硒化低聚氨基多糖对RAW264.7细胞的免疫调节作用
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摘要
本实验旨在研究硒化低聚氨基多糖对小鼠巨噬细胞RAW264.7免疫功能的影响。用噻唑蓝比色法分别考察硒化低聚氨基多糖、Na2Se O3对巨噬细胞RAW264.7增殖能力的影响,并观察巨噬细胞形态的变化。通过中性红法、酶联免疫吸附测定法及实时荧光定量反转录聚合酶链式反应法分别检测巨噬细胞的吞噬能力、肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-6及IL-10细胞因子的分泌量及其m RNA表达水平。结果表明:硒质量浓度为100~1 000μg/L时,硒化低聚氨基多糖对细胞的相对增殖率无显著影响;而Na2Se O3的硒质量浓度超过200μg/L即对巨噬细胞RAW264.7产生细胞毒性作用。硒化低聚氨基多糖能增强巨噬细胞吞噬活性,显著提高RAW264.7细胞TNF-α、IL-6及IL-10分泌量及其m RNA表达水平,且效果比Na2Se O3处理组、低聚氨基多糖处理组及Na2Se O3+低聚氨基多糖复合添加组好。实验结果提示硒化低聚氨基多糖对小鼠巨噬细胞RAW264.7具有一定的免疫调节作用。
The aim of the present study was to investigate the immunomodulatory effect of low-molecular-mass selenoaminopolysaccharide(LSA) in RAW264.7 cells. The effects of LSA and sodium selenite(Se) on the proliferation of RAW264.7 cells were detected by MTT assay. Neutral red test was used to investigate the effect of LSA on phagocytosis and cell morphology in RAW264.7 cells. Enzyme-linked immunoabsorbent assay and real-time fluorescent quantitative reverse transcription-polymerase chain reaction were used to explore the effects of different selenium concentrations of LSA on the secretion and m RNA expression of cytokines(tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-10) in RAW264.7 cells. The results revealed that LSA did not affect cell viability at selenium concentrations between 100 and 1 000 μg/L. Sodium selenite significantly inhibited cell viability at selenium concentrations more than 200 μg/L. LSA contributed significantly to phagocytic activity in RAW264.7 cells. In addition, LSA could significantly increase the secretion and gene expression levels of TNF-α, IL-6 and IL-10 in RAW264.7 cells. Taken together, these findings suggested that LSA has immunomodulatory activity on macrophage cell line RAW264.7.
The aim of the present study was to investigate the immunomodulatory effect of low-molecular-mass selenoaminopolysaccharide(LSA) in RAW264.7 cells. The effects of LSA and sodium selenite(Se) on the proliferation of RAW264.7 cells were detected by MTT assay. Neutral red test was used to investigate the effect of LSA on phagocytosis and cell morphology in RAW264.7 cells. Enzyme-linked immunoabsorbent assay and real-time fluorescent quantitative reverse transcription-polymerase chain reaction were used to explore the effects of different selenium concentrations of LSA on the secretion and m RNA expression of cytokines(tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-10) in RAW264.7 cells. The results revealed that LSA did not affect cell viability at selenium concentrations between 100 and 1 000 μg/L. Sodium selenite significantly inhibited cell viability at selenium concentrations more than 200 μg/L. LSA contributed significantly to phagocytic activity in RAW264.7 cells. In addition, LSA could significantly increase the secretion and gene expression levels of TNF-α, IL-6 and IL-10 in RAW264.7 cells. Taken together, these findings suggested that LSA has immunomodulatory activity on macrophage cell line RAW264.7.
引文
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[31]CINEL I,OPAL S M.Molecular biology of inflammation and sepsis:a primer[J].Critical Care Medicine,2009,37(1):291-304.DOI:10.1097/CCM.0b013e31819267fb.
[2]张国财,赵博,刘春延,等.响应面法优化超声波-微波协同提取富硒蛹虫草硒多糖工艺[J].食品科学,2016,37(12):33-39.DOI:10.7506/spkx1002-6630-201612006.
[3]WANG J L,LI Q Y,BAO A J,et al.Synthesis of selenium-containing Artemisia sphaerocephala,polysaccharides:solution conformation and anti-tumor activities in vitro[J].Carbohydrate Polymers,2016,152(5):70-78.DOI:10.1016/j.carbpol.2016.06.090.
[4]WANG Y,CHEN J,ZHANG D,et al.Tumoricidal effects of a selenium(Se)-polysaccharide from Ziyang green tea on human osteosarcoma U-2 OS cells[J].Carbohydrate Polymers,2013,98(1):1186-1190.DOI:10.1016/j.carbpol.2013.07.022.
[5]商龙臣,吴少魏,张驰,等.南瓜硒多糖的制备表征及活性分析[J].食品科学,2016,37(19):48-53.DOI:10.7506/spkx1002-6630-201619008.
[6]REN D Y,HU Y Y,LUO Y Y,et al.Selenium-containing polysaccharides from Ziyang green tea ameliorate high-fructose diet induced insulin resistance and hepatic oxidative stress in mice[J].Food&Function,2015,6(10):3342-3350.DOI:10.1039/C5F000557D.
[7]GAO Z,CHEN J,QIU S,et al.Optimization of selenylation modification for garlic polysaccharide based on immune-enhancing activity[J].Carbohydrate Polymers,2016,136(7):560-569.DOI:10.1016/j.carbpol.2015.09.065.
[8]HAMED I,OZOGUL F,REGENSTEIN J M.Industrial applications of crustacean by-products(chitin,chitosan,and chitooligosaccharides):a review[J].Trends in Food Science&Technology,2016,12(48):40-50.DOI:10.1016/j.tifs.2015.11.007.
[9]YANG S J,LIN F H,TSAI K C,et al.Folic acid-conjugated chitosan nanoparticles enhanced protoporphyrin IX accumulation in colorectal cancer cells[J].Bioconjugate Chemistry,2010,21(4):679-689.DOI:10.1021/bc9004798.
[10]HOSSEINI S F,REZAEI M,ZANDI M,et al.Development of bioactive fish gelatin/chitosan nanoparticles composite films with antimicrobial properties[J].Food Chemistry,2016,194:1266-1274.DOI:10.1016/j.foodchem.2015.09.004.
[11]HYUNG J H,AHN C B,IL K B,et al.Involvement of Nrf2-mediated heme oxygenase-1 expression in anti-inflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages[J].European Journal of Pharmacology,2016,793:43-48.DOI:10.1016/j.ejphar.2016.11.002.
[12]王志华,江阳阳,余晓华,等.壳聚糖及其水溶性衍生物对小鼠免疫功能的影响[J].食品科学,2016,37(1):198-202.DOI:10.7506/spkx 1002-6630-201601035.
[13]郭向阳,王友明,高阳,等.硒化低聚氨基多糖制备工艺优化及其抗氧化活性研究[J].浙江海洋学院学报(自然科学版),2015,34(3):216-221.
[14]GORDON S,MARTINEZ F O.Alternative activation of macrophages:mechanism and functions[J].Immunity,2010,32(5):593-604.DOI:10.1016/j.immuni.2010.05.007.
[15]JANEWAY C A,MEDZHITOV R.Innate immune recognition[J].Immunology,2002,20:197-216.
[16]KIYOHARA H,UCHIDA T,TAKAKIWA M,et al.Different contributions of side-chains inβ-D-(1→3,6)-galactans on intestinal Peyer's patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge[J].Phytochemistry,2009,71(2/3):280-293.DOI:10.1016/j.phy tochem.2009.10.001.
[17]YIN X L,CHEN L,LIU Y,et al.Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression[J].Biochemical&Biophysical Research Communications,2010,397(2):232-238.DOI:10.1016/j.bbrc.2010.05.090.
[18]LI R,CHEN W C,WANG W P,et al.Extraction,characterization of Astragalus,polysaccharides and its immune modulating activities in rats with gastric cancer[J].Carbohydrate Polymers,2009,78(4):738-742.DOI:10.1016/j.carbpol.2009.06.005.
[19]LI S G,ZHANG Y Q.Characterization and renal protective effect of a polysaccharide from Astragalus membranaceus[J].Carbohydrate Polymers,2009,78(2):343-348.DOI:10.1016/j.carbpol.2009.04.013.
[20]TAPIERO H,TOWNSEND D M,TEW K D.The antioxidant role of selenium and seleno-compounds[J].Biomedicine&Pharmacotherapy,2003,57(3/4):134-144.DOI:10.1016/S0753-3322(03)00035-0.
[21]BECKETT G J,AETHUR J R.Selenium and endocrine systems[J].Journal of Endocrinology,2005,184(3):455-465.
[22]ARTHUR J R,MCKENZIE R C,BECKETT G J.Selenium in the immune system[J].Journal of Nutrition,2003,133(1):1457-1459.
[23]KIM Y Y,MAHAN D C.Comparative effects of high dietary levels of organic and inorganic selenium on selenium toxicity of growingfinishing pigs[J].Journal of Animal Science,2001,79(4):942-948.
[24]BIRK R W,GRATCHEV A,HAKIY N,et al.Alternative activation of antigen-presenting cells:concepts and clinical relevance[J].Hautarzt,2001,52(3):193-200.
[25]BARBARA J A,VAN OSTADE X,LOPEZ A.Tumour necrosis factor-alpha(TNF-alpha):the good,the bad and potentially very effective[J].Immunology&Cell Biology,1996,74(5):434-443.
[26]KOBAYASHI K S,FLAVELL R A.Shielding the double-edged sword:negative regulation of the innate immune system[J].Journal of Leukocyte Biology,2004,75(3):428-433.
[27]WANG N,LIANG H W,ZEN K.Molecular mechanisms that influence the macrophage Ml-M2 polarization balance[J].Frontiers in Immunology,2014,28(5):614-622.DOI:10.3389/fimmu.2014.00614.
[28]DAVIS M J,TSANGS T M,QIU Y,et al.Macrophage M1/M2polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection[J].mBio,2013,4(3):264-270.DOI:10.1128/mBio.00264-13.
[29]NISHIMOTO N.Interleukin-6 as a therapeutic target in candidate inflammatory diseases[J].Clinical Pharmacology&Therapeutics,2010,87(4):483-487.DOI:10.1038/clpt.2009.313.
[30]IVANOVA-TODOROVA E,BOCHEV I,DIMITROV R,et al.Conditioned medium from adipose tissue-derived mesenchymal stem cells induces CD4+FOXP3+cells and increases IL-10 secretion[J].Journal of Biomedicine&Biotechnology,2012,2012:1-8.DOI:10.1155/2012/295167.
[31]CINEL I,OPAL S M.Molecular biology of inflammation and sepsis:a primer[J].Critical Care Medicine,2009,37(1):291-304.DOI:10.1097/CCM.0b013e31819267fb.