| Sea cucumber,as a nourishing delicacy,has high edible and medicinal value.Sea cucumbers contain a variety of compounds,including collagen,polysaccharides and saponins,which have been shown to have a variety of effects.Sea cucumber peptide is a polypeptide obtained by enzymolysis of sea cucumber protein.Sea cucumber peptide has been proved to have various biological activities,but its mechanism is not clear,which affects its application.The basic composition,molecular weight distribution and oligopeptide composition of sea cucumber peptides were analyzed in this study.The immunosuppressive model induced by cyclophosphamide was used to detect the immuneenhancing function of sea cucumber peptide,and the relieving effect of sea cucumber peptide on UC was tested in the mouse ulcerative colitis model induced by sodium glucan sulfate(DSS).The antitumor activity of sea cucumber peptide was examined by cell model and animal model.On this basis,the biological activity mechanism of sea cucumber peptide was further discussed by molecular biology experiment and intestinal microbial sequencing experiment.The experimental content and results are as follows:(1)Determination of basic components,molecular weight distribution and oligopeptide composition of sea cucumber peptidesThe basic components,molecular weight distribution and oligopeptide composition of sea cucumber were determined by chemical and instrumental analysis.The results showed that the protein content was 94.71%± 4.69%,the moisture content was 2.95%± 0.11%and the ash content was 0.05 ± 0.00%.The molecular weight distribution test results showed that there were four main components in sea cucumber peptide,whose molecular weights were 1589.13 Da,1113.46 Da,569.29 Da and 193.05 Da,respectively,accounting for about 90%in sea cucumber peptide.Among them,the proportion of components whose molecular weight is 193.05 Da is 39.80%,the proportion of components whose molecular weight is less than 1000 Da is about 70%,and the proportion of components whose molecular weight is less than 2000 Da is about 90%.The results of amino acid automatic analyzer showed that there were 16 kinds of amino acids in sea cucumber peptide,among which glycine(7.77%± 0.07%)and glutamic acid(6.49%± 0.19%)accounted for the most.The oligopeptide sequences of sea cucumber peptides were detected by LC-MS,and the oligopeptide sequences were scored by PeptideRanker website.A total of 26 oligopeptides with activity scores greater than 0.5 were obtained.(2)Effect of sea cucumber peptide on immunosuppressed miceThe immunomodulatory effect of sea cucumber peptide was detected by immunosuppressive mouse model.The results showed that sea cucumber peptide could increase thymus index and spleen index of mice.Compared with model group,thymus index in SCPL group was significantly increased(p<0.05);Spleen index of mice in SCPM group was significantly increased(p<0.01).Sea cucumber peptide can reduce the content of ALT and AST in serum of mice.Compared with model group,ALT content in serum of sea cucumber peptide groups was significantly decreased(P<0.05);Serum AST content in SCPM group was significantly decreased(P<0.01).Sea cucumber peptide can increase the contents of IgG in serum and sIgA in small intestine of mice.Compared with model group,the IgG level in serum of mice in each dose group of sea cucumber peptide was significantly increased(p<0.01);The level of sIgA in small intestine of SCPL and SCPM groups was significantly increased(p<0.01).Sea cucumber peptide alleviates tissue damage in small intestine and colon of mice.Sea cucumber peptide can increase the protein contents of TRAF6 and IRAK1 and the protein phosphorylation levels of IκBα and p65 in small intestine of mice.Compared with model group,the content of TRAF6 in SCPM group was significantly increased(p<0.05);The content of IRAK1 in SCPL group was significantly increased(p<0.01);Phosphorylation levels of IκBα and p65 in SCPL and SCPM groups were significantly increased(p<0.05).Intestinal microbiota sequencing results showed that:Sea cucumber peptide alleviated intestinal microbiome imbalance in immunosuppressed mice.At the genus level,sea cucumber peptide increased the relative abundance of Dubosiella,Lachnospiraceae and Ligilactobacillus in mice intestinal microbiome.And lower the relative abundance of Lactobacillus,Bacteroides and Turicibacter.These results indicate that sea cucumber peptide can improve the immune regulatory capacity of immunosuppressed mice by increasing the immune organ index and immunoglobulin level,alleviating the damage of liver and intestinal tissue,alleviating the imbalance of intestinal microbiome and activating the nuclear factor-κB(NF-κB)pathway.(3)Relieving effect of sea cucumber peptide on UC in miceThe relieving effect of sea cucumber peptide on UC was detected by DSS induced mouse UC model.The results showed that sea cucumber peptide could reduce the Disease activity index(DAI)of UC mice during the experiment.Sea cucumber peptide alleviated colon shortening in UC mice,and colon length was significantly increased in SPL group compared with model group(P<0.01),colon length in SPM group was significantly increased(P<0.05).Sea cucumber peptide can alleviate the pathological injury and goblet cell reduction of colon in mice.Compared with model group,the colon injury and pathological score of mice in different dose groups of sea cucumber peptide were significantly reduced(P<0.01).Sea cucumber peptide could decrease spleen index of UC mice,and spleen index of SPL and SPM groups was significantly lower than that of model group(P<0.05).Sea cucumber peptide can improve the expression of tight junction proteins Occludin and Claudin-1,and compared with model group,Claudin-1 protein expression in SPL group was significantly increased(P<0.01),the expression of Occludin in SPM group was significantly increased(P<0.05).Sea cucumber peptide can increase the content of short-chain fatty acids in feces of mice,and the contents of three short-chain fatty acids in SPL group were significantly higher than those in model group(P<0.05).miR-155/SOCS1 axis was involved in relieving the UC effect of sea cucumber peptide in mice.Compared with model group,the content of miR-155 in SPM group was significantly decreased(P<0.01);SOCS1 protein content in SPM group was significantly increased(P<0.01).Intestinal microbiota sequencing results showed that sea cucumber peptide alleviated intestinal microbiota imbalance caused by DSS.At the phylum level,sea cucumber peptide reduced the relative abundance of Proteobacteria in the intestinal tract of mice.At the genus level,sea cucumber peptide reduced the relative abundance of Bacteroides and Eubacterium ruminantium in the intestinal microbiome of mice.The relative abundance of Lachnospiraceae NK4A136 group,Prevotellaceae UCG-001 and Ligilactobacillus was increased.The correlation analysis results between UC related indicators and intestinal microbiota showed that:The relative abundance of Bacteroides,Eubacterium ruminantium group,Dubosiella,Bifidobacterium,Akkermansia,Clostridium sensu stricto 1,Escherichia.Shigella,Eubacterium fissicatena group and Turicibacter are positively correlated with the severity of UC.The relative abundance of Lachnospiraceae NK4A136 group and Muribaculum was inversely correlated with the severity of UC in mice.(4)Study on antitumor activity of sea cucumber peptideThe effects of sea cucumber peptide on lung cancer cell A549 were detected by cell proliferation assay,cell scratch assay and cell invasion assay.The results showed that sea cucumber peptide effectively inhibited the proliferation,migration and invasion of lung cancer cell A549.The effect of sea cucumber peptide on lung cancer mice was detected by malignant pleural effusion(MPE)model.The results showed that sea cucumber peptide could reduce the volume of pleural effusion in mice.Compared with model group,the volume of pleural effusion in SCP-M and SCP-H groups was significantly reduced(P<0.01).Sea cucumber peptide can reduce the contents of ALT,AST and SCR in serum of mice.Compared with model group,the content of ALT in serum of mice in SCP-H group was significantly decreased(P<0.05);Serum AST content in SCP-M and SCP-H groups was significantly decreased(P<0.01);Serum SCR content in SCP-H group was significantly decreased(P<0.05).Sea cucumber peptide can increase the contents of IL-2 and IL-12 and decrease the contents of IL-6 and TNF-α in serum of mice.Compared with model group,the contents of IL-2 in serum of mice in SCP-H group were significantly increased(P<0.01);Serum IL-6 content in SCP-M and SCP-H groups was significantly decreased(P<0.01);Serum IL-12 content in SCP-H group was significantly increased(P<0.05);Serum TNF-α content in SCP-H group was significantly decreased(P<0.05).The results showed that sea cucumber peptide can inhibit the development of lung cancer in MPE mice and prolong the survival of experimental animals by reducing pleural effusion,alleviating liver and kidney injury,increasing the level of immune factors and decreasing the level of cancer promoting factors.The effect of sea cucumber peptide on lung cancer mice was tested by mouse lung cancer tumor bearing model.The results showed that sea cucumber peptide could inhibit the tumor growth of mice,and the tumor mass of mice in SCP-H group was significantly decreased compared with model group(P<0.01).Sea cucumber peptide can decrease the contents of ALT,AST and SCR in serum of mice.Compared with model group,the content of ALT in serum of mice in SCP-M group was significantly decreased(P<0.05);Serum AST content in SCP-H group was significantly decreased(P<0.01);Serum SCR content in SCP-L and SCP-M groups was significantly decreased(P<0.05).Sea cucumber peptide can increase the contents of IL-2 and IL-12 and decrease the contents of IL-6 and TNF-α in serum of mice.Compared with model group,the contents of IL-2 in serum of mice in SCPH group were significantly increased(P<0.01);The content of IL-6 in serum of SCP-H group was significantly decreased(P<0.01).Serum IL-12 content in SCP-M and SCP-H groups was significantly increased(P<0.05);Serum TNF-α content in SCP-L and SCP-M groups was significantly decreased(P<0.05).The results showed that sea cucumber peptide inhibited the development of lung cancer in tumor-bearing mice by delaying tumor growth,alleviating liver and kidney damage,increasing the level of immune factors and decreasing the level of cancer promoting factors.The mechanism of sea cucumber peptide inhibiting lung cancer was studied by miRNA sequencing,target gene prediction of differentially expressed miRNA and target protein expression in tumor tissues of tumor-bearing mice.The results showed that sea cucumber peptide could reduce the content of miR-378a-5p in mouse tumor tissue.The target gene set of miR-378a-5p was obtained by using miRDB,miRanda,TargetScan and RNAHybrid databases to predict the target gene of miR-378a-5p,and the target gene of miR-378a-5p was identified as the tumor suppressor TUSC2.Sea cucumber peptide can increase the content of TUSC2 protein in mouse tumor tissue,and the content of TUSC2 in SCP-M and SCP-H groups is significantly higher than that in model group(P<0.01).The results indicated that sea cucumber peptide inhibited lung cancer by enhancing the expression of TUSC2 by targeting miR-378a-5p. |
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