| Sea cucumber is a high-quality seafood rich in protein,with high nutritional and medicinal value.The effective regulation in anti-oxidation,anti-fatigue,anti-coagulation and other aspects has been confirmed.However,few studies have been reported on the efficacy in medical,health care or functional foods based on the characteristics of sea cucumbers that are rich in nutrients and high in collagen.Nutrient supply and energy are needed during the healing process of skin wound,which is one of the most common body injuries,and collagen is also essential the skin composition.Therefore,the potential effects of sea cucumber hydrolysate(SCH)in promoting wound healing were detected and verified based on the rich nutrition and high collagen concentration of sea cucumbers,and the pathways and absorption mechanism for promoting wound healing were also explored and investigated.This work aimed to give a new direction for the high-value utilization of sea cucumber resources,and to provide a theoretical basis and experimental reference for the in-depth development of sea cucumber resources in medical nutrition.In this paper,the nutritional composition of sea cucumbers was determined first.Then SCH was obtained by enzymatic hydrolysis and used as the raw material in animal experiments.The effect of SCH in promoting wound healing was subsequently investigated via three batches of animal experiments.The results of enzymatic hydrolysis showed that the enzymatic hydrolysis efficiency was optimized at the ratio of aquatic protease to flavour protease of 6:4 with the total enzyme amount of 10000 U/(g prot),and the molecular weight of product hydrolyzed for 1 hour was less than 500 Da,accounting for 70.73 %.The effects of SCH on wound healing promotion were confirmed in the first batch of animal experiments though the model rats with total cortical excision.The animal experimental groups investigated included the vehicle group(Vehicle),the high-dose group(H-SCH),the middle-dose group(M-SCH),the low-dose group(L-SCH)and the positive group(YNBY).The results of wound healing rate showed that SCH insignificantly affected the normal growth of rats and effectively promoted their wound healing.The healing rate of M-SCH group with protein concentration of 0.50 g/kg by intragastric administration was significantly higher than that of Vehicle group(P<0.05)on the 8th day after injection,and the healing rate was 88.15%.The results of hematoxylin-eosin staining(H&E)and Masson's trichrome staining(Masson),inflammatory markers,Hyp and growth factors showed that SCH could reduce inflammatory cell infiltration,and promote angiogenesis,fibroblast proliferation,collagen deposition and re-epithelialization.The regulatory process might be related to the significant up-regulation of Hyp,vascular endothelial growth factor(VEGF),basic fibroblast growth factor(b FGF)and epidermal growth factor(EGF)(P<0.05).In addition,the M-SCH group showed the best morphological characteristics in the late healing stage,with the orderly and dense arrangement of newly formed collagen and the thinnest scar epidermis.After the effect of SCH on promoting wound healing were determined,three groups of SCH,i.e.,SCH with high,medium and low molecular weights,were prepared after the optimization of enzymatic hydrolysis time,and their composition differences were further analyzed.A second animal experiment was subsequently conducted to explore the effect of molecular weight of SCH on the wound healing efficacy based on the rat model of cortical resection trauma.At the same time,the inflammation and oxidative stress of traumatic rats were further measured,and the expression of pathway factors was combined to elucidate the role of SCH in promoting wound healing.Animals were divided into several groups,including the blank group(Blank),the model group(Vehicle),the group fed with SCH of high molecular weight(H-Mw),the group fed with SCH of middle molecular weight(M-Mw),the group fed with SCH of low molecular weight(L-Mw)and the positive group fed with Yunnan Baiyao(YNBY).The main difference among three groups with high,medium and low molecular weight SCH in amino acid and mineral composition was the content of Pro,Hyp and small peptides(<500 Da).The results of wound healing rate further confirmed that SCH could promote wound healing.The results of H&E,Masson staining and Hyp showed that the wound healing effect of L-Mw group was the best with insignificant difference with YNBY group(P > 0.05).In addition,the interleukin-6(IL-6),interleukin-8(IL-8),tumor necrosis factor-?(TNF-?)and malondialdehyde(MDA)(P < 0.05)were significantly down-regulated in L-Mw group,while the interleukin-10(IL-10),catalase(CAT),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)(P < 0.05)were up-regulated.Meanwhile,the secretion of VEGF,EGF and b FGF were significantly promoted in L-Mw group,and the m RNA expression of transforming growth factor-?(TGF-?),transforming growth factor receptor type ?(T?R?)and Smad family member 3(Smad3)were also stimulated(P<0.05).Next,the TGF-?/Smad pathway was activated to improve inflammation and oxidative stress and to promote collagen synthesis,ultimately accelerating the wound healing.The third animal experiment aimed to explore the difference in absorption of SCH with high and low molecular weight in model animals.In the experiment,the plasma,stomach,intestine and skin tissues of rats were collected under 6 different time intervals.The changes of gastrointestinal contents were observed,and the total contents of free/peptide-Pro,free/peptide-Hyp and 5 kinds of Hyp-binding peptides in blood and skin were determined.The expression of Oligopeptide transporter 1(Pep T1)m RNA in intestinal tract was measured.The influence of SCH molecular weight on its absorption in vivo was analyzed,and the absorption mechanism of SCH on wound healing promotion was explained.The results showed that the faster gastric emptying rate and intestinal absorption rate were observed in SCH with low molecular weight,in which more peptide-Pro and peptide-Hyp were exsited.More absorption rate of Ala-Hyp-Gly,Pro-Hyp,Gly-Pro-Hyp,Hyp-Gly and Ile-Hyp was observed in SCH with low molecular weight,and their contents were higher than that in SCH with high molecular weight.The expression of Pep T1 in the jejunum was more rapidly in SCH with low molecular weight,while the continuous expression of Pep T1 in the jejunum was stimulated for 2 hours.In conclusion,the effect of SCH with high,medium and low molecular weight on the wound healing promotion in SD rats was compared,and the best effect was observed in L-Mw-S group.The preparation conditions of L-Mw-S included substrate concentration of20%,enzyme addition of 10000 U/(g prot),enzyme ratio of aquatic protease to flavourase of6?4,hydrolysis temperature of 55?,p H 7.0,and 5 h hydrolysis.The absorption mechanism of SCH on rats wound healing promotion studied in animal experiments was related to the complete absorption of active Hyp-binding peptides in SCH by the intestine.These small peptides were completely absorbed through the Pep T1 pathway in the small intestinal epithelium,thereby inhibiting the inflammatory response and oxidative stress,promoting the secretion of growth factors,activating the TGF-?/Smad pathway to induce procollagen synthesis,and finally accelerating wound healing. |
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