Exploring The Immunomodulatory Effect Of Sulfated Yam Polysaccharide On RAW264.7 Macrophages Based On RNA-seq And The Development Of Yam Polysaccharide Oral Liquid

Yam(Dioscorea opposite Thunb.)is a long-established dual-use plant resource for food and medicine,and one of its most important active components is yam polysaccharide,which has various biological activities such as immunomodulation,antioxidant,antitumor and hypoglycemia,so it is necessary to study it thoroughly.Sulfation modification can improve the biological activity of natural polysaccharides and is commonly used to modify polysaccharides.Most of the current studies on the biological activity of yam polysaccharides are based on natural polysaccharides,and few studies have investigated their modified biological activity.Therefore,in this thesis,we extracted refined yam polysaccharide named CYP from yam in Ruichang,Jiangxi province,and modified CYP with sulfation to obtain sulfated derivatives of yam polysaccharide named S-CYP.The physicochemical properties of the above two yam polysaccharides were analyzed,the immunomodulatory activity of sulfated yam polysaccharides was explored by an in vitro RAW264.7 macrophage model,and the transcriptome information of S-CYP-treated RAW264.7 macrophages was screened by RNA-seq technology.Based on the results of RNA-seq,the mechanism of action of S-CYP on the immunomodulatory activity of RAW264.7 macrophages was further investigated.The main study contents and results are as follows:1.The effect of sulfated modification on the physicochemical properties of CYP was investigated,and the effect of S-CYP on the immunomodulatory activity of RAW264.7 macrophages was investigated using RAW264.7 macrophages as a model.The sulfated modification increased the molecular weight of CYP,and the incorporation of the sulfate group into the structure of CYP was also confirmed by the infrared spectrogram,and the degree of substitution of S-CYP was measured to be0.44.The neutral sugar of CYP was 35.17 ± 0.09%,glyoxylate was 33.40 ± 3.2%,and protein was 2.87 ± 0.17%;the neutral sugar of S-CYP was 33.27 ± 0.04%,glyoxylate was 21.90 ± 2.15%,and protein was 2.87 ± 0.10%.The results of monosaccharide composition showed that sulfation modification changed the proportion of monosaccharide composition,but not its species;scanning electron micrographs showed that S-CYP was mostly in fine fragments with more dispersed and curled surfaces.S-CYP treatment upregulated the expression of i NOS in RAW264.7macrophages and induced NO production;it also induced ROS,TNF-α and IL-6secretion in RAW264.7 macrophages.2.The effect of S-CYP on the immunomodulatory effect gene expression of RAW264.7 cells was investigated in depth based on RNA-seq technology,and the mechanism of immunomodulatory effect of S-CYP on RAW264.7 cells was explored by the information of genomic changes in them.Transcriptional information obtained from S-CYP-treated RAW264.7 macrophages was analyzed by RNA-seq,and 401up-regulated genes and 1363 down-regulated genes were screened out of 1764 differential genes.GO enrichment analysis suggested that the stimulation of RAW264.7 macrophages by S-CYP could be related to the regulation of signal transduction,cell surface receptor signaling pathway,and cellular response to chemical stimulation;KEGG enrichment analysis suggested that the stimulation of RAW264.7 macrophages by S-CYP immunomodulatory activity of macrophages was associated with MAPK signaling pathway,cytokine receptor interaction,Toll-like receptor signaling pathway,NF-kappa B signaling pathway and other pathways.3.The mechanism of S-CYP on macrophage immune function was explored based on the TLR4-MAPK/NF-κB signaling pathway.S-CYP treatment can upregulate the protein expression levels of TLR4 and three subunits of MAPK(ERK,JNK,p38),and immunofluorescence experiments have shown that S-CYP treatment can promote the transport of NF-κB p65 subunit translocation to the nucleus.Further addition of TLR4,three subunits of MAPK(ERK,JNK,p38)and specific inhibitors of NF-κB treated RAW264.7 macrophages,the secretion of cytokines(TNF-α and IL-6)produced by S-CYP stimulation was significantly inhibited.4.Based on the above research,yam polysaccharide was used as raw material to develop yam polysaccharide oral liquid products.The formulation of yam polysaccharide oral liquid was optimized by single-factor experiment and response surface,and its optimal process conditions were obtained as follows: yam polysaccharide 15 mg/m L,white sugar 3%,honey 3%,and citric acid 0.12%.The influence on the oral solution was in the order of yam polysaccharide addition >honey addition > citric acid addition > white granulated sugar gum addition,which provides technical support for the high value utilization of yam resources.