Study Of Sipunculus Nudus Accelerating Wound Healing Of Mice Skin And Its Mechanism

Wound healing was a complex and highly coordinated bioremediation activity that involves stages such as hemostatic,inflammatory,proliferative,and remodeling phases.When the skin was traumatized,and its continuity was destroyed,the blood vessels in the injury site were broken,causing blood loss and local inflammatory reaction.At the same time,environmental pollutants were easy to cause infection,delay healing and leave scars,which caused serious impact of patients physically and mentally.In China,more than 10 million people suffered from skin wounds per year.The development of new preparations for promoting wound healing and preventing abnormal scar formation possessed high clinical application value.Sipunculus nudus was nutritious and delicious.In China,its processing and utilization were less,mainly for fresh marketing and processing into dry products.Currently,its deep processing value had been paid more and more attention,and its activity research and application were also increasingly widespread.Modern medical research showed that Sipunculus nudus possessed functions of anti-oxidation,anti-fatigue,anti-viral,immune-modulating,anti-bacteria,anti-inflammatory and peripheral analgesia,among which functions of immune regulation,anti-bacteria and anti-inflammatory were closely related to wound healing.In addition,The Sipunculus nudus possessed a considerable content of arginine which possessed function of promoting wound healing.Other studies had shown that collagen from Phascolosoma esculenta could promote the repair of skin wounds,and as another member of Sipuncula,Sipunculus nudus might also possess the same effects.In this paper,Sipunculus nudus was used as raw material,and its efficacy and mechanism of promoting wound healing were discussed from the several periods of wound healing,so as to provide theoretical basis for its development into related products for promoting skin wound healing.The research contents were as follows:1.Enzymatic hydrolysis and full-thickness skin excisional model in mice were employed to evaluate the effect of Sipunculus nudus hydrolysate(SNH)on wound healing by smearing SNH ointment and orally administrating SNH solution.Compared with the NT group,smearing SNH ointment significantly shortened the bleeding time and accelerated wound healing,inhibited the secretion of IL-1?,TNF-? and TGF-?1 at 7 days post-wounding,and significantly increased the hydroxyproline content at 14 days post-wounding.The results showed that smearing SNH possessed the effect of wound repair.However,SNH by oral administration possessed no effect on wound healing.Consequently,the manner of external smear was employed to further screen for pro-healing components and to explore its mechanism.2.In order to screen out the best pro-healing components and explore the pro-healing mechanism,the method of extracting Sipunculus nudus collagen(SNC)by acetic acid and pepsin,using enzymatic hydrolysis of animal protease and flavor enzyme to prepare Sipunculus nudus collagen peptide(SNCP),using hot water extraction to prepare Sipunculus nudus extraction(SNE)and full-thickness skin excisional model in mice were employed.The wound closure,histopathology,quantitative real-time PCR(q RT-PCR)and transmission electron microscope(TEM)analysis were used to evaluate the effects of SNCP,SNE and SNH on pro-healing and scar formation prevention.In terms of wound closure,time of scab removal,epidermal remodeling anti-inflammation,promotion of collagen production and remodeling,regulation of diverse factors involved in pathological scar formation and diameter distribution of collagen fibrils,SNCP,SNE and SNH group were significantly better than none-treatment group(NT)(P<0.05).The results indicated that all three groups possessed activity of accelerating wound healing and inhibiting scar formation,and their mechanism were closely related to hemostatic function,regulation of inflammatory factors,collagen deposition,collagen fiber remodeling and expression of scar formation related factors.Among them,SNCP possessed the best effect,followed by SNH and SNE.Collagen peptide plays an important role in it,the higher content of it,the better effect of the pro-healing.3.The physical and chemical properties of SNC were analyzed by UV spectroscopy,SDS-PAGE gel electrophoresis,fourier transform infrared spectroscopy and differential scanning calorimetry(DSC),and the molecular weight distribution and free amino acids of SNCP were analyzed by high performance liquid chromatography(HPLC)and amino acid analyzer.The results showed that SNC was consisted of ?1 and ?2 chains,and possessed a triple helix structure,and its denaturation temperature was 32.8 °C.The molecular weight distribution of SNCP peptide chains below 5 000 Da,3 000 Da and 1 000 Da,accounted for 82.00%,67.13% and 34.36%,respectively.The contents of free amino acid accounted for 4.16% of SNCP.Arginine and proline accounted for 27.5% of the free amino acid,which indicated that the contents of amino acids related to wound healing were relatively abundant.4.SNCP was isolated and purified by Sephadex G25 gel column,and two components named F1 and F2 were obtained.The healing activity of SNCP,F1 and F2 was evaluated by cell proliferation assay.The results showed that both SNCP and F2 promoted the proliferation of HUVEC,Hacat and HSF.At the same concentration,F2 could greatly promote the proliferation of three cells.