TLRs Mediated Immune Enhancement Effects Of Selenized Locust Bean Polysaccharide In Vitro

Selenium（Se）is recognized as the most relevant trace element to human health,which displays various bioactivities.As a kind of important organic form of selenium supplements,Se-polysaccharides possess the characteristics of high bioactivity and low toxicity.Although the researches on the bioactivities of Se-polysaccharides have been a hot topic,little attention has been paid to the relationship between Se content and immunomodulatory activities.In addition,the potential mechanism of immunomodulatory activity of Se-polysaccharides is unclear.Locust bean gum（LBG）is a kind of galactomannan withβ-（1→4）-D-mannose as the main chain andα-（1→6）-D-galactose as the side chain,which has been widely employed as an important material in food,medicine,and cosmetics.It is reported that the bioactivities of polysaccharides can be significantly enhanced by selenylation modification.In order to expand the application of LBG,the SeLBGs were effectively synthesized by deep eutectic solvents（DES）.Therefore,the Se content-related immunomodulatory activities of SeLBGs and the potential mechanism were detected under multiple comparisons among Na₂SeO₃,selenomethionine（Se-Met）,Se-nanoparticles（Nano-Se）,and LBG to dictate the correlation between Se content and immunomodulatory activities,which will provide valuable information for the structure-activity relationship of Se-polysaccharides.1.The immunomodulatory activities of SeLBGs against macrophage RAW264.7 in vitroThe immunomodulatory activities of SeLBGs were detected by multiple comparisons among Na₂SeO₃,selenomethionine（Se-Met）,Se-nanoparticles（Nano-Se）,and LBG.It was found that SeLBG_H displayed more potent immunomodulatory activities against RAW 264.7 cells as compared with Nano-Se,Se-Met,and LBG,which had been confirmed by the much higher cell viability,phagocytic activity,NO production,TNF-α,IL-1β,and IL-6 secretion in SeLBG_H group.As compared with Na₂SeO₃ group,SeLBGs did not exhibit cytotoxicity against RAW 264.7 cells under high Se equivalent treatment（20μg/m L）,suggesting Se-polysaccharides possessed much lower cytotoxicity than inorganic Se（Na₂SeO₃）.In addition,it could be found that SeLBG_H with high Se content（4926.54μg/g）exhibited much higher immunomodulatory activities than SeLBG_L（1049.57μg/g）,which suggested that Se content might be an important factor affecting immunomodulatory activities of Se-polysaccharides,and it is positively correlated to Se content.Similarly,Nano-Se and Se-Met incubation RAW 264.7 cells could obviously enhance its immune activities in a Se-content dependent manner,which is positively correlated to Se content.2.SeLBGs obviously increase RAW264.7 cells immunomodulatory activities via TLRs-MAPKs/NF-κB signaling pathwayAccording to the above results,it was found that SeLBGs could obviously increase RAW264.7 cells immunomodulatory activities in vitro.In order to investigate the potential mechanisms for the potent immunomodulatory effects of SeLBGs,TLR2/4specific receptor inhibitors were employed to determine the effect of SeLBGs specific recognition with pattern recognition receptors.In addition,the proteins expression level both in MAPKs and NF-κB signaling pathways（ERK1/2,p-ERK1/2,JNK1/2,p-JNK1/2,p38,p-p38,p65,and p-p65）were detected by western blotting analysis.Results showed that SeLBGs could specific recognition with TLR2/4,activated MAPKs and NF-κB signaling pathways,which effectively enhanced RAW264.7 cells proliferation and stimulated the release of NO,TNF-α,IL-1β,and IL-6.These results confirmed that the TLRs-MAPKs/NF-κB signaling pathways were the potential mechanisms for the potent immunomodulatory effects of SeLBGs.In conclusion,SeLBG_H with high Se content was more effectively activated TLRs-MAPKs/NF-κB signaling pathways in RAW264.7 cells,which could significantly enhance its immune activities.Our results will provide valuable information for the structure-activity relationship of Se-polysaccharides.