元蘑多糖的结构特征及其调节巨噬细胞免疫活性机制

doi:10.13560/j.cnki.biotech.bull.1985.2023-0483

摘要/Abstract

摘要：

【目的】为明确元蘑多糖的结构特征，并探究其调节巨噬细胞免疫活性机制。【方法】采用水提醇沉法获得元蘑多糖粗提物，并通过DEAE-52离子交换层析及Sephacryl S-400葡聚糖凝胶过滤层析对该多糖粗提物进行分离纯化，获得多糖组分SEP-0a。分别采用高效凝胶渗透色谱法、离子交换色谱法、傅立叶红外光谱法检测了SEP-0a的理化性质。进一步采用CCK-8法、ELISA法、qPCR法、Western blot等方法考察了元蘑多糖SEP-0a对巨噬细胞RAW264.7的免疫调节作用及机制。【结果】元蘑多糖SEP-0a由半乳糖、甘露糖、葡萄糖和岩藻糖组成，相对分子量为4.23×10⁴ Da，并且具有α构型与β构型。体外免疫活性结果表明，元蘑多糖SEP-0a可显著增强RAW264.7细胞增殖和吞噬能力，促进该细胞活性氧（reactive oxygen species，ROS）、一氧化氮（nitric oxide，NO）的分泌，提高肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素（interleukin，IL）-1β、IL-6的表达，Western blot检测发现，该多糖可显著提高核转录因子-κB（nuclear factor-κB，NF-κB）信号通路p65蛋白磷酸化表达。【结论】元蘑多糖SEP-0a可通过NF-κB信号通路调控巨噬细胞的免疫活性。

关键词: 元蘑多糖, 结构特征, RAW264.7细胞, NF-κB信号通路

Abstract:

【Objective】In order to clarify the structural characteristics of Sarcomyxa edulis polysaccharides and explore its mechanism of regulating macrophage immunomodulatory activity. 【Method】The crude polysaccharide of S. edulis was obtained by water extraction and ethanol precipitation, and the purified polysaccharide component SEP-0a was obtained by DEAE-52 ion exchange column chromatography and Sephacryl S-400 glucan gel column chromatography. The physicochemical properties of SEP-0a were investigated by high performance gel permeation chromatography evaporative light-scattering detector, ion exchange chromatography high performance liquid chromatography and Fourier transform infrared spectrometer. On this basis, the immunomodulatory effect of polysaccharide SEP-0a from S. edulis on macrophage cell RAW264.7 was further studied by CCK-8, ELISA, qPCR, Western Blot and other methods. 【Result】The S. edulis polysaccharide SEP-0a was composed of galactose, mannose, glucose and fucose with a relative molecular weight of 4.23×10⁴ Da, and had α and β configurations. The results of in vitro immunocompetence showed that S. edulis polysaccharide SEP-0a significantly increased the proliferation and phagocytic ability of RAW264.7 cells, promoted the secretion of reactive oxygen species（ROS）, nitric oxide（NO）and increased the expressions of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, and IL-6 in these cells. Western blot results showed that this polysaccharide significantly increased p65 protein phosphorylation expression in the nuclear factor-κB（NF-κB）signaling pathway. 【Conclusion】The polysaccharide SEP-0a of S. edulis may regulates the immune activity of macrophages by NF-κB signal pathway.

Key words: Sarcomyxa edulis polysaccharide, structural characteristics, RAW264.7 cells, NF-κB signal pathway

王子璇, 隋玉, 尚学钰, 马思嘉, 吴天香, 刘洋, 王琦. 元蘑多糖的结构特征及其调节巨噬细胞免疫活性机制[J]. 生物技术通报, 2024, 40(1): 308-321.

WANG Zi-xuan, SUI Yu, SHANG Xue-yu, MA Si-jia, WU Tian-xiang, LIU Yang, WANG Qi. Structural Characteristics of Sarcomyxa edulis Polysaccharide and Its Mechanism of Regulating Macrophage Immunomodulatory Activity[J]. Biotechnology Bulletin, 2024, 40(1): 308-321.

图/表 15

表1 引物序列

Table 1 Primer sequences

基因 Gene	上游Forward primer（5'-3'）	下游Reverse primer（5'-3'）
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

表2 引物序列

Table 2 Primer sequences

基因 Gene	上游 Forward primer（5'-3'）	下游 Reverse primer（5'-3'）
NO	CTGGTGAAGGAACGGGTCAG	CCGATCATTGACGGCGAGAAT
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

图1 元蘑多糖洗脱曲线 A: DEAE-52离子交换层析；B: Sephacryl S-400葡聚糖凝胶过滤层析

Fig. 1 Elution curve of S. edulis polysaccharide A: DEAE-52 ion exchange column chromatography; B: sephacryl S-400 glucan gel column chromatography

图2 SEP-0a高效凝胶渗透色谱洗脱曲线

Fig. 2 Elution curve of SEP-0a high performance gel permeation chromatography

图3 SEP-0a离子色谱图 A：标品；B：SEP-0a

Fig. 3 SEP-0a ion chromatogram A: Standard sample; B: SEP-0a

图4 SEP-0a红外光谱图

Fig. 4 Infrared spectrum of SEP-0a

图5 不同浓度SEP-0a对RAW264.7细胞增殖的影响 **P<0.01，与空白组相比（n=6）

Fig. 5 Effects of SEP-0a at different concentrations on the proliferation of RAW264.7 cells **P<0.01, compared with blank group（n=6）

图6 不同浓度SEP-0a对RAW264.7细胞吞噬作用的影响 *P<0.05；**P<0.01，与空白组相比（n=3）

Fig. 6 Effects of SEP-0a at different concentrations on the phagocytosis of RAW264.7 cells *P<0.05; **P<0.01, compared with blank group（n=3）

图7 不同浓度SEP-0a对RAW264.7细胞分泌ROS的影响 **P<0.01，与空白组相比（n=6）

Fig. 7 Effects of SEP-0a at different concentrations on the ROS secretion of RAW264.7 cells **P<0.01, compared with blank group（n=6）

图8 不同浓度SEP-0a对RAW264.7细胞分泌NO的影响 **P<0.01，与空白组相比（n=4）

Fig. 8 Effects of SEP-0a at different concentrations on NO secretion by RAW264.7 cells **P<0.01, compared with blank group（n=4）

图9 不同浓度SEP-0a对RAW264.7细胞分泌TNF-α（A）、IL-1β（B）、IL-6（C）的影响 **P<0.01，与空白组相比（n=4）

Fig. 9 Effects of SEP-0a at different concentrations on TNF-α（A）, IL-1β（B）and IL-6（C）secretion by RAW264.7 cells **P<0.01, compared with blank group（n=4）

图10 不同浓度SEP-0a对RAW264.7细胞中TNF-α（A）、IL-1β（B）、IL-6（C）基因表达的影响 **P<0.01，与空白组相比（n=4）

Fig. 10 Effects of SEP-0a at different concentrations on TNF-α（A）, IL-1 β（B）and IL-6（C）gene expressions by RAW264.7 cells **P<0.01, compared with blank group（n=4）

图11 SEP-0a对RAW264.7细胞蛋白磷酸化的影响 **P<0.01，与空白组相比

Fig. 11 Effects of SEP-0a on protein phosphorylation in RAW264.7 cells **P<0.01, compared with blank group

图12 NF-κB抑制剂对SEP-0a诱导RAW264.7分泌NO（A）、TNF-α（B）、IL-1β（C）和IL-6（D）的影响 **P<0.01，与空白组相比；##P<0.01，与SEP-0a组相比（n=3）

Fig. 12 Effects of NF-κB inhibitors on the secretions of NO（A）, TNF-α（B）, IL-1β（C）and IL-6（D）induced by SEP-0a in RAW264.7 cells **P<0.01, compared with blank group; ##P<0.01, compared with SEP-0a（n=3）

图13 NF-κB抑制剂对SEP-0a诱导RAW264.7中NO（A）、TNF-α（B）、IL-1β（C）和IL-6（D）基因表达的影响 **P<0.01，与空白组相比；##P<0.01，与SEP-0a组相比（n=4）

Fig. 13 Effects of NF-κB inhibitors on SEP-0a on the gene expressions of NO（A）, TNF-α（B）, IL-1β（C）and IL-6（D）by RAW264.7 cells **P<0.01, compared with blank group; ##P<0.01, compared with SEP-0a（n=4）

参考文献 40

[1]	田风华. 中国东北元蘑种质资源评价及其三萜合成途径相关基因研究[D]. 长春: 吉林农业大学, 2019.
	Tian FH. Evaluation of germplasm resources and genes related to triterpene synthetic pathway of Sarcomyxa edulis in Northeast China[D]. Changchun: Jilin Agricultural University, 2019.
[2]	宋振康, 张海悦, 尹家乐, 等. 响应面法优化元蘑多糖提取工艺及抗疲劳活性的研究[J]. 食品科技, 2020, 45(6): 254-260.
	Song ZK, Zhang HY, Yin JL, et al. Study on optimization of extraction technology and antifatigue activity of Hohenbuehelia serotinao polysaccharide by response surface method[J]. Food Sci Technol, 2020, 45(6): 254-260.
[3]	王翠翠, 崔成伟, 陈屏, 等. 金针菇化学成分及药理活性研究进展[J]. 菌物研究, 2021, 19(3): 207-216.
	Wang CC, Cui CW, Chen P, et al. Research advances on chemical constituents and pharmacological activities of Flammulina filiformis[J]. J Fungal Res, 2021, 19(3): 207-216.
[4]	Yelithao K, Surayot U, Lee CS, et al. Studies on structural properties and immune-enhancing activities of glycomannans from Schizophyllum commune[J]. Carbohydr Polym, 2019, 218: 37-45. doi: 10.1016/j.carbpol.2019.04.057 URL
[5]	Chen Q, Che CC, Yang SS, et al. Anti-inflammatory effects of extracellular vesicles from Morchella on LPS-stimulated RAW_264.7 cells via the ROS-mediated p38 MAPK signaling pathway[J]. Mol Cell Biochem, 2023, 478(2): 317-327. doi: 10.1007/s11010-022-04508-y
[6]	陈诺, 席文杰, 胡美芬, 等. 多糖免疫调节作用与结构关系研究进展[J]. 中国中药杂志, 2023, 48(10): 2667-2678.
	Chen N, Xi WJ, Hu MF, et al. Relationship between immune regulation and structure of polysaccharides[J]. China J Chin Mater Med, 2023, 48(10): 2667-2678.
[7]	Zhou P, Wang L, An SY, et al. Fabrication of quercetin-loaded nanoparticles based on Hohenbuehelia serotina polysaccharides and their modulatory effects on intestinal function and gut microbiota in vivo[J]. Innov Food Sci Emerg Technol, 2022, 78: 102993. doi: 10.1016/j.ifset.2022.102993 URL
[8]	Wang L, Li XY. Preparation, physicochemical property and in vitro antioxidant activity of zinc-Hohenbuehelia serotina polysaccharides complex[J]. Int J Biol Macromol, 2019, 121: 862-869. doi: S0141-8130(18)34107-2 pmid: 30342126
[9]	Liu QH, Wu J, Wang P, et al. Neutral polysaccharides from Hohenbuehelia serotina with hypoglycemic effects in a type 2 diabetic mouse model[J]. Front Pharmacol, 2022, 13: 883653. doi: 10.3389/fphar.2022.883653 URL
[10]	陆艳娟, 马岩, 李晓梅, 等. 黄蘑多糖对衰老模型小鼠单胺类神经递质的影响[J]. 中国老年学杂志, 2007, 27(12): 1124-1125.
	Lu YJ, Ma Y, Li XM, et al. The effect of Chinese mushroom(Huangmo)polysaccharides on monoamine neurotransmitter in the aging mice[J]. Chin J Gerontol, 2007, 27(12): 1124-1125.
[11]	隋玉, 舒昉, 崔帅, 等. 元蘑多糖提取工艺优化、结构特征和抗氧化活性[J]. 食用菌学报, 2021, 28(4): 48-56.
	Sui Y, Shu F, Cui S, et al. Extraction process optimization, structural characteristics and antioxidant activities of polysaccharides from Sarcomyxa edulis[J]. Acta Edulis Fungi, 2021, 28(4): 48-56.
[12]	秦子芳, 谭晓妍, 宁慧娟, 等. 不同生长年限铁皮石斛多糖含量与特性分析[J]. 食品科学, 2018, 39(6): 189-193. doi: 10.7506/spkx1002-6630-201806030
	Qin ZF, Tan XY, Ning HJ, et al. Quantification and characterization of polysaccharides from different aged Dendrobium officinale stems[J]. Food Sci, 2018, 39(6): 189-193.
[13]	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254. pmid: 942051
[14]	李瑾, 刘冲英, 张婷婷, 等. 微波合成金银花硒多糖工艺及免疫活性研究[J]. 中国中药杂志, 2022, 47(14): 3773-3780.
	Li J, Liu CY, Zhang TT, et al. Synthesis of seleno-polysaccharides from Lonicerae japonicae Flos via microwave and its immunological activity[J]. China J Chin Mater Med, 2022, 47(14): 3773-3780.
[15]	马高兴, 王晗, 杨文建, 等. 不同提取工艺对杏鲍菇多糖结构特征及免疫活性的影响[J]. 食品科学, 2022, 43(17): 42-49.
	Ma GX, Wang H, Yang WJ, et al. Effects of different extraction processes on structural characteristic and immunomodulatory activity of Pleurotus eryngii polysaccharide[J]. Food Sci, 2022, 43(17): 42-49.
[16]	Yang Y, Ye HQ, Zhao CH, et al. Value added immunoregulatory polysaccharides of Hericium erinaceus and their effect on the gut microbiota[J]. Carbohydr Polym, 2021, 262: 117668. doi: 10.1016/j.carbpol.2021.117668 URL
[17]	黄菊青, 齐睿婷, 张英. 竹茹多糖的化学结构及体外免疫活性研究[J]. 中国食品学报, 2017, 17(7): 34-40.
	Huang JQ, Qi RT, Zhang Y. Structure and in vitro immunomodulatory activity of polysaccharides from bamboo(Phyllostachys pubescens mazel)shavings[J]. J Chin Inst Food Sci Technol, 2017, 17(7): 34-40.
[18]	胡彦波, 王思琪, 石曾卉, 等. 水溶性豆渣酸性多糖对RAW264.7细胞的免疫调节作用及其机制[J]. 食品科学, 2022, 43(3): 169-177.
	Hu YB, Wang SQ, Shi ZH, et al. Immunomodulatory effect and mechanism of soluble acidic polysaccharides from soybean dregs on RAW264.7 cells[J]. Food Sci, 2022, 43(3): 169-177.
[19]	张壮, 李琼, 黄丽萍, 等. 粉葛与葛根多糖对脂多糖诱导RAW264.7细胞的抗炎作用[J]. 现代食品科技, 2022, 38(7): 1-10.
	Zhang Z, Li Q, Huang LP, et al. Anti-inflammatory effect of Pueraria lobata(Willd.) ohwi and P.thomsonii Benth. polysaccharides on LPS-induced RAW264.7 cells[J]. Mod Food Sci Technol, 2022, 38(7): 1-10.
[20]	Meng Y, Yan JM, Yang G, et al. Structural characterization and macrophage activation of a hetero-galactan isolated from Flammulina velutipes[J]. Carbohydr Polym, 2018, 183: 207-218. doi: 10.1016/j.carbpol.2017.12.017 URL
[21]	Tabarsa M, You SG, Yelithao K, et al. Isolation, structural elucidation and immuno-stimulatory properties of polysaccharides from Cuminum cyminum[J]. Carbohydr Polym, 2020, 230: 115636. doi: 10.1016/j.carbpol.2019.115636 URL
[22]	王丽波, 高婧宇, 李腾飞, 等. 硒化蒲公英多糖的制备、结构表征及益生菌促增殖活性[J]. 食品科学, 2021, 42(7): 169-175.
	Wang LB, Gao JY, Li TF, et al. Preparation, structural characterization and probiotics proliferation-promoting activity of selenized dandelion polysaccharide[J]. Food Sci, 2021, 42(7): 169-175.
[23]	Yang J, Cao MX, Hu WY, et al. Sophorasubprosrate polysaccharide suppress the inflammatory reaction of RAW264.7 cells infected with PCV₂ via regulation NF-κB/MAPKs/c-Jun signal pathway and histone acetylation modification[J]. Int J Biol Macromol, 2020, 159: 957-965. doi: 10.1016/j.ijbiomac.2020.05.128 URL
[24]	宋林珍, 朱丽云, 高永生, 等. 茶多糖的结构特征与降血糖活性[J]. 食品科学, 2018, 39(19): 162-168. doi: 10.7506/spkx1002-6630-201819025
	Song LZ, Zhu LY, Gao YS, et al. Structural characteristics and hypoglycemic activity of polysaccharides from green tea leaves[J]. Food Sci, 2018, 39(19): 162-168. doi: 10.1111/jfds.1974.39.issue-1 URL
[25]	Li XY, Wang ZY, Wang L. Polysaccharide of Hohenbuehelia serotina as a defense against damage by whole-body gamma irradiation of mice[J]. Carbohydr Polym, 2013, 94(2): 829-835. doi: 10.1016/j.carbpol.2013.02.015 URL
[26]	Li XY, Wang L. Effect of extraction method on structure and antioxidant activity of Hohenbuehelia serotina polysaccharides[J]. Int J Biol Macromol, 2016, 83: 270-276. doi: 10.1016/j.ijbiomac.2015.11.060 URL
[27]	赵炳杰, 郭岩彬. 食用菌多糖的提取纯化及生物活性研究进展[J]. 中国生物工程杂志, 2022, 42(S1): 146-159.
	Zhao BJ, Guo YB. Advances in extraction, purification and bioactivity of polysaccharides from edible fungi[J]. China Biotechnol, 2022, 42(S1): 146-159.
[28]	Yu Q, Nie SP, Li WJ, et al. Macrophage immunomodulatory activity of a purified polysaccharide isolated from Ganoderma atrum[J]. Phytother Res, 2013, 27(2): 186-191. doi: 10.1002/ptr.v27.2 URL
[29]	杨许花, 赵梦潇, 陈红, 等. 植物多糖免疫调节作用机制研究进展[J]. 食品安全质量检测学报, 2021, 12(13): 5349-5355.
	Yang XH, Zhao MX, Chen H, et al. Advances in research on the mechanism of immunomodulatory effects of plant polysaccharides[J]. J Food Saf Qual, 2021, 12(13): 5349-5355.
[30]	Yang G, Ham I, Choi HY. Anti-inflammatory effect of prunetin via the suppression of NF-κB pathway[J]. Food Chem Toxicol, 2013, 58: 124-132. doi: 10.1016/j.fct.2013.03.039 pmid: 23597450
[31]	黄平, 洪静霞, 米杰, 等. 羊栖菜多酚通过核转录因子-κB/丝裂原活化蛋白激酶通路缓解脂多糖诱导的RAW264.7细胞炎症反应[J]. 食品科学, 2022, 43(23): 141-148.
	Huang P, Hong JX, Mi J, et al. Polyphenols extracted from Hizikia fusiformis relieves lipopolysaccharide-induced inflammation in RAW264.7 Cells via the nuclear factor-κB/mitogen-activated protein kinase signaling pathways[J]. Food Sci, 2022, 43(23): 141-148. doi: 10.1111/jfds.1978.43.issue-1 URL
[32]	Bode JG, Albrecht U, Häussinger D, et al. Hepatic acute phase proteins—regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-κB-dependent signaling[J]. Eur J Cell Biol, 2012, 91(6-7): 496-505. doi: 10.1016/j.ejcb.2011.09.008 URL
[33]	苗月, 任桂红, 甄东, 等. 蛹虫草多糖调节小鼠巨噬细胞RAW264.7免疫活性的分子机制[J]. 食品科学, 2019, 40(9): 188-194. doi: 10.7506/spkx1002-6630-20180122-294
	Miao Y, Ren GH, Zhen D, et al. Molecular mechanism of Cordyceps militaris polysaccharides in regulating the immune function of macrophage RAW264.7 cells[J]. Food Sci, 2019, 40(9): 188-194.
[34]	Ren Z, Qin T, Qiu FA, et al. Immunomodulatory effects of hydroxyethylated Hericium erinaceus polysaccharide on macrophages RAW_264.7[J]. Int J Biol Macromol, 2017, 105(Pt 1): 879-885. doi: 10.1016/j.ijbiomac.2017.07.104 URL
[35]	郝正祺, 刘靖宇, 孟俊龙, 等. 绣球菌子实体单组分多糖结构表征及其免疫活性[J]. 中国食品学报, 2021, 21(10): 46-55.
	Hao ZQ, Liu JY, Meng JL, et al. Structural characterization and immunomodulatory activities of a single-component polysaccharide from the fruiting body of Sparassis crispa[J]. J Chin Inst Food Sci Technol, 2021, 21(10): 46-55.
[36]	Yuan R, Huang LT, Du LJ, et al. Dihydrotanshinone exhibits an anti-inflammatory effect in vitro and in vivo through blocking TLR4 dimerization[J]. Pharmacol Res, 2019, 142: 102-114. doi: 10.1016/j.phrs.2019.02.017 URL
[37]	王琴琴, 韩珊, 李新星, 等. 毛蕊花苷对脂多糖诱导RAW_264.7细胞炎症模型的作用及其机制研究[J]. 中草药, 2020, 51(16): 4217-4222, 4250.
	Wang QQ, Han S, Li XX, et al. Anti-inflammatory effect and mechanism of verbascoside on LPS-induced RAW264.7 cells[J]. Chin Tradit Herb Drugs, 2020, 51(16): 4217-4222, 4250.
[38]	王悦. 左归丸对5/6肾切除血管钙化模型大鼠IL-1β与p65蛋白的影响[D]. 武汉: 湖北中医药大学, 2021.
	Wang Y. Effect of Zuogui pill on IL-1β and P65 protein in 5/6 nephrectomy model rats with vascular calcification[D]. Wuhan: Hubei University of Chinese Medicine, 2021.
[39]	Liao WZ, Luo Z, Liu D, et al. Structure characterization of a novel polysaccharide from Dictyophora indusiata and its macrophage immunomodulatory activities[J]. J Agric Food Chem, 2015, 63(2): 535-544. doi: 10.1021/jf504677r URL
[40]	Wang DD, Pan WJ, Mehmood S, et al. Polysaccharide isolated from Sarcodon aspratus induces RAW264.7 activity via TLR4-mediated NF-κB and MAPK signaling pathways[J]. Int J Biol Macromol, 2018, 120(Pt A): 1039-1047. doi: 10.1016/j.ijbiomac.2018.08.147 URL