Modulation of immunol-properties by chito-oligosaccharide

2 191 doi 10. 3969 /j. issn. 1000-484X. 2013. 02. 017 1 2 266003 R392. 12 A 1000-484X 2013 02-0191-06 3-7 COS HPLC FITC FITC-COS Toll 4 TLR4 TNF-α IgG IgM HPLC COS 3-7 FITC-COS COS TLR4 1 FITC-COS COS COS TNF-α COS IgG IgM 3-7 TLR4 FITC Modulation of immunol-properties by chito-oligosaccharide ZHANG Pei HAN Bao-Qin CHEN Lie-Huan PENG Yan-Fei LIU Wan-Shun ZHENG Han-Dong YANG Yan. Department of Marine Life Sciences Ocean University of China Qingdao 266003 China Abstract Objective To study the immunol-regulation properties of chito-oligosaccharides COS with 3-7 degree of polymerization which are prepared by enzymatic hydrolysis. Methods Chitosanase founded by our research group was used to prepare chito-oligosaccharides through the enzyme hydrolysis method and HPLC method was used to identify the composition of COS. COS was then labeled with fluorescein isothiocyanate FITC to get FITC-COS. Phagocytosis of COS by mouse peritoneal macrophages' and its relationship with TOLL-like receptor 4 TLR4 was investigated. The effects of different concentrations of COS on the proliferation neutral red phagocytosis ability and tumor necrosis factor TNF-alpha secretion of mouse peritoneal macrophages were then tested. Mice were administrated with different doses of COS to elucidate the impact of COS on serum IgG and IgM levels and on the thymus spleen index of mouse. Results HPLC analysis results showed that COS prepared by chitosanase hydrolysis mainly consist of 3-7 degree of polymerization of monosaccharide. Fluorescence microscopy results showed that FITC-COS could be phagocytized by mouse peritoneal macrophages in a time-dependent manner. Pretreatment of macrophages with TLR4 monoclonal antibody for 1hr phagocytosis of COS was almost completely inhibited. The phagocytosis and TNF-α secretion abilities of macrophage were all significantly enhanced after COS was phagocytized. The results of in vivo studies showed that administration of mice with COS could significantly increase the spleen index and serum IgG levels. While the thymus index and serum IgM levels were not significantly changed. Conclusion COS with 3-7 polymerization degree can be phagocytized by macrophage and then activate macrophages. COS has good in vitro and in vivo immunological function. The activation of macrophages by COS is mediated by the TLR4 receptor on cell surface. Key words Chito-oligosaccharide FITC Macrophage Immune function 1 12-1-4-1- 23 -jch 2 222000 1989 - E-mail zhangxiaobei88@ 163. com 1976 - E-mail yany@ ouc. edu. cn. COS 2 ~ 10 β-1 4-1 2

192 2013 29 TLR4 Cell Signaling 3 SPF 4 5 RT-2100C Rayto 5% CO 2 37 HF240 HEAL FORCE 6 HEAL FORCE Nikon 7 8 TS100 Costar 96 IL-18 Costar 6 Costar TNF-α IL-1β IL-18 IL-6 5 9-11 10 1 ml A 10% 10 Han 12 0. 5 ml 2% NaOH 0. 2 ml TNF-α 2% NaOH 10 ml Ca 2 + 1. 3 Feng 10 HPLC LPS β- Wu Waters 13 TLR4 CD14 CR3 301 RAW264. 7 1% / 75 25 30 20 μl 1. 0 ml / min TLR4 CD14 CR3 NF-кB 1. 4 14 25 ml FITC- 1 mg /ml 10 ml - 15% m /V ph 9 FITC- 3 60 1 3-7 FITC-COS 1. 5 FITC-COS COS 1% 3 1 5 10 4 /ml 48 5% CO 2 1. 1 LPS Sigma 37 4 D-Hank's GENray FITC D-Hank's 2 ml 48 MTT 1 ml FITC-COS 1 mg /ml DMSO HyClone DMEM Solarbio Beijing Solarbio Science & Technology Co. Ltd IgG IgM 1. 2 100 ml 1% ph 5. 5 45 1 5 15 FICT-COS PBS 2 α COS TLR4 FITC-COS TLR4

2 193 1 10 37 1 1. 8 x ± s 1 ml FITC-COS 1 mg /ml SPSS t 15 FICT-COS PBS 2 P < 0. 05 2 1. 6 PMφ 2. 1 HPLC 1. 6. 1 MTT COS PMφ 1 1% 3 3 ~ 7 5 8 10 4 /ml 96 200 μl 5% 2. 2 CO 2 37 4 2 D-Hank's 6 LPS 10 ng /ml COS 10 20 50 100 μg /ml 8 24 48 72 MTT 1. 6. 2 PMφ PMφ 1. 6. 1 6 LPS 10 ng /ml COS 10 20 50 100 μg /ml 8 24 200 μl 0. 7 mg /ml 1 3 0. 1 mol /L 1 1 200 μl 4 D-Hank's 492 nm 1. 6. 3 PMφ TNF-α 1 HPLC Fig. 1 HPLC result of enzyme hydrolysis product of PMφ 1. 6. 1 6 LPS 10 ng /ml COS 10 20 50 100 μg /ml 8 24-20 ELISA TNF-α 1. 7 100 mg /kg 250 mg /kg 500 mg /kg 10 20 8 3 000 r /min 15-20 IgG chitosan IgM mg 2 Fig. 2 Phagocytosis of chito-oligosaccharides by macrophage 10 mg /10 g

194 2013 29 3 Fig. 3 PMφ Effects of COS on the proliferation ability of mice PMφ Note A. 24 h B. 48 h C. 72 h. 4 24 24 Fig. 5 Effect of COS on TNF-α secretion ability of Fig. 4 Effect of COS on the neutral red phagocytosis a- bility of mice PMφ at different concentrations 24 h mice PMφ at different concentrations 24 h Note *. P < 0. 05 vs control. Note *. P < 0. 05 vs control. 5 PMφ TNF-α 20 μg /ml FITC-COS 15 2. 5 COS PMφ TNF-α TLR4 TNF-α 1 5 LPS FITC-COS 15 TNF-α PMφ 2. 3 COS PMφ COS TNF-α 20 μg /ml 3 50 μg /ml PMφ TNF-α 2. 6 IgG IgM PMφ COS 10 20 IgG 2. 4 COS PMφ IgM 6 4 LPS 10 ~ 100 μg /ml

2 195 6 Fig. 6 IgG IgM 10 20 Effect of different doses of COS on mice serum IgG and IgM content 10 20 d Note *. P < 0. 05 vs control. Toll 4 TLR4 15 TLR4 B 16 TLR4 NFкB 17 LPS 7 TLR4 10 20 Fig. 7 Effect of different doses of COS onmice thymus MAPK-NF-кB 18 Wu 13 and spleen index 10 20 d TLR4 Note *. P < 0. 05 vs control. FITC FITC- G IgG COS TLR4 TLR4 500 μg /kg M IgM COS 10 20 7 3 3 ~ 7 3 ~ 7 TLR4 CD14 CR3 10 12 13 Toll 2. 7

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