Study On The Immunomodulatory Effects Of The Active Peptides From The Solenocera Crassicornis Head

Solenocera crassicornis is a kind of marine economic shrimp,and the annual catch in Zhejiang Province is more than 20,000 tons.Most of S.crassicornis are made into shelled shrimps,while 30%-50%of the byproducts of shrimps'heads are mostly discarded,causing water pollution and resource waste.Bioactive peptides derived from natural proteins play a unique physiological role in coordinating body functions.Studies have shown that the bioactive peptide obtained by enzymatic hydrolysis has high bioavailability,easy absorption and high safety.As a potential protein source,S.crassicornis head can be used to prepare biologically active peptides,so as to improve the high value-added utilization of processing by-products,improve the economic benefits of enterprises and reduce environmental pollution.In this study,S.crassicornis head was used as the raw material,and the active peptides(SCHPs-F1)with a molecular weight of less than 1 k Da were prepared by in vitro simulated gastrointestinal digestion.Combining chromatography and spectroscopy,the basic physical,chemical properties and composition sequence of SCHPs-F1 are explored.Furthermore,the effects of SCHPs-F1 on the cell proliferation and differentiation,phagocytosis,cytokine release and related signaling pathway regulation of RAW 264.7 macrophages have also been studied.The effects of SCHPs-F1 on the immune functions of model mice and related mechanisms were explored with an immunocompromised model induced by cyclophosphamide(CTX).Moreover,the Illumina Mi Seq high-throughput sequencing technology was used to evaluate the diversity and composition of the mice fecal microbial community.The results showed that the proportion of SCHPs-F1 less than 1 k Da which digested by in vitro simulating gastrointestinal tract accounted for 84.38%.The essential amino acids of SCHPs-F1 accounted for 38.07%of the total amino acids.The limit value of toxic heavy metals meets national standards,and endotoxins have no interference effect on this study.SCHPs-F1 has a good scavenging rate for DPPH·,·OH,O₂^-·,ABTS+·free radicals.Through LC-MS/MS peptide matching,71 special peptides in SCHPs-F1 were identified,and the sequence of these active peptides was ranged from 2 to 9 amino acids.The immunomodulatory activity of different concentrations of SCHPs-F1 on RAW264.7 cells was evaluated.The results showed that the cell proliferation rate,neutral red phagocytic ability,NO content and pro-inflammatory cytokine secretion were increased in a dose-dependent manner by treating with SCHPs-F1.Moreover,the expression of NF-?B p65,IKK?and p-I?B protein could be significantly up-regulated by SCHPs-F1.The nuclear translocation of NF-?B p65 was activated,and the protein levels of p-P38,p-JNK and p-ERK were also significantly up-regulated.The results of the effect of SCHPs-F1 on the immune regulation ability of mice showed that the state of the spleen and thymus of the mice was improved by treating with SCHPs-F1.The NO secretion capacity of mouse peritoneal macrophages and the proliferative activity of splenic lymphocytes,and the content of serum pro-inflammatory factors were also increased after treating with SCHPs-F1 In addition,SCHPs-F1 significantly increased the phosphorylation and expression levels of NF-?B and MAPK pathway-related proteins in the spleen of immunosuppressed mice.Moreover,the results of high-throughput sequencing of intestinal flora showed that the diversity and abundance of intestinal microbes were restored after treating with SCHPs-F1,and the abundance of beneficial bacteria was also increased.In summary,the low molecular weight peptide SCHPs-F1 which obtained by simulating gastrointestinal digestion in vitro is rich in essential amino acids.In vitro experiments,the proliferation and phagocytic capacity of RAW 264.7 macrophages and the promotion of the release of pro-inflammatory cytokines were enhanced by treating with SCHPs-F1.The NF-?B and MAPK signaling pathways may be activated by SCHPs-F1 to exhibt its immunomodulatory effects.In vivo animal experiments,the activity of mouse peritoneal macrophages and splenic lymphocytes,and humoral immunity were enhanced by treating with SCHPs-F1.The tissue damage of mouse immune organs and the diversity of mouse intestinal flora were also repaired by treating with SCHPs-F1.The spleen NF-?B and MAPK signaling pathways may be activated by SCHPs-F1 to exert its immunomodulatory effects on mice.Our research will be expected to provide a basis for the high value-added utilization of S.crassicornis heads and provide a new direction for the development of immune-regulating functional foods in future.