| Two homogenous polysaccharides LPG1 and LPG2 were isolated and purified from longan pulp.Its structure properties were characterized using gel permeation laser light scattering(GPC-LLS),infrared spectroscopy(IR)and nuclear magnetic resonance(NMR).Its immunoregulatory molecular mechanisms were analyzed on macrophages with related pathway inhibitors using transcriptome sequencing,protein expression(Western Blot,WB).Moreover,degrade LPG1 and LPG2 with 1,4-?-glucan cellulase,comparing the structure and immunoregulatory properties between the two polysaccharides before and after degradation.And the main results are as follows:1.Structural characterization of polysaccharidesThe structure properties of LPG1 and LPG2 were characterized by GPC-LLS,IR,ion chromatography and NMR.LPG1 is composed of fucose,galactose,arabinose,mannose,glucose,galacturonic acid and glucuronic acid in a molar ratio of 0.86%:18.64%:16.43%:25.62%:35.42%:2.44%:0.59%.Its Mn is 1.148 × 105,Mw is 2.386 × 105,Rz is 47.5nm.While LPG2 is composed of fucose,galactose,arabinose,mannose,glucose,galacturonic acid and glucuronic acid in a molar ratio of 4.54%:28.63%:17.31%:12.83%:32.52%:1.73%:2.45%.Its Mn is 1.574 × 104,Mw is 7.008×104,Rz is 6.3 nm.2.Polysaccharide activates the macrophagesLPG1 and LPG2 could stimulate macrophage proliferation and phagocytosis in the concentration range of 2.5?100 ?g/mL.For LPG1 and LPG2,the relative phagocytosis rate of macrophages at 2.5 ?g/mL was 148.94±4.23%(P<0.05)and 156.10 ± 1.04%(p<0.05).At 25 ?g/mL,the phagocytosis rate of macrophages was 112.62± 0.65%(p<0.05)and 135.14 ± 4.10%(p<0.05),respectively.At the same time,LPG1 and LPG2 significantly increased the secretion of NO,TNF-? and IL-1?in macrophages.At 100 ?g/mL,the secretion on NO,TNF-? and IL-1? of LPG1 treatment group was 115.33 ± 1.74%(p<0.05)and 8139.56±150.90%(7988.98 pg/mL,p<0.05)and 368.19±142.80%(66.79 pg/mL,p<0.05)of the control group.The secretion on NO,INF-?a and IL-1? of the LPG2 treatment group was 125.71 ±2.18%(p<0.05),701.45±19.90%(992.13 pg/mL,p<0.05)? 231.46±22.36%(47.38 pg/mL,P<0.05)of the control group.3.Transcriptome analysis of macrophagesThere were 84 significantly up-regulated genes and 201 significantly down-regulated genes in LPG2 group.Analyze the differentially expressed genes with the KEGG enrichment pathway analysis and GO functional annotation analysis.The differentially expressed genes were mainly enriched in Toll-like receptors,NOD-like receptors,NF-kappa B,TNF,JAK,MAPK and other immune-related signaling pathways.Compare the differentially expressed genes to the KEGG pathway,it was found that LPG2 regulated macrophage activation by up-regulating LPG2 mainly up-regulated expression of MyD88?TAB2?IKK??P150?Tp12?MEK1/2?AP-1 in TLR4?MyD88?IRAK1?TRAF6?TAK1?IKK??p105?MEK1/2?ERK?AP-1?IL-1? signaling pathway,up-regulated expression of each gene in TLR4?TRAM?RIP1?TAB?IKK??IkB??p50?TNF-? signaling pathway,up-regulated expression of NOD2?RIP2?TAB?JNK?AP-1 in NOD2?RIP2?TAB2?JNK?AP-1?IL-1? signaling pathway.At the same time,up-regulated expression of JAK?STAT?IRF9 in JAK?STAT?IRF9?SLIM?Bcl-2 signaling pathway,up-regulated expression of JAK?GRB?SOS?Ras?Raf in JAK?GRB?SOS?Ras?Raf signaling pathway,up-regulated expression of JAK?PI3K?AKT?mTOR in JAK?PI3K?AKT?mTOR signaling pathway.LPG2 up-regulated expression of GRB2?SOS?Ras?RafB?MEK2?MNK1/2?CREB in RTK?GRB2?SOS?Ras?RafB?MEK2?ERK?MNK1/2?CREB signaling pathway,up-regulated expression of TRAF2/5?TAB1/2/3?MKK4/7?JNK1/2?AP-1 in TNFR?TRADD?TRAF2/5?TAB1/2/3?MKK4/7?JNK1/2?AP-1?IL-1?signaling pathway,up-regulated expression of TRAF2/5?NIK?IKK??I?B??NF-?B in TNFR?TRADD?TRAF2/5?NIK?IKK??I?Ba?NF-?B signaling pathway.LPG2 mainly down-regulated expression of TRAF3?IRF3/7 in NOD2?RIP2?TRAF3?IKKe?IRF3/7?IFNa/p signaling pathway,down-regulated expression of TRADD?MKK3/6?P38?c/EBPp in TNFR?TRADD?TRAF2/5?TAB1/2/3?MKK3/6?p38?c/EBPp signaling pathway.Among the 13 immunoregulation-related genes(Cd40,Cpeb4,Cxcl10,Cxcl11,Cxcl2,Hmmr,Ifih1,Irgl,Isg15,Isg20,Mapk9,Mat2b,Zfp62)screened out from the genes with significantly differentially expressed genes,there were 5 in toll-like signaling pathway:Cd40,Spp1,Mapk9,Cxcl11,Cxcl10,and 2 in NOD-like signaling pathway:Cxcl2 and Mapk9.Based on the analysis of differentially expressed genes and enrichment pathways,LPG2 mainly mediates the activation of macrophages by regulating Toll-like and NOD-like and other immune pathways.4.Effect of cellulase degradation on the immunoregulatory activity of polysaccharideCellulase was used to hydrolyze LPG1 and LPG2.Immunoregulatory activity of longan polysaccharides before and after degradation and the genes in related immune pathways were analyzed.The results showed that the two polysaccharides obtained after degradation showed a more significant(p<0.05)activation on macrophages at 25 to 100 ?g/mL.At 100 ?g/mL,The relative phagocytic rates of the degradation polysaccharides of LPGI and LPG2 groups were increased to 129.79%and 130.03%of the original polysaccharide group.At 100 ?g/mL,NO,TNF-? and IL-1? secretion levels of LPGI and LPG2 after degradation treatment groups were 104.74 ± 2.71%and 116.51 ± 1.16%of the control group,13281.27 pg/mL and 11156 pg/mL,148.57 pg/mL.and 160.19 pg/mL.TLR4 and NOD2 receptor inhibitors could significantly inhibit polysaccharide-induced macrophage phagocytosis and cytokine secretion.It showed that TNF-? and IL-1? secretion of four polysaccharides treatment groups decreased to 30.86%to 81.26%(p<0.05)and 22.83%to 94.16%(p<0.05)when pre-incubated with TLR4 inhibitor TAK-242.TNF-? and IL-1? secretion were reduced to 38.31%to 69.19%(p<0.05).and 76.00%to 92.04%under the incubation of TLR4 inhibitor IRAK-4.TNF-? and IL-1? secretion of NOD2 inhibitor GSK583 group decreased from 34.95%to 69.28%(p<0.05)and 36.18%to 119.50%.Combined with cellular immunofluorescence,four polysaccharides presented the same receptors and signaling pathway mechanisms:mainly via TLR4 signaling pathway and NOD2 signaling pathway.Protein expressions of polysaccharide after degradation showed that the phosphorylation levels of STAT,NF-?B and p38MAPK in the downstream pathways of TLR4 and NOD2 were 1.18,1.28 and 1.53 times of the original polysaccharide group.These results suggested that for polysaccharides after degradation,the molecular mechanisn of significantly activating macrophages might be phosphorylation expression up-regulation of STAT,NF-?B and p38MAPK in TLR4 and NOD2 pathways. |
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