrolysates of the mantle of Pinctada martensii(EHMPM)was prepared by enzymatic method,and the full-thickness skin defects model of mice was established,and its healing-promoting activity was evaluated by smearing and oral administration.The protein content of EHMPM was 55.66 g/100 g,among,the peptides content accounted for 21.56 % of the protein content.The essential amino acid accounted for 63.02 % of the total amino acid,whereas the branched chain amino acidand the hydrophobic amino acid accounted for 32.92 % and 59.75 %,respectively.Compared with the negative control group,the application of EHMPM significantly shortened the hemostasis time and non-toxicity;both ways of administration of EHMPM significantly shortened the epithelialization time and improved the wound healing rate.On the 7th day,compared with the two control groups,smear administration could inhibit the production of inflammatory factor IL-6.Compared with the negative control group,EHMPM could significantly promote the secretion of IL-10.Moreover,compared with the negative control group,smear administration promoted the secretion of TGF-? and CCND1.Timewhile,TGF-? was significantly increased in the three dose groups of oral administration,CCND1 of the middle and high dose groups,and EGF of the low and high dose groups were obviously promoted.On the 14 th day,the hydroxyproline content in skin tissue was significantly increased in the three dose groups of oral administration,and the scar residue was significantly inhibited in the low and high dose groups.The results demonstrated that smear and oral administration had the effects of inhibiting inflammation,promoting growth factors to regulate cell proliferation and facilitatting collagen formation to accelarate skin wound healing in mice.(2)The EHMPM were preliminarily separated through sequencely ceramic membrane and ultrafiltration system to obtain > 5 KDa,3-5 KDa and < 3 KDa components(The protein content was 71.75?62.85?54.69 %,respectively;polypeptide content was 64.10?78.92?50.40 %,respectively).The hemostatic and antimicrobial activities of each ultrafiltration component were determined by in vitro experiments;the full-thickness skin defects model of mice was established,and the expression of cytokines and related proteins,collagen formation were detected by ELISA,H&E staining,immunohistochemical staining and other techniques to evaluate the healing-promoting activity and related mechanisms.The results showed that < 3 KDa ultrafiltration component had a certain procoagulant effect;smearing and oral enzymatic hydrolysis ultrafiltration products of Pinctada martensii mantle were significantly superior to the negative control group in hemostasis,scab removal and epithelialization time,wound healing rate,anti-inflammation,angiogenesis,epithelialization,fibroblast differentiation,promoting collagen generation and remodeling,inhibiting scar formation,etc.Among them,smearing and oral administration of < 3 KDa component could promote wound healing and inhibit scar formation,which was related to its ability to significantly shorten the time of hemostasis and epithelialization,and secrete antiinflammatory factors during the inflammatory phase.It was closely related to the secretion of anti-inflammatory factor IL-10 during the inflammatory phase to inhibit the inflammatory response,promote the expression of CD31,FGF and EGF,and secretion of TGF-?1,thus effectively promoting epithelialization and contraction of wounds,promoting collagen deposition and partially inhibiting superficial scar hyperplasia were closely related.< 3 KDa ultrafiltration component was the best active component to promote skin wound healing in mice.Therefore,this study separated and purified < 3 KDa ultrafiltration components,and preliminarily explored the mechanism of function through cell experiments.(3)After physical and chemical analysis and mass spectrometry analysis of < 3 KDa ultrafiltration component,the component was separated by gel chromatography(Sephadex G25),and four components(F1,F2,F3 and F4)were obtained.Ha Ca T and HSF cells were selected for proliferation experiments to evaluate the proliferation activities of < 3 KDa ultrafiltration components and four components.The results revealed that the < 3 KDa ultrafiltration component consisted of polypeptides with molecular weights ranging from 302.17-2936.43 Da.It also contained two characteristic peptide fragments of Gln-Leu and Asp-Leu.Polypeptides containing 2-15 amino acids accounted for 73.87% and hydrophobic amino acids accounted for 56.51 %.Both the < 3 KDa ultrafiltration component and the F4 component have the activity of promoting the proliferation of Ha Ca T and HSF.Among them,the F4 component was the optimum component,and the optimal concentration was 12.5 ?g/m L.That was correspond to the research results in animal experiments and enriches the mechanism of promoting the healing of active peptides in the mantle of Pinctada martensii.(4)In this study,natural healing-promoting ointment with marine characteristics was preliminarily developed with the core healing-promoting active constituent from < 3 KDa ultrafiltration component of the mantle of Pinctada martensii,and a full-thickness skin defects model of mice was established to evaluate its healing-promoting activity by smearing administration.Compared with the negative control group,the ointment had shortened the epithelialization time(10.80 ± 1.10 days),improved wound healing rate,inhibited inflammation,promoted fibroblast differentiation and granulation tissue formation.Mechanistically,it had been affected collagen synthesis through TGF-?/Smad signaling pathway,promoted CCND1 secretion and CD31,EGF and FGF proliferation,thereby inhibited scar formation,and increased the speed and quality of wound healing... |
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