The Study On Immunomodulatory Effect Of Meretrix Meretrix Oligopeptide

Ocean,as a natural treasure trove,contains abundant resources.In recent years,the exploitation of marine living resources has become a hot spot.It is found that the extracts of marine organisms have the functions of anticancer,anti-oxidation,lowering blood lipid and immunomodulation.Meretrix,as one of the four marine cultured shellfish,has high edible and medicinal value.There are few reports on immunomodulation of M.meretrix oligopeptides(MMO)with amino acid sequence of Gln-Leu-Asn-Trp-Asp extracted in our laboratory.Therefore,in this study,the immunomodulatory effect of MMO was investigated by RAW264.7 cells and cyclophosphamide(Cy)immunosuppressive mice that were respectively used as cell model in vitro and animal model in vivo.Control group,positive group(LPS),MMO low dose group,MMO medium dose group and MMO high dose group were set up in vitro experiment.MTT colorimetric assay was used to detect the activity of RAW264.7 macrophages.Inverted microscope,HE staining and transmission electron microscopy were used to observe the morphological changes of the cells.Flow cytometry was used to detect the cell cycle changes,and neutral red phagocytosis assay was used to detect the phagocytosis ability of the cells.The expression of iNOS was observed by immunofluorescence labeling method.The contents of NO,IL-6 and IL-1β in the supernatant of cell culture were detected by nitrate reductase method and ELISA method.Western Blot assay was used to detect the expression of mitogen-activated protein kinase(MAPK)signaling pathyway related proteins in the cell culture supernatant which to explore the immunomodulatory effect and the potential mechanism of MMO on RAW264.7 cells.The control group,model group(Cy),positive drug group(levamisole),MMO low dose group,MMO middle dose group,MMO high dose group were established in vivo animal experiment.The mice were treated by intraperitoneal injection of normal saline,MMO,levamisole and Cy.The immune organ index was calculated,the blood of mice were collected from the eyeball,then serum was isolated.The contents of IgG and hemolysin in serum were measured.The spleen,thymus and liver were stained by HE.Spleen T lymphocytes were stimulated by Con A to measure the proliferation and transformation ability of T lymphocytes,and the expression of NLRP3 in spleen was detected by immunohistochemistry.The expression of NF-κB signaling pathyway related proteins in spleen of mice were detected by Western Blot which to explore the immunomodulatory effect and potential mechanism of MMO on Cy immunosuppressive mice.Results: 1.MTT showed that MMO had a dose-dependent effect on the proliferation of RAW264.7 cells from 10 μg/mL to 250 μg/mL.2.MMO high dose group and LPS group,compared with the control group,the percentage of G1/G0 phase cells in RAW264.7 cell cycle increased,that in S phase were decreased,and that in G2/M phase were increased.3.The number of RAW264.7 cells in the MMO dose groups were increased under light microscope,the mitosis of chromosomes in the nucleus were increased,many mitochondria and bacteriophages could be seen in the cytoplasm under electron microscope,and the surface deformation of the cell membrane extended outward to form pseudopod.An inward depression forms a phagocytic vesicle.The phagocytosis test of neutral red also showed that the phagocytosis index of MMO high dose group was higher than that of LPS group.4.The expression of iNOS and the content of NO,IL-6,IL-1β in the supernatant of RAW264.7 cells were increased in MMO dose groups.5.MMO can alleviate weight loss,atrophy of spleen and thymus,increase spleen index and thymus index in immunosuppressive mice.The content of IgG and hemolysin in serum were increased.6.The results of HE staining showed that the red and white marrow boundaries in the spleen of the model group were confused,the cortex and medulla of the thymus could not be distinguished,the hepatic cord in the liver was broken and the hepatocytes were damaged.The spleen body structure of the mice in the MMO group was restored,and the structure of the spleen body in the model group was restored.The white medulla marginal zone was increased,the thymus T lymphocytes and thymus corpuscles were increased,the hepatic cord in the liver recovered into radiate shape,and the morphology of hepatocytes returned to normal.7.Immunohistochemistry showed that the expression of NLRP3 in spleen of MMO group was decreased.8.The results of Western Blot showed that the phosphorylation of Erk and JNK proteins were activated in RAW264.7 cells,and the phosphorylation of IKKα and NF-κB p65 proteins in spleen of immunosuppressive mice were inhibited.Conclusion: the immunomodulatory effects of MMO include that activation of proliferation and differentiation of RAW264.7 cells,phosphorylation of intracellular Erk and JNK proteins,acceleration of protein synthesis in cell cycle,stimulation of the formation of pseudopod on the surface of cell membrane and phagocytosis of vesicles.The phagocytosis ability of RAW264.7 cells was enhanced.The synthesis of iNOS,NO,IL-6 and IL-1β in RAW264.7 cells were increased.It can reduced the rate of weight loss in immunosuppressive mice,alleviate the injury of spleen,thymus and liver,increased the content of IgG,hemolysin in serum,decreased the expression of NLRP3,and inhibited the phosphorylation of IKKα and NF-κB p65 proteins.MMO can reduced the immune damage and inflammatory reaction of Cy immunosuppressive mice.Therefore,MMO has the potential to develop into a safe,efficient and economical immunomodulator.