Immunomodulatory Activity Of Chlorella Sp.polysaccharide CEP4 On RAW264.7 Macrophages

Microalgal polysaccharides,which have important research value,are widely used in food,feed,medicine,sewage treatment and other fields due to their antioxidant,anti-tumor,anti-viral,and anti-immune functions.In this study,Chlorella sp.was used as the research object.The DEAE-52 cellulose anion exchange column was used to separate and purify the extracellular polysaccharides of Chlorella sp.The structure of purified fraction CEP4 was characterized and the immunomodulatory activity of CEP4 against macrophages was studied.Transcriptome sequencing technology was used to analyze the potential molecular mechanism of CEP4 on macrophage.The purified fraction of exopolysaccharide CEP4 was isolated and obtained by DEAE-52.Characterization results showed that CEP4 was a sulfated heteropolysaccharide compose with two molecular weights of 2394.889 k Da and67.759 k Da.The monosaccharide components of CEP4 are rhamnose,arabinose,glucosamine,hydrochloride,galactose,N-acetyl-D-glucosamine,xylose and glucuronic acid with a molar ratio of 0.037:0.083:0.408:0.026:0.077:0.086:0.210.In vitro immune experiments showed that CEP4 could activate the immune activity of macrophages.Within the range of 6 μg/m L,CEP4 not only promoted the proliferation of macrophages,induced the secretion of NO,IL-6 and TNF-α in a dose-dependent manner,but also reduced the content of IL-10 in the supernatant of macrophages.4824 differentially expression genes(DEGs)were obtained from the result of RNA-seq.GO enrichment analysis showed that CEP4 mainly affected the biological process(BP)of macrophages and some DEGs such as Ccr2、Ccl3、Cx Cr3 、 Gpr183 、 Il27 、 Tlr7 were related to immune function.KEGG enrichment analysis indicated that 19 metabolic pathways were significantly enriched,among which Toll-like receptor signaling pathway,Cytosolic DNAsensing pathway and C-type lectin receptor signaling pathway were related to the immune system.These results indicated that macrophages play immunomodulatory effect through several pathways.The protein interaction network was constructed by DEGS and the PPI network was consisted of 137 nodes with 20 hub genes.Most of the hub genes,such as HSP90AB1,RBX1,ISG15,SNRPD2,POLR2 F,UQCRH,PSMB8,PSMB6,RPSA and ORC1,are related to immune function.The extracellular polysaccharide CEP4 obtained in this study has immunomodulatory effect,and RNA-Seq results preliminarily revealed the potential mechanism of CEP4 stimulating macrophages,which provides a scientific basis for the development and application of extracellular polysaccharide from Chlorella as an immunomodulatory agent.