蛹虫草多糖调节小鼠巨噬细胞RAW264.7免疫活性的分子机制
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摘要
多种真菌多糖近年来因其免疫调节活性成为保健食品领域研究的热点。本实验以食药用真菌蛹虫草提取物蛹虫草多糖(Cordyceps militaris polysaccharides,CMP)为研究材料,初步探究CMP对小鼠巨噬细胞RAW264.7免疫活性的调节机制。噻唑蓝实验结果显示CMP无细胞毒性,且100、200μg/mL CMP可以明显增强RAW264.7细胞活性;中性红法、Griess法和酶联免疫吸附检测结果表明25～200μg/mL的CMP以剂量依赖方式增强RAW264.7细胞吞噬活性并增加一氧化氮(nitric oxide,NO)和白介素-1β(interleukin-1β,IL-1β)分泌,肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)的分泌呈先升高后降低的趋势,在100μg/mL时达到最高值;抑制剂中和实验结果显示当Toll样受体4(Toll-like receptors 4,TLR4)和甘露糖受体(mannose receptor,MR)受到抑制时,CMP诱导的RAW264.7免疫反应显著降低,这表明TLR4和MR均为CMP激活巨噬细胞的受体;此外,Western blot结果显示丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路也参与CMP诱导的RAW264.7细胞分泌NO、TNF-α和IL-1β。表明TLR4和MR/MAPK信号传导途径在CMP诱导巨噬细胞RAW264.7产生免疫应答时发挥了重要作用。
In recent years, the immunomodulatory activity of fungal polysaccharides has become a hot spot in the field of health foods. In the present study, Cordyceps militaris polysaccharides(CMP) were used to investigate the mechanism for its immunoregulatory effect on macrophage RAW264.7 cells. Thiazolyl blue assay results showed that CMP had no cytotoxicity and at doses of 100 and 200 μg/m L could significantly promote the proliferation of RAW264.7 cells. The neutral red staining, Griess and ELISA results showed that CMP at a concentration of 25–200 μg/mL increased the phagocytic activity and the secretion of nitric oxide(NO) and interleukin-1β(IL-1β) in RAW264.7 cells in a dose-dependent manner;tumor necrosis factor-α(TNF-α) secretion increased first and then decreased after reaching the highest value at 100 μg/mL.Inhibitor neutralization assay showed that CMP was significantly less able to induce immune response in RAW264.7 cells when Toll-like receptors 4(TLR4) and mannose receptor(MR) were inhibited, indicating that both TLR4 and MR were CMP-activated macrophage receptors. Western blot results showed MAPK signaling was also involved in CMP-induced secretion of NO, TNF-α and IL-1β in RAW264.7 cells. Therefore, TLR4 and MR/MAPK signal transduction pathways play an important role in CMP-induced immune response in macrophage RAW264.7 cells.
In recent years, the immunomodulatory activity of fungal polysaccharides has become a hot spot in the field of health foods. In the present study, Cordyceps militaris polysaccharides(CMP) were used to investigate the mechanism for its immunoregulatory effect on macrophage RAW264.7 cells. Thiazolyl blue assay results showed that CMP had no cytotoxicity and at doses of 100 and 200 μg/m L could significantly promote the proliferation of RAW264.7 cells. The neutral red staining, Griess and ELISA results showed that CMP at a concentration of 25–200 μg/mL increased the phagocytic activity and the secretion of nitric oxide(NO) and interleukin-1β(IL-1β) in RAW264.7 cells in a dose-dependent manner;tumor necrosis factor-α(TNF-α) secretion increased first and then decreased after reaching the highest value at 100 μg/mL.Inhibitor neutralization assay showed that CMP was significantly less able to induce immune response in RAW264.7 cells when Toll-like receptors 4(TLR4) and mannose receptor(MR) were inhibited, indicating that both TLR4 and MR were CMP-activated macrophage receptors. Western blot results showed MAPK signaling was also involved in CMP-induced secretion of NO, TNF-α and IL-1β in RAW264.7 cells. Therefore, TLR4 and MR/MAPK signal transduction pathways play an important role in CMP-induced immune response in macrophage RAW264.7 cells.
引文
[1]LI Y,MENG T,HAO N,et al.Immune regulation mechanism of Astragaloside IV on RAW264.7 cells through activating the NF-kappaB/MAPK signaling pathway[J].International Immunopharmacology,2017,49:38-49.DOI:10.1016/j.intimp.2017.05.017.
[2]LEE S B,LEE W S,SHIN J S,et al.Xanthotoxin suppresses LPS-induced expression of iNOS,COX-2,TNF-alpha,and IL-6 via AP-1,NF-kappaB,and JAK-STAT inactivation in RAW 264.7macrophages[J].International Immunopharmacology,2017,49:21-29.DOI:10.1016/j.intimp.2017.05.021.
[3]ZHENG X,ZOU S,XU H,et al.The linear structure of beta-glucan from baker’s yeast and its activation of macrophage-like RAW264.7cells[J].Carbohydrate Polymers,2016,148:61-68.DOI:10.1016/j.carbpol.2016.04.097.
[4]DU Y Q,LIU Y,WANG J H.Polysaccharides from Umbilicaria esculenta cultivated in Huangshan Mountain and immunomodulatory activity[J].International Journal of Biological Macromolecules,2015,72:1272-1276.DOI:10.1016/j.ijbiomac.2014.09.057.
[5]ZHANG X,QI C,GUO Y,et al.Toll-like receptor 4-related immunostimulatory polysaccharides:Primary structure,activity relationships,and possible interaction models[J].Carbohydrate Polymers,2016,149:186-206.DOI:10.1016/j.carbpol.2016.04.097.
[6]MARTINEZ-SOTO D,RUIZ-HERRERA J.Functional analysis of the MAPK pathways in fungi[J].Revista Iberoamericana de Micologia,2017,34(4):192-202.DOI:10.1016/j.riam.2017.02.006.
[7]李倩,李顺,陈善泽.巨噬细胞极化与炎症性疾病[J].国外医药抗生素分册,2018,39(1):80-85.DOI:10.13461/j.cnki.wna.005106.
[8]李婧文,周长林.多糖的免疫调节作用及多糖药物的研究进展[J].中国生化药物杂志,2016,36(4):24-28.DOI:10.3969/j.issn.1005-1678.2016.04.06.
[9]YOON J Y,KIM J H,BAEK K S,et al.A direct protein kinase B-targeted anti-inflammatory activity of cordycepin from artificially cultured fruit body of Cordyceps militaris[J].Pharmacognosy Magazine,2015,11(43):477-485.DOI:10.4103/0973-1296.160454.
[10]钟文,郭丽新,齐彦.蛹虫草多糖提取及其生物活性研究进展[J].辽宁中医药大学学报,2017,19(9):219-221.DOI:10.13194/j.issn.1673-842x.2017.09.062.
[11]王米,张丽芳,费陈忠,等. 蛹虫草多糖对小鼠腹腔巨噬细胞免疫功能的影响[J]. 中国生化药物杂志,2015,35(4):10-12.DOI:1005-1678(2015)04-0010-03.
[12]LEE J S,KWON D S,LEE K R,et al.Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth[J].Carbohydrate Polymers,2015,120:29-37.DOI:10.1016/j.carbpol.2014.11.059.
[13]LEE J S,HONG E K.Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris[J].International immunopharmacology,2011,11(9):1226-1233.DOI:10.1016/j.intimp.2011.04.001.
[14]ZONG A,CAO H,WANG F.Anticancer polysaccharides from natural resources:a review of recent research[J].Carbohydrate Polymers,2012,90(4):1395-1410.DOI:10.1016/j.carbpol.2012.07.026.
[15]ZHAO L H,MA Z X,ZHU J,et al.Characterization of polysaccharide from Astragalus radix as the macrophage stimulator[J].Cellular Immunology,2011,271(2):329-334.DOI:10.1016/j.cellimm.2011.07.011.
[16]许海林,吴小勇,聂少平,等.黑木耳多糖提取工艺优化及其对小鼠巨噬细胞功能的影响[J].食品科学,2016,37(10):100-104.DOI:10.7506/spkx1002-6630-201610017.
[17]HAN C,YANG J,SONG P,et al.Effects of Salvia miltiorrhiza polysaccharides on lipopolysaccharide-induced inflammatory factor release in RAW264.7 cells[J].Journal of Interferon&Cytokine Research,2018,38(1):29-37.DOI:10.1089/jir.2017.0087.
[18]VIDYA M K,KUMAR V G,SEJIAN V,et al.Toll-like receptors:significance,ligands,signaling pathways,and functions in mammals[J].International Reviews of Immunology,2017,37(1):1-17.DOI:10.1080/08830185.2017.1380200.
[19]SHIRJIANG S,MANSOORI B,SOLALI S,et al.Toll-like receptors as a key regulator of mesenchymal stem cell function:an up-to-date review[J].Cellular Immunology,2017,315:1-10.DOI:10.1016/j.cellimm.2016.12.005.
[20]黄蓉,吴青,李茂军,等.Toll样受体4在免疫相关细胞中的作用[J].中华临床医师杂志(电子版),2016,10(8):1178-1182.DOI:10.3877/cma.j.issn.1674-0785.2016.08.029.
[21]YU Q,NIE S P,WANG J Q,et al.Toll-like receptor 4-mediated ROSsignaling pathway involved in Ganoderma atrum polysaccharideinduced tumor necrosis factor-alpha secretion during macrophage activation[J].Food and Chemical Toxicology,2014,66:14-22.DOI:10.1016/j.fct.2014.01.018.
[22]XIE S Z,HAO R,ZHA X Q,et al.Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways[J].Carbohydrate Polymers,2016,146:292-300.DOI:10.1016/j.carbpol.2016.03.059.
[23]WANG Z,DONG B,FENG Z,et al.A study on immunomodulatory mechanism of polysaccharopeptide mediated by TLR4 signaling pathway[J].BMC Immunology,2015,16:1-9.DOI:10.1186/s12865-015-0100-5.
[24]ZHANG S,NIE S,HUANG D,et al.Polysaccharide from Ganoderma atrum evokes antitumor activity via Toll-like receptor 4-mediated NF-kappaB and mitogen-activated protein kinase signaling pathways[J].Journal of Agricultural and Food Chemistry,2013,61(15):3676-3682.DOI:10.1021/jf4004225.
[25]LI X,XU W.TLR4-mediated activation of macrophages by the polysaccharide fraction from Polyporus umbellatus(pers.)Fries[J].Journal of Ethnopharmacology,2011,135(1):1-6.DOI:10.1016/j.jep.2010.06.028.
[26]LI Wenjuan,TANG Xiaofang,SHUAI Xiaoxue,et al.Mannose receptor mediates the immune response to Ganoderma atrum polysaccharides in macrophages[J].Journal of Agricultural and Food Chemistry,2017,65(2):348-357.DOI:10.1021/acs.jafc.6b04888.
[27]WANG Changlu,CUI Haiyan,WANG Yurong,et al.Bidirectional immunomodulatory activities of polysaccharides purified from Pleurotus nebrodensis[J].Inflammation,2014,37(1):83-93.DOI:10.1007/s10753-013-9714-z.
[28]VARFOLOMEEV E,GONCHAROV T,MAECKER H,et al.Cellular inhibitors of apoptosis are global regulators of NF-kappaB and MAPKactivation by members of the TNF family of receptors[J].Science Signaling,2012,5(216):ra22.DOI:10.1126/scisignal.2001878.
[29]FANG Q,WANG J F,ZHA X Q,et al.Immunomodulatory activity on macrophage of a purified polysaccharide extracted from Laminaria japonica[J].Carbohydrate Polymers,2015,134:66-73.DOI:10.1016/j.carbpol.2015.07.070.
[30]XU Duoduo,WANG Haiyang,ZHENG Wei,et al.Charaterization and immunomodulatory activities of polysaccharide isolated from Pleurotus eryngii[J].International Journal of Biological Macromolecules,2016,92:30-36.DOI:10.1016/j.ijbiomac.2016.07.016.
[31]LIAO W,LUO Z,LIU D,et al.Structure characterization of a novel polysaccharide from Dictyophora indusiata and its macrophage immunomodulatory activities[J].Journal of Agricultural and Food Chemistry,2015,63(2):535-544.DOI:10.1021/jf504677r.
[2]LEE S B,LEE W S,SHIN J S,et al.Xanthotoxin suppresses LPS-induced expression of iNOS,COX-2,TNF-alpha,and IL-6 via AP-1,NF-kappaB,and JAK-STAT inactivation in RAW 264.7macrophages[J].International Immunopharmacology,2017,49:21-29.DOI:10.1016/j.intimp.2017.05.021.
[3]ZHENG X,ZOU S,XU H,et al.The linear structure of beta-glucan from baker’s yeast and its activation of macrophage-like RAW264.7cells[J].Carbohydrate Polymers,2016,148:61-68.DOI:10.1016/j.carbpol.2016.04.097.
[4]DU Y Q,LIU Y,WANG J H.Polysaccharides from Umbilicaria esculenta cultivated in Huangshan Mountain and immunomodulatory activity[J].International Journal of Biological Macromolecules,2015,72:1272-1276.DOI:10.1016/j.ijbiomac.2014.09.057.
[5]ZHANG X,QI C,GUO Y,et al.Toll-like receptor 4-related immunostimulatory polysaccharides:Primary structure,activity relationships,and possible interaction models[J].Carbohydrate Polymers,2016,149:186-206.DOI:10.1016/j.carbpol.2016.04.097.
[6]MARTINEZ-SOTO D,RUIZ-HERRERA J.Functional analysis of the MAPK pathways in fungi[J].Revista Iberoamericana de Micologia,2017,34(4):192-202.DOI:10.1016/j.riam.2017.02.006.
[7]李倩,李顺,陈善泽.巨噬细胞极化与炎症性疾病[J].国外医药抗生素分册,2018,39(1):80-85.DOI:10.13461/j.cnki.wna.005106.
[8]李婧文,周长林.多糖的免疫调节作用及多糖药物的研究进展[J].中国生化药物杂志,2016,36(4):24-28.DOI:10.3969/j.issn.1005-1678.2016.04.06.
[9]YOON J Y,KIM J H,BAEK K S,et al.A direct protein kinase B-targeted anti-inflammatory activity of cordycepin from artificially cultured fruit body of Cordyceps militaris[J].Pharmacognosy Magazine,2015,11(43):477-485.DOI:10.4103/0973-1296.160454.
[10]钟文,郭丽新,齐彦.蛹虫草多糖提取及其生物活性研究进展[J].辽宁中医药大学学报,2017,19(9):219-221.DOI:10.13194/j.issn.1673-842x.2017.09.062.
[11]王米,张丽芳,费陈忠,等. 蛹虫草多糖对小鼠腹腔巨噬细胞免疫功能的影响[J]. 中国生化药物杂志,2015,35(4):10-12.DOI:1005-1678(2015)04-0010-03.
[12]LEE J S,KWON D S,LEE K R,et al.Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth[J].Carbohydrate Polymers,2015,120:29-37.DOI:10.1016/j.carbpol.2014.11.059.
[13]LEE J S,HONG E K.Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris[J].International immunopharmacology,2011,11(9):1226-1233.DOI:10.1016/j.intimp.2011.04.001.
[14]ZONG A,CAO H,WANG F.Anticancer polysaccharides from natural resources:a review of recent research[J].Carbohydrate Polymers,2012,90(4):1395-1410.DOI:10.1016/j.carbpol.2012.07.026.
[15]ZHAO L H,MA Z X,ZHU J,et al.Characterization of polysaccharide from Astragalus radix as the macrophage stimulator[J].Cellular Immunology,2011,271(2):329-334.DOI:10.1016/j.cellimm.2011.07.011.
[16]许海林,吴小勇,聂少平,等.黑木耳多糖提取工艺优化及其对小鼠巨噬细胞功能的影响[J].食品科学,2016,37(10):100-104.DOI:10.7506/spkx1002-6630-201610017.
[17]HAN C,YANG J,SONG P,et al.Effects of Salvia miltiorrhiza polysaccharides on lipopolysaccharide-induced inflammatory factor release in RAW264.7 cells[J].Journal of Interferon&Cytokine Research,2018,38(1):29-37.DOI:10.1089/jir.2017.0087.
[18]VIDYA M K,KUMAR V G,SEJIAN V,et al.Toll-like receptors:significance,ligands,signaling pathways,and functions in mammals[J].International Reviews of Immunology,2017,37(1):1-17.DOI:10.1080/08830185.2017.1380200.
[19]SHIRJIANG S,MANSOORI B,SOLALI S,et al.Toll-like receptors as a key regulator of mesenchymal stem cell function:an up-to-date review[J].Cellular Immunology,2017,315:1-10.DOI:10.1016/j.cellimm.2016.12.005.
[20]黄蓉,吴青,李茂军,等.Toll样受体4在免疫相关细胞中的作用[J].中华临床医师杂志(电子版),2016,10(8):1178-1182.DOI:10.3877/cma.j.issn.1674-0785.2016.08.029.
[21]YU Q,NIE S P,WANG J Q,et al.Toll-like receptor 4-mediated ROSsignaling pathway involved in Ganoderma atrum polysaccharideinduced tumor necrosis factor-alpha secretion during macrophage activation[J].Food and Chemical Toxicology,2014,66:14-22.DOI:10.1016/j.fct.2014.01.018.
[22]XIE S Z,HAO R,ZHA X Q,et al.Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways[J].Carbohydrate Polymers,2016,146:292-300.DOI:10.1016/j.carbpol.2016.03.059.
[23]WANG Z,DONG B,FENG Z,et al.A study on immunomodulatory mechanism of polysaccharopeptide mediated by TLR4 signaling pathway[J].BMC Immunology,2015,16:1-9.DOI:10.1186/s12865-015-0100-5.
[24]ZHANG S,NIE S,HUANG D,et al.Polysaccharide from Ganoderma atrum evokes antitumor activity via Toll-like receptor 4-mediated NF-kappaB and mitogen-activated protein kinase signaling pathways[J].Journal of Agricultural and Food Chemistry,2013,61(15):3676-3682.DOI:10.1021/jf4004225.
[25]LI X,XU W.TLR4-mediated activation of macrophages by the polysaccharide fraction from Polyporus umbellatus(pers.)Fries[J].Journal of Ethnopharmacology,2011,135(1):1-6.DOI:10.1016/j.jep.2010.06.028.
[26]LI Wenjuan,TANG Xiaofang,SHUAI Xiaoxue,et al.Mannose receptor mediates the immune response to Ganoderma atrum polysaccharides in macrophages[J].Journal of Agricultural and Food Chemistry,2017,65(2):348-357.DOI:10.1021/acs.jafc.6b04888.
[27]WANG Changlu,CUI Haiyan,WANG Yurong,et al.Bidirectional immunomodulatory activities of polysaccharides purified from Pleurotus nebrodensis[J].Inflammation,2014,37(1):83-93.DOI:10.1007/s10753-013-9714-z.
[28]VARFOLOMEEV E,GONCHAROV T,MAECKER H,et al.Cellular inhibitors of apoptosis are global regulators of NF-kappaB and MAPKactivation by members of the TNF family of receptors[J].Science Signaling,2012,5(216):ra22.DOI:10.1126/scisignal.2001878.
[29]FANG Q,WANG J F,ZHA X Q,et al.Immunomodulatory activity on macrophage of a purified polysaccharide extracted from Laminaria japonica[J].Carbohydrate Polymers,2015,134:66-73.DOI:10.1016/j.carbpol.2015.07.070.
[30]XU Duoduo,WANG Haiyang,ZHENG Wei,et al.Charaterization and immunomodulatory activities of polysaccharide isolated from Pleurotus eryngii[J].International Journal of Biological Macromolecules,2016,92:30-36.DOI:10.1016/j.ijbiomac.2016.07.016.
[31]LIAO W,LUO Z,LIU D,et al.Structure characterization of a novel polysaccharide from Dictyophora indusiata and its macrophage immunomodulatory activities[J].Journal of Agricultural and Food Chemistry,2015,63(2):535-544.DOI:10.1021/jf504677r.