Physicochemical Properties And Immunomodulatory Activity Of Mung Bean Skin Polysaccharide

Mung bean is an annual erect herb crop and traditional food grown throughout the world,which is rich in nutrients and has a high medicinal value that promotes digestive and detoxifying properties.The skin of mung beans,as a protective tissue surrounding the outside of the embryo and endosperm,is often discarded in the production and processing of products such as mung bean cakes and mung bean cakes,accounting for 7% to 10% of the total weight of mung beans.Mung bean skin contains many active substances,mainly proteins,dietary fibre,polysaccharides and polyphenols,which make it anti-tumour,hypolipidemic and hypoglycaemic.Polysaccharides,as one of the main active components of mung bean skin,have been shown to have immunomodulatory activity,but the mechanism by which mung bean skin polysaccharides exert immunomodulatory effects is still unclear.In this study,a water-soluble polysaccharide(MBP)was extracted from mung bean skin and its structure was initially characterized by determining the chemical composition,molecular weight,monosaccharide composition and apparent morphology.The immunomodulatory activity of MBP was evaluated using RAW264.7 cells as a cell model.In addition,the immunomodulatory mechanism of MBP on macrophages was investigated by RNA-seq sequencing,and the mechanism of MBP activation on macrophages was verified by western blotting and inhibitor assay.The main findings and results of this paper were as follows:1.A water-soluble polysaccharide(MBP)was extracted from mung bean skin by hot water extraction and its structure was characterized.The MBP was determined to contain 63.69 ± 0.13% total sugars,27.21 ± 0.16% glyoxylates and 8.36 ± 0.14%proteins.Chemical structure analysis showed that MBP has a molecular weight of362.66 k D and is mainly composed of glucose,galactose,galacturonic acid,xylose and arabinose,corresponding to a molar ratio of 1.00 : 4.67 : 3.29 : 1.51 : 28.45.Infrared spectroscopy showed that MBP has characteristic absorption peaks of polysaccharides and confirmed the presence of galacturonic acid and pyran rings,while scanning electron microscopy revealed that it is composed of smooth and irregular lamellar structures.XRD and TGA thermogravimetric analysis showed that MBP is a semi-crystalline polymer with weak thermal stability.Particle size and Zeta potential analysis revealed an average particle size of 107.0 ± 3.4 nm and a Zeta potential of-11.6 ± 0.11 m V,while rheological analysis showed that MBP solution system tends to form a weak gel structure.2.RAW264.7 cells were used as a cell model to investigate the effect of MBP on the immunomodulatory activity of the cells.The results showed that MBP was not toxic to RAW264.7 cells in the concentration range(25 ～ 200 μg/m L)and could effectively promote cell proliferation.Moreover,MBP significantly promoted the phagocytosis of RAW264.7 cells and induced the production of NO,cytokines(TNF-α,IL-1β and IL-6)and ROS,and showed a significant dose-dependent effect.Above findings suggested that MBP has immunomodulatory effects on RAW264.7cells.3.RNA-seq sequencing technology was used to analyse the effect of MBP on the transcription of RAW264.7 cells in order to explore the mechanism of effect of MBP in regulating the immune activity of RAW264.7 cells.The transcriptional information of RAW264.7 in MBP-stimulated RAW264.7 macrophages was successfully obtained,in which 927 differentially expressed genes were screened,including 196up-regulated genes and 731 down-regulated genes.GO enrichment analysis of the differentially expressed genes revealed that "cytokines" were significantly enriched in a number of functional groups,suggesting that cytokines play an important role in the regulation of RAW264.7 immune function by MBP.The KEGG enrichment analysis showed that among the significantly enriched pathways,the signaling pathways that may be associated with the regulation of RAW264.7 immunomodulatory activity by MBP are "Toll-like receptor signaling pathway","MAPK signaling pathway" and "NF-κB signaling pathway".4.On the basis of the RNA-seq sequencing results,the activation of TLR4-MAPK/NF-κB signaling pathway by MBP in RAW264.7 cells was verified using western blotting and inhibitor assays.The results showed that MBP could promote the expression of proteins related to TLR4,MAPK and NF-κB signalling pathways in a dose-dependent manner,indicating that MBP could activate MAPK and NF-κB signalling pathways through activation of TLR4 receptors and indirectly exert immunomodulatory effects.Moreover,the production of NO and TNF-α cytokine in MBP-stimulated cells was significantly reduced after treatment of RAW264.7 cells with TLR4,MAPKs and NF-κB inhibitors respectively,indicating that activation of both MAPK and NF-κB signalling pathways mediated by MBP-activated TLR4 receptors was inhibited.This provides a good validation with the RNA-seq sequencing results.