Immunomodulatory Activity And Mechanism Of Mung Bean Peptides On Macrophage

Bioactive peptides are the generic terms of peptides that possesses regulating functions of life activities and some physiological activities.Protein hydrolysates(peptides)is an important value-added food ingredients due to its easily absorption,biological function and highly safety,which has been proved to have some bioactivities,inculding anti-tumor,anti-oxidantion,anti-hypertension,immune regulation and improving cholesterol,which depends on its particular amino acid composition.MBPs derived from its protein have been reported to possess immunomodulatory activity,however,its immunomodulatory mechanisms have not been studied clearly.The research is designed to investigate the immunomodulatory activity of MBPs in vitro on macrophages,the relationship of MBPs structure and its immune activity by using chromatographic and spectral,which could conclude the peptides size,amino acid composition,molecular weight and sequence.In addition,inorder to illustrate the possible mechanism of MBPs,the expression of cytokines and the signaling pathways of MBPs-treated RAW264.7 cell and abdominal macrophage in mice were analyzed,and the immunomodulatory activity and signaling pathways of macrophages with treated MBPs were also evaluated in vivo.1.We determined the immune activities on the RAW264.7 macrophages by using cell and molecular biology technologies.The results indicated that MBPs significantly increased cell proliferation rate,SOD activity,NO production and cytokine secretion content in a dose-dependent manner(p < 0.05).These results illustrated that MBPs are able to effectively enhance the immune defense of host cells by activating macrophages,enhancing their own metabolism enzyme activity and releasing cytokines.The results of cytokines produced with MBPs on LPS-stimulated RAW 264.7 cells showed that MBPs could significant inhibited the TNF-??IL-1? and IL-6.This finding suggested that MBPs exerts immunomodulatory activity by anti-inflammatory action.MBPs affected the expression of autophagy-related protein(LC3 and p62)on LPS-induced macrophage,it reduced the content of theintracelluar LC3 and LC3-I transformation to LC3-II,down-regulated the expression of LC3-II/LC3-I protein,up-regulated the expression of p62 protein.The results proved that MBPs could regulate immune response by inhibiting autophagy.These finding may indicate the potential utility of the mung bean protein peptides as an ingredient of functional foods in food industry.2.The stronger immune activity of MBPs was separated by Sephadex G-15 chromatography,which has analyzed by chromatography,spectrum,amino acid analysis technologies and immune molecular biology technologies.The third grade of MBPs(P3)possessed the strongest immune activity due to the higher hydrophobicity,molecular size and amino acid composition,which increased the level glycogen and nucleic acid metabolism activities,updated the cytokines such as IL-1?,IL-6 and IL-10 secretion level.Then,the amino acid sewuences of P3 was determined by 2D-nono liquid chromatography mass spectrometry,the results showed that the amino acid sequence of isolated stronger immune peptides were Phe-Leu-Val-Asn-Arg-Ile(885.1 Da),Phe-Leu-Val-Asn-Pro-Asp-Asp(961.06 Da),and Lys-Asp-Asn-Val-Ile-Ser-Glu-Leu(1043.13Da).The secondary structure of MBPs had a certain relationship with the immune activity,in other words,the more ?-helix structure,the more immune activity.3.TLR1,TLR 2 and TLR4 m RNA level of the different groups were determined by using PCR technology,the results showed that TLR1,TLR 2 and TLR4 m RNA level of LPS-treated group significantly increased compared with the control group,TLR1 and TLR4 m RNA level of MBPs-treated group changed,and TLR2 m RNA level has no change.In addition,TLR1,TLR2 and TLR4 m RNA level were inhibited on LPS+MBPs induced cell.In order to demonstrate whether TLR receptors was activated by MBPs,and then regulated the signaling pathways for immune response,this research has designed the group which was the anti-TLR1 and anti-TLR4 plus MBPs group.The results showed that the production of IL-1?,IL-6 and IL-10 of the anti-TLR1-and anti-TLR4-treated groups decreased compared with the MBPs-treated group.But the cytokines production of anti-TLR1-treated group were lower than the anti-TLR4-treated group,and the inhibition rate reached more than 50%.These results suggested that TLR1 was the main receptor of MBPs to activate macrophages.4.In order to explore signalling pathways,the phosphorylation expression level of the main subunits of MAPK and NF-?B pathway in macrophages were analyzed by Western Blotting.The results showed that the relative protein levels of JNK,ERK 1/2 and p38 MAPK of LPS-treated group increased about 4.84,3.72 and 9.20 times compared with the control group.The levels of MBPs-treated group with different concentration groups were higher about 1.98,3.11 and 8.4 times than the control group,and decreased about 2.44,1.20 and 1.10 times than the control group respectively.The stimulation of the MAPK signal transduction pathways by MBPswas studied in macrophages using inhibitors for p38(SB203580),ERK1/2(PD98059)and JNK(SP600125).The cytokines results showed that the expression of cytokines activated by MBPs depends on MAPK signaling pathways,and the inhibition effects of p38 MAPK and ERK1/2 for TNF-? and IL-6 were higher than JNK1/2.In addition,the relative level of p65 in nucleus and I?B? in cytoplasm of LPS-treated group increased,and the different concentration of MBPs-treated groups were lower than the LPS group.When the NF-?B pathway was treated with specific inhibitors,which lead to the secretion of TNF-? and IL-6 of MBPs-treated groups were lower than the control group.The above results showed that macrophages was activated by MBPs dependent on the p38 MAPK,JNK and NF-?B pathway.5.The immune activities and the related signalling pathways of MBPs on abdominal macrophage in BALB/c mice were analyzed by using ELISA,PCR and Western blotting technologies,the results showed that the cytokines of MBPs-treated group increased,however,the anti-TLR1 + MBPs group were lower than the control group.The effect of the MBPs on cytokines were evoked by p38 MAPK,ERK1/2,JNK and NF-?B pathway in abdominal macrophage.This research conclude that MBPs has immunomodulatory effects on macrophages activating NF-?B and MAPK dependent pathways,and predominantly inducing cytokines production.6.MBPs were found to possess immunomodulatory activity in vitro in our previous study.The objective of this study was to investigate the nutritional immunity effects of MBPs in vivo in a cyclophosphamide(CY)-treated immunosuppressed mouse model.MBPs exert nutritional protection effects in CY-treated immunosuppressed mice.They enhanced both LDH and Ac Pase activities,and improved the splenic histopathology in immunocompromised mice,increased the phagocytosis of peritoneal macrophages and enhanced the secretion of cytokines such as TNF-?and IL-6,and MBPs upregulated cytokines production in peritoneal macrophages through TLR1 receptor and MAPK,NF-?B pathways.MBPs possessed strong anti-stress activities,which increased with the dose.MBP-treated groups exhibited stronger recovery effects than other groups.The serum cortisol and glucose concentrations of the MBP-treated groups were significantly decreased compared to the model group,and there was no significant difference between the high-dose MBP group and the control group.In addition,the MBP-treated groups also showed strong superoxide dismutase(SOD)activity and low glutamic oxalacetictransaminase(GOT)activity,and the effects were close to those of the control group.The results demonstrated that MBPs can act as a nutritional protective material to improve the immunity of immunosuppressed mice and increase their anti-stress activities.MBPs can be a valuable nutritional functional ingredient for food products.Based on these above results,it can be concluded that MBPs have immunomodulatory effects which were related to the peptides? molecular weight,amino acid content,hydrophobicity and secondary structure.According to the resarches results of in vitro and in vivo,MBPs upregulated TNF-?,IL-1? and IL-6 production in macrophages,inhibited the cytokines production in LPS-induced cell,and the possible mechanism of MBPs were that MBPs activated the macrophages via TLR1,then regulated p38 MAPK,ERK1/2 and NF-?B signaling pathway.