动物营养学报 >

2022 , Vol. 34 >Issue 3: 1996 - 2008

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.03.058

研究论文 RESEARCH PAPER

桑叶多糖对免疫抑制小鼠肠道损伤和微生物多样性的调节作用

  • 陈晓兰 ,
  • 宣嘉颖 ,
  • 王婧 ,
  • 冒玉娟 ,
  • 徐向萍 ,
  • 杨海峰
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  • 江苏农牧科技职业学院, 泰州 225300
陈晓兰(1979-),女,江苏海安人,副教授,博士,研究方向为兽医药理毒理学和中兽医药理学。E-mail:cxl7972563@163.com

收稿日期: 2021-07-18

  网络出版日期: 2022-03-14

基金资助

国家自然科学基金项目（31702286）；2021年江苏省高校青蓝工程学术带头人资助项目；安徽省科技厅重点研发计划（S202104g01020016）；泰州市科技支撑计划（农业）项目（TN202006）；江苏农牧科技职业学院动物药品科技创新团队（NSF2021TC02）

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Regulating Effects of Mulberry Leaf Polysaccharide on Intestinal Damage and Microbial Diversity of Immunosuppressed Mice

  • CHEN Xiaolan ,
  • XUAN Jiaying ,
  • WANG Jing ,
  • MAO Yujuan ,
  • XU Xiangping ,
  • YANG Haifeng
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  • Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China

Received date: 2021-07-18

  Online published: 2022-03-14

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摘要

本试验旨在揭示桑叶多糖（MLP）对免疫抑制小鼠肠道损伤和微生物多样性的调节作用，初步阐明其作用机制。选择体重（20.0±0.5）g的5周龄雄性BALB/c小鼠60只，随机分为正常对照组（NC组）、环磷酰胺模型组（MC组）、桑叶多糖低剂量组（MLPL组）、桑叶多糖中剂量组（MLPM组）、桑叶多糖高剂量组（MLPH组）和药物对照组（LM组），每组10只小鼠。各组小鼠腹腔注射80 mg/kg BW环磷酰胺（除NC组外），每天1次，连续3 d，以诱导免疫抑制。第4天开始MLPL、MLPM、MLPH和LM组小鼠每天分别灌胃20、40、80 mg/kg BW的MLP和40 mg/kg BW的左旋咪唑，连续给药9 d；NC和MC组小鼠每天灌胃等量饮用水。试验期13 d。采用苏木精-伊红（HE）染色法、定量逆转录PCR（RT-qPCR）法、微生物扩增子法分别测定各组小鼠空肠组织形态学，结肠上皮紧密连接蛋白[闭合蛋白（Occludin）、闭锁小带蛋白-1（ZO-1）、封闭蛋白-5（Claudin-5）]、黏液素2（MUC2）、免疫相关因子[肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-10（IL-10）、转化生长因子-β（TGF-β）]、免疫信号通路相关蛋白[Toll样受体4（TLR4）、髓样分化因子88（MyD88）、核因子-κB p65亚基（p65）]的mRNA表达以及盲肠微生物多样性。结果表明：1）与MC组比较，MLPL、MLPM和MLPH组小鼠空肠病理损伤明显减轻。2）与NC组相比，MC组Claudin-5、ZO-1、Occludin、MUC2的mRNA相对表达量显著降低（P<0.05）。与MC组相比，MLPM、MLPH和LM组Claudin-5、MUC2的mRNA相对表达量显著升高（P<0.05），MLPM和MLPH组Occludin的mRNA相对表达量显著升高（P<0.05），MLPH组ZO-1的mRNA相对表达量显著升高（P<0.05）。3）与NC组相比，MC组TNF-α、IL-1β的mRNA相对表达量显著降低（P<0.05），IL-10、TGF-β的mRNA相对表达量显著升高（P<0.05）。与MC组相比，MLPM、MLPH和LM组TNF-α、IL-1β的mRNA相对表达量升高（P<0.05），MLPL、MLPM、MLPH和LM组IL-10和TGF-β的mRNA相对表达量显著降低（P<0.05）。4）与NC组相比，MC组TLR4、MyD88、p65的mRNA相对表达量显著降低（P<0.05）。与MC组相比，MLPM、MLPH和LM组TLR4、MyD88、p65的mRNA相对表达量显著升高（P<0.05）。5）与NC组相比，MC组拟杆菌门的相对丰度显著降低（P<0.05）；与MC组比较，MLPH组拟杆菌门的相对丰度显著增加（P<0.05）。与NC组相比，MC组免疫抑制相关菌如丁酸弧菌属（Butyricimonas）和真杆菌属（Eubacterium）的相对丰度显著增加（P<0.05），而MLPL、MLPM、MLPH组丁酸弧菌属和真杆菌属的相对丰度显著降低（P<0.05）。由此可见，MLP可通过修复肠道屏障功能、调节免疫细胞因子、改变肠道微生物群落的空间结构来调节免疫应答，拮抗免疫抑制，其调控机制可能与TLR4/MyD88/p65信号通路的激活有关。

关键词： 桑叶多糖; 肠道损伤; 微生物多样性; 免疫抑制; 环磷酰胺

本文引用格式

陈晓兰 , 宣嘉颖 , 王婧 , 冒玉娟 , 徐向萍 , 杨海峰 . 桑叶多糖对免疫抑制小鼠肠道损伤和微生物多样性的调节作用[J]. 动物营养学报, 2022 , 34(3) : 1996 -2008 . DOI: 10.3969/j.issn.1006-267x.2022.03.058

Abstract

This experiment was conducted to reveal the regulating effects of mulberry leaf polysaccharide (MLP) on intestinal damage and microbial diversity of immunosuppressed mice, and to preliminarily clarify its mechanism. Sixty 5-week-old male BALB/c mice with body weight of (20.0±0.5) g were randomly divided into normal control group (NC group), cyclophosphamide model group (MC group), mulberry leaf polysaccharide low-dose group (MLPL group), mulberry leaf polysaccharide medium-dose group (MLPM group), mulberry leaf polysaccharide high-dose group (MLPH group) and drug control group (LM group), with 10 mice in each group. Mice in all groups except NC group were injected intraperitoneally with 80 mg/kg BW cyclophosphamide once a day for three days to establish immunosuppressive model. From the fourth day, mice were administered intragastrically with 20, 40 and 80 mg/kg BW of MLP in MLPL, MLPM and MLPH groups and 40 mg/kg BW of levamisole in LM group, continuous administration for 9 days; while the mice in MC and NC groups were administered intragastrically instead with equivalent drinking water. The jejunal histomorphology, colonic epithelium tight junction protein[(Occludin, zonula occludens protein-1 (ZO-1) and Claudin-5)], mucin 2 (MUC2), immune related factors[tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)] and immune signaling pathway related proteins[Toll-like receptor 4 (TLR4), myeloid differentiation protein 88 (MyD88) and nuclear factor-κB p65 subunit (p65)] mRNA expression and cecal microbial diversity were measured by hematoxylin-eosin (HE) staining, quantitative reverse transcription PCR (RT-qPCR) and microbial amplicon sequencing. The results showed as follows:1) compared with the MC group, the jejunal pathological injury of mice of MLPL, MLPM and MLPH groups was obvious reduced. 2) Compared with the NC group, the mRNA relative expression levels of Claudin-5, ZO-1, Occludin and MUC2 of the MC group were significantly decreased (P<0.05). Compared with the MC group, the mRNA relative expression levels of Claudin-5 and MUC2 of MLPM, MLPH and LM groups were significantly increased (P<0.05), the mRNA relative expression levels of Occludin of MLPM and MLPH groups were significantly increased (P<0.05), and the mRNA relative expression level of ZO-1 of MLPH group was significantly increased (P<0.05). 3) Compared with the NC group, the mRNA relative expression levels of TNF-α and IL-1β of the MC group were significantly decreased (P<0.05), while the mRNA relative expression levels of IL-10 and TGF-β were significantly increased (P<0.05). Compared with the MC group, the mRNA relative expression levels of TNF-α and IL-1β of MLPM, MLPH and LM groups were significantly increased (P<0.05), while the mRNA relative expression levels of IL-10 and TGF-β of MLPL, MLPM, MLPH and LM groups were significantly decreased (P<0.05). 4) Compared with the NC group, the mRNA relative expression levels of TLR4, MyD88 and p65 of the MC group were significantly decreased (P<0.05). Compared with the MC group, the mRNA relative expression levels of TLR4, MyD88 and p65 of MLPM, MLPH and LM groups were significantly increased (P<0.05). 5) Compared with the NC group, the relative abundance of Bacteroidetes of the MC group was significantly decreased (P<0.05); compared with the MC group, the relative abundance of Bacteroidetes of the MLPH group was significantly increased (P<0.05). Compared with the NC group, the relative abundances of immunosuppressive related bacteria such as Butyricimonas and Eubacterium of the MC group were significantly increased (P<0.05), while the relative abundances of Butyricimonas and Eubacterium of MLPL, MLPM and MLPH groups were significantly decreased (P<0.05). In conclusion, MLP can regulate immune response and antagonize immune suppression by repairing intestinal barrier function, regulating immune cytokines and changing the spatial structure of intestinal microbiota, the mechanism can be related to the activation of TLR4/MyD88/p65 signaling pathway.

Key words： mulberry leaf polysaccharide; intestinal damage; microbial diversity; immunosuppression; cyclophosphamide

参考文献

[1] 黄佳铭,杨冬雪,李晓曦,等.肠道黏膜免疫与炎症小体的研究进展[J].微生物与感染,2019,14(2):113-123. HUANG J M,YANG D X,LI X X,et al.Inflammasome and intestinal mucosal immunity[J].Journal of Microbes and Infections,2019,14(2):113-123.(in Chinese)
[2] 刘昌孝.肠道菌群与健康、疾病和药物作用的影响[J].中国抗生素杂志,2018,43(1):1-14. LIU C X.Gut microbiota:the effects of health,illness and medicines[J].Chinese Journal of Antibiotics,2018,43(1):1-14.(in Chinese)
[3] LI C X,LIU H Y,LIN Y X,et al.The gut microbiota and respiratory diseases:new evidence[J].Journal of Immunology Research,2020,2020:2340670.
[4] XU X F,YANG J G,NING Z X,et al.Lentinula edodes-derived polysaccharide rejuvenates mice in terms of immune responses and gut microbiota[J].Food & Function,2015,6(8):2653-2663.  
[5] TANG C,SUN J,ZHOU B,et al.Effects of polysaccharides from purple sweet potatoes on immune response and gut microbiota composition in normal and cyclophosphamide treated mice[J].Food & Function,2018,9(2):937-950.  
[6] 韦芳媚,陈春,李超,等.桑叶提取物、茶多酚及其复配物的抗氧化和降血糖活性[J].食品工业科技,2018,39(21):299-305. WEI F M,CHEN C,LI C,et al.Antioxidant and hypoglycemic activities of mulberry leaves extract,tea polyphenols and their compounds[J].Science and Technology of Food Industry,2018,39(21):299-305.(in Chinese)
[7] CHEN X L,SHENG Z C,QIU S L,et al.Purification,characterization and in vitro and in vivo immune enhancement of polysaccharides from mulberry leaves[J].PLoS One,2019,14(1):e0208611.
[8] CHEN X,YANG H,JIA J,et al.Mulberry leaf polysaccharide supplementation contributes to enhancing the respiratory mucosal barrie immune response in newcastle disease virus vaccinated chicks[J]. Poultry Science,2020,100:592-602.
[9] DING Y,YAN Y M,CHEN D,et al.Modulating effects of polysaccharides from the fruits of Lycium barbarum on the immune response and gut microbiota in cyclophosphamide-treated mice[J].Food & Function,2019,10(6):3671-3683.  
[10] SHI H J,CHANG Y G,GAO Y,et al.Dietary fucoidan of Acaudina molpadioides alters gut microbiota and mitigates intestinal mucosal injury induced by cyclophosphamide[J].Food & Function,2017,8(9):3383-3393.  
[11] 廖吕燕,马玉芳,李健,等."芪苓"制剂多糖对环磷酰胺免疫损伤小鼠肠道菌群的影响[C]//中国畜牧兽医学会2010学术年会暨第二届中国兽医临床大会论文集.长春:中国畜牧兽医学会,2010:1452-1455. LIAO L Y,MA Y F,LI J,et al.Effect of QiLing preparations polysaccharide on intestinal flora of cyclophosphamide inducedimmune suppression mice[C]//Proceedings of the 2010 Academic Annual Meeting of China Society of Animal Husbandry and Veterinary Medicine and the Second China Veterinary Clinical Conference.Changchun:Chinese Association of Animal Science and Veterinary Medicine,2010:1452-1455.(in Chinese)
[12] 谢俊华,聂少平,丁翘,等.植物乳杆菌NCU116发酵胡萝卜浆对免疫抑制小鼠肠道黏膜免疫的影响[J].食品科学,2015,36(21):201-206. XIE J H,NIE S P,DING Q,et al.Effect of carrot slurry fermented with Lactobacillus plantarum NCU116 on intestinal mucosal immunity in immune suppressed mice[J].Food Science,2015,36(21):201-206.(in Chinese)
[13] 胡彩虹,钱仲仓,刘海萍,等.高锌对早期断奶仔猪肠黏膜屏障和肠上皮细胞紧密连接蛋白表达的影响[J].畜牧兽医学报,2009,40(11):1638-1644. HU C H,QIAN Z C,LIU H P,et al.Effect of high level of zinc oxide on tight junction protein expression in intestinal epithelial cells and intestinal mucosal barrier in early weaning piglets[J].Acta Veterinaria et Zootechnica Sinica,2009,40(11):1638-1644.(in Chinese)
[14] 黄则华,曾悦,黄春兰.肠道菌群对黏蛋白2的影响及其作用机制[J].胃肠病学,2019,24(2):111-114. HUANG Z H,ZENG Y,HUANG C L.Effect and mechanism of intestinal flora on mucin 2[J].Chinese Journal of Gastroenterology,2019,24(2):111-114.(in Chinese)
[15] 薛然.火麻仁多糖对环磷酰胺致小鼠肠道黏膜损伤的保护作用研究[D].硕士学位论文.舟山:浙江海洋大学,2019:36-42. XUE R.Protective effect of hemp seed polysaccharide on intestinal mucosal injury induced by cyclophosphamide in mice[D].Master's Thesis.Zhoushan:Zhejiang Ocean University,2019:36-42.(in Chinese)
[16] 牛佳卉,袁静,魏然,等.红枣多糖对小鼠肠道免疫屏障的保护作用及机制研究[J].食品工业科技,2021,42(4):295-300,306. NIU J H,YUAN J,WEI R,et al.Protective effect and mechanism study of jujube polysaccharides on intestinal immune barrier in mice[J].Science and Technology of Food Industry,2021,42(4):295-300,306.(in Chinese)
[17] MA X L,MENG M,HAN L R,et al.Immunomodulatory activity of macromolecular polysaccharide isolated from Grifola frondosa[J].Chinese Journal of Natural Medicines,2015,13(12):906-914.  
[18] 赵晓芳.板党多糖对溃疡性结肠炎大鼠的防治作用及机制研究[D].硕士学位论文.武汉:湖北中医药大学,2016:27-31. ZHAO X F.The study on prevention and mechanism of BDP against the ulcerative colitis on rats[D].Master's Thesis.Wuhan:Hubei University of Traditional Chinese Medicine,2016:27-31.(in Chinese)
[19] 王昕旭,邹凯,张晓慧,等.沙棘多糖对DSS诱导的溃疡性结肠炎的影响[C]//第十二届全国免疫学学术大会摘要汇编.天津:中国免疫学会,2017:421-422. WANG X X,ZOU K,ZHANG X H,et al.Effect of seabuckthorn polysaccharide on ulcerative colitis induced by DSS[C]//Summary of the 12th National Conference on Immunology.Tianjin:Chinese Society for Immunology,2017:421-422.(in Chinese)
[20] 王磊,刘懿,张志军,等.TLR4mAb对急性期溃疡性结肠炎小鼠结肠黏膜中细胞因子IL-17、IL-10及TGF-β的影响[J].复旦学报(医学版),2008,35(6):892-896. WANG L,LIU Y,ZHANG Z J,et al.Preventive effects of TLR4 monocional antibodies on the gut mucosal cytokines (IL-17,IL-10 and TGF-β) expression in mice with DSS-induced acute ulcerative colitis[J].Fudan University Journal of Medical Sciences,2008,35(6):892-896.(in Chinese)
[21] BROOKS D G,TRIFILO M J,EDELMANN K H,et al.Interleukin-10 determines viral clearance or persistence in vivo[J].Nature Medicine,2006,12(11):1301-1309.  
[22] 张竞舟,胡楠,汤佳琦,等.胃癌中TGF-β、PD-L1表达与肿瘤免疫表型的关系及其临床意义[J].中国现代应用药学, 2021,38(9):1025-1030. ZHANG J Z,HU N,TANG J Q,et al.Correlation of the expression of TGF-β and PD-L1 and cancer-immune phenotypes in gastric cancer and its clinical significance[J].Chinese Journal of Modern Applied Pharmacy,2021,38(9):1025-1030.(in Chinese)
[23] 杨锦涛,程希,陈红伟,等.白术多糖免疫调节作用机制的研究进展[J].中国兽药杂志,2018,52(6):80-85. YANG J T,CHENG X,CHEN H W,et al.Advances in the mechanism of polysaccharide immunization in Atractylodes macrocephala Koidz[J].Chinese Journal of Veterinary Drug,2018,52(6):80-85.(in Chinese)
[24] 彭颖,李宗军.菌物多糖对巨噬细胞和树突状细胞的免疫刺激作用及信号通路[J].食品科学,2012,33(15):318-323. PENG Y,LI Z J.Immunostimulating effect and signal pathway of fungal polysaccharides on macrophages and dendritic cells[J].Food Science,2012,33(15):318-323.(in Chinese)
[25] 李燕华,梁月琴,王崇静,等.粗根荨麻多糖对环磷酰胺诱导的免疫抑制小鼠肠道派氏结细胞表型及TLR4的影响[J].中成药,2017,39(6):1272-1276. LI Y H,LIANG Y Q,WANG C J,et al.Effect of nettle polysaccharide on phenotype and TLR4 of Parry's node cells in immunosuppressed mice induced by cyclophosphamide[J].Chinese Traditional Patent Medicine,2017,39(6):1272-1276.(in Chinese)
[26] 刘海鑫,葛旭影,曹丽艳,等.NF-κB p65在仔猪体内各组织脏器中的表达与分布[C]//中国畜牧兽医学会兽医病理学分会第二十四次学术研讨会,中国病理生理学会动物病理生理学专业委员会第二十三次学术研讨会,中国实验动物学会实验病理学专业委员会第三次学术研讨会,中国兽医病理学家第三次学术研讨会论文集.青岛:中国畜牧兽医学会兽医病理学分会,中国病理生理学会动物病理生理学专业委员会,中国实验动物学会实验病理学专业委员会,中国兽医病理学家,2018:102. LIU H X,GE X Y,CAO L Y,et al.Expression and distribution of NF-κB p65 in various tissues and organs of piglets[C]//The 24th Academic Seminar of Veterinary Pathology Branch of Chinese Society of Animal Husbandry and Veterinary Medicine,the 23rd Academic Seminar of Animal Pathophysiology Professional Committee of Chinese Society of Pathophysiology,the Third Academic Seminar of Experimental Pathology Professional Committee of Chinese Society of Experimental Animals,and the Proceedings of the Third Academic Seminar of Chinese Veterinary Pathologists.Qingdao:Veterinary Pathology Branch of Chinese Society of Animal Husbandry and Veterinary Medicine,Professional Committee of Animal Pathophysiology of Chinese Society of Pathophysiology,Professional Committee of Experimental Pathology of Chinese Society of Experimental Animals,and Chinese Veterinary Pathologist,2018:102.(in Chinese)
[27] 邹川,吴禹池,罗丽,等.中药大黄复方灌肠对慢性肾脏病5期(非透析)肠道菌群和肠道屏障功能影响的临床研究[J].辽宁中医杂志,2012,39(7):1309-1311. ZOU C,WU Y C,LUO L,et al.Clinical study on the effect of rhubarb compound enema on intestinal flora and intestinal barrier function of chronic kidney disease stage 5(non-dialysis)[J].Liaoning Journal of Traditional Chinese Medicine,2012,39(7):1309-1311.(in Chinese)
[28] XUE R,DU M,ZHOU T Y,et al.Polysaccharides from hemp seed protect against cyclophosphamide-induced intestinal oxidative damage via Nrf2-Keap1 signaling pathway in mice[J].Oxidative Medicine and Cellular Longevity,2020,2020:1813798.
[29] YING M X,YU Q,ZHENG B,et al.Cultured Cordyceps sinensis polysaccharides attenuate cyclophosphamide-induced intestinal barrier injury in mice[J].Journal of Functional Foods,2019,62:103523.
[30] TANG C,DING R X,SUN J,et al.The impacts of natural polysaccharides on intestinal microbiota and immune responses-a review[J].Food & Function,2019,10(5):2290-2312.  
[31] TANG C,SUN J,ZHOU B,et al.Effects of polysaccharides from purple sweet potatoes on immune response and gut microbiota composition in normal and cyclophosphamide treated mice[J].Food & Function,2018,9(2):937-950.  
[32] 罗青,禄璐,闫亚美,等.枸杞粉及多糖对环磷酰胺致免疫低下小鼠的影响及其对肠道菌群的调节作用[J/OL].食品科学,2021:1-14.(2021-06-21)[2021-07-01].http://kns.cnki.net/kcms/detail/11.2206.TS.20210618.1729.044.html. LUO Q,LU L,YAN Y M,et al.Effects of spray drying powder of Goji (Lycium barbarum L.) and polysaccharides on immunosuppressive mice induced by cyclophosphamide and its regulation on gut microbiota[J/OL].Food Science,2021:1-14.(2021-06-21)[2021-07-01].http://kns.cnki.net/kcms/detail/11.2206.TS.20210618.1729.044.html. (in Chinese)
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