Alleviating Mechanism Of Alkali-extracted Polysaccharides From Mung Bean Skin On H₂O₂-induced Oxidative Stress Through MAPK/Nrf2 And NF-κB Pathways

Mung bean is an important legume crop,which is widely cultivated because of its rich nutrients and even medicinal value.During the processing of mung bean products,a large amount of mung bean skin as by-product is produced,which accounts for about7-10%of the total mass of mung bean.They are either processed into cheap feed,or simply discarded and not used effectively,resulting in a huge waste of resources.Mung bean skin contain a variety of bioactive chemicals such as polysaccharides,proteins and flavonoids.As one of the main bioactive components of mung bean skin,mung bean skin polysaccharide has been proved to have good antibacterial,immunomodulatory and antioxidant activities.Alkali solution extraction is an effective method to obtain cell wall polysaccharide.Different concentration of alkali solution can effectively extract a variety of polysaccharide components,so it is easier to obtain polysaccharide components existing in different layers of plant cells.At present,the research on the antioxidant capacity of Alkali-extracted polysaccharides from mung bean skin is still in a preliminary stage,and its mechanism of alleviating oxidative damage in cells is still unclear.Therefore,in this paper,mung bean skin polysaccharide was extracted with different alkali concentrations,and its physicochemical properties,rheological properties and antioxidant properties were investigated.Using IEC-6 as cell model,oxidative damage was induced by H₂O₂ and alleviated by Alkali-extracted Mung bean skin polysaccharide.The regulatory mechanism of the polysaccharide alleviating H₂O₂-induced oxidative damage in IEC 6 cells was investigated by RNA-seq technique.Western blotting and inhibitor experiments were used to verify the relevant regulatory mechanisms of alkali-extracted mung bean skin polysaccharides.The main research contents and results of this paper are as follows:1.Three kinds of mung bean skin polysaccharides(MBP_0.5,MBP_1.0 and MBP_2.0)were extracted from mung bean skin with different alkali concentrations（Na OH:H₂O,0.5%,1.0%,2.0%,w/v）.The effects of alkali concentration on extraction yield,microstructure,rheological properties and antioxidant activity of MBP were investigated.The results showed that the ester bond and hydrogen bond could be destroyed by alkali extraction.Therefore,the extraction yield of those three polysaccharides was higher than that of the hot water extraction（MBP_W）,and the extraction yield of MBP_1.0 was 3.64%.In addition,compared with MBP_W,the molecular weight,particle size and thermal stability of the three base polysaccharides were smaller.MBP_0.5 has good antioxidant activity because of its high galacturonic acid content and low molecular weight.MBP_1.0 and MBP_2.0 have tight structure and smooth surface,forming a strong gel network structure.2.The effect of MBP on H₂O₂-induced oxidative damage in IEC-6 cells was investigated using IEC-6 cells as cell model.The results showed that MBP had no toxicity to IEC-6 cells in the concentration range（10-200μg/m L）,and could effectively promote cell proliferation.In addition,MBP could significantly relieve the H₂O₂-induced decline in IEC-6 cell viability.MBP could relieve the oxidative stress of IEC-6 cells caused by H₂O₂,restore SOD activity to normal level,and reduce the content of intracellular lipid peroxide MDA.H₂O₂ treatment can abnormally increase ROS levels in cells,while MBP can reduce ROS levels and protect cells from oxidative damage.Western blotting results showed that MBP could alleviate oxidative damage through NF-κB signaling pathway,MAPK signaling pathway and Nrf2 signaling pathway.3.MBP could alleviate H₂O₂-induced oxidative damage in IEC-6 cells.To further explore the mechanism of action,the effect of MBP on H₂O₂ induced transcription in IEC-6 cells was analyzed by RNA-seq technology.The transcriptional information in cells was obtained successfully.Compared with the control group,the model group had408 differentially expressed genes,among which were 169 up-regulated and 239 down-regulated.And the polysaccharide group had 2422 differentially expressed genes,including 946 up-regulated and 1476 down-regulated.Between model group and polysaccharide group,there were 2670 differentially expressed genes,covered 959 up-regulated and 1711 down-regulated genes.KEGG enrichment analysis indicated that among the significantly enriched pathways,MAPK signaling pathway and NF-κB signaling pathway may be related to MBP’s regulation of H₂O₂-induced oxidative damage in IEC-6 cells.4.Based on the results of RNA-seq,Western blotting and inhibitor experiments were used to verify the role of MAPK and NF-κB signaling pathways on MBP in alleviating H₂O₂-induced oxidative damage of IEC 6 cells.After addition of specific inhibitors of key proteins of MAPK pathway（P38,Erk1/2 and JNK）.It was found that SOD activity increased,MDA level and protein expression decreased in the H₂O₂ group with inhibitor added,comparing with the H₂O₂ group.Compared with the polysaccharide group,SOD activity,MDA content and related protein expression could be regulated in polysaccharide group with inhibitor added,alleviating oxidative damage to normal level.alleviating oxidative damage to normal level.The same results were seen with the addition of key protein inhibitors of NF-κB pathway.These results demonstrated that MBP could alleviate H₂O₂-induced oxidative damage of IEC 6 cells through MAPK and NF-κB signaling pathways.Which was a good verification of RNA-seq results.