Structural Characterisation And Immunostimulatory Activity Of Polysaccharides From Caulerpa Lentillifera

This study compared the nutritional composition of C.lentillifera from different regions and used single-factor and response surface experiments to optimize the extraction rate of C.lentillifera polysaccharides(CLP).A component with immunostimulatory activity of C.lentillifera polysaccharide(CLP-P1)was isolated and purified,and using methylation,nuclear magnetic resonance(NMR)and other technologies to identify its structural information.The molecular mechanism of CLP-P1 immunostimulatory activity was studied by ELISA,flow cytometry,q RT-PCR,Western blot and transcriptomics.The main results are as follows:1.Nutritional analysis of C.lentilliferaIn this study,the nutrients in C.lentillifera collected from Hainan and Shandong provinces were analyzed comprehensively.They were compared each other as well as with two other species of seaweed reported previously.It's found that the polysaccharide contents in C.lentillifera from Hainan province and Shandong province were higher than those in E.cottonii and S.polycystum.The essential amino acid composition of C.lentillifera from Shandong province was more in line with the ideal FAO/WHO model.The respective unsaturated fatty acid contents in C.lentillifera from Hainan province and Shandong province were 44.35% and 36.34%,of which the PUFA content was higher than in samples from Semporna,Sabah,and S.polycystum.C.lentillifera from Hainan province had a higher fiber content than those from Shandong province,Sempoma,Sabah,and Petchburi.The ash content of all of the C.lentillifera samples was lower than that of S.polycystum and E.cottonii.2.Optimization of Extraction Conditions of CLPIn this study,the extraction rate of CLP was used as an indicator to optimize the extraction process such as ultrasonification,extraction time,extraction temperature and ratio of water to raw material.The best extraction conditions were obtained through data analysis: ultrasonification for 30 min,extraction time of 9 h,extraction temperature of 100°C,and a ratio of water to raw material of 40:1.Under these conditions,the extraction rate of CLP was 3.215%,which was 0.269% worse than the theoretical value of the model.3.Isolation,Purification and Structural Analysis of CLP-P1Polysaccharide components CLP-P1 and CLP-P2 were separated by anion chromatography column and gel exclusion chromatography column.The molecular weight of CLP-P1 was 1583.9 k Da.CLP-P1 is a homogeneous heteropolysaccharide mainly composed of Gal,Glc,Xyl and Man,and contains a small amount of Gal-UA.The results of methylation and nuclear magnetic resonance analysis of CLP-P1 revealed that it may be composed of ?-D-Galp-(1 ?,? 3)?-D(4SO4)-Galp-(1 ?,? 4)?-D-Galp-(1 ?,? 4)-?-D-Xylp-(1 ?.The polysaccharide has a sulfated group modification,and its modification site is presumed to be located at the C-4 of ? 3)?-D-Galp-(1 ?,and there is a pyruvate modification group.The content of t-Man,3,4-Gal,2,3-Man(p)is low,and there is no obvious nuclear magnetic signal.4.Immunostimulatory activity of CLP-P1 on macrophage RAW264.7The in vitro cell model was used to evaluate the immunostimulatory activity of CLPP1 on macrophages RAW264.7.The results showed that CLP-P1(6.25,12.5,25,and 50 ?g/m L)can significantly increase the cell viability and enhance phagocytosis of RAW 264.7 cells in a dose-dependent manner.CLP-P1-induced macrophages significantly increased the secretion of ROS,NO/i NOS,IL-6,IL1?,and TNF-? and related cytokine gene expression.We show that CLP-P1 can improve the immune function of macrophage RAW 264.7 by promoting cell activity and increasing the secretion and gene expression of NO/i NOS,IL-6,IL-1? and TNF-?.5.Effect of CLP-P1 on TLR4 receptor of macrophage RAW264.7TLR4 receptor is a key receptor for contractile macrophage immunostimulatory activity.Flow cytometry was used to determine the binding rate of Try-CLP-P1-FITC to macrophage RAW264.7 and the competition of Anti-TLR4 at different concentrations.The results showed that with the increase of the concentration of Try-CLP-P1-FITC,the binding rate with RAW264.7 cells increased significantly(p<0.05).Compared with the Try-CLP-P1-FITC group,the Anti-TLR4 group showed a significant decrease.TLR4 expression was measured by q RT-PCR and Western Blot,the expression level of TLR4 gene increased significantly with the increase of CLP-P1 concentration and was dose-dependent.Compared with the blank group,the expression of TLR4 protein increased slightly with the increase of CLP-P1 concentration,so CLP-P1 can activate the TLR4 signaling pathway of macrophages RAW264.7.6.The mechanism of immunostimulatory activity of CLP-P1 on macrophage RAW264.7.Transcriptome sequencing was used to analyze the effects of C.lentillifera polysaccharide on the differentially expression gene and signaling pathway of macrophage RAW264.7.Using volcano map and cluster analysis,it was found that compared with the blank group,the CLP-P1(50 ?g/m L)group significantly increased the expression of 378 differentially expressed genes,significantly reduced the expression of 592 differentially expressed genes,and partially increased the differential expression Genes are involved in immune regulation.Combining GO enrichment and KEGG enrichment pathways,CLP-P1 significantly up-regulates 209 signaling pathways and significantly down-regulates 220 signaling pathways.The main immunostimulatory signaling pathways include Toll-like receptor signaling pathway,TNF signaling pathway,NOD-like receptor signaling pathway,Natural killer cell mediated cytotoxicity,NF-?B signaling pathway,JAK-STAT signaling pathway and MAPK signaling pathway,etc.,and Up-regulated related gene expression.