黑灵芝多糖对免疫抑制小鼠肠道黏膜形态及肠道黏膜免疫的影响
|
摘要
目的:探究黑灵芝多糖对免疫抑制小鼠肠道黏膜形态及肠道黏膜免疫的影响。方法:腹腔注射环磷酰胺构建免疫抑制小鼠模型,将小鼠分为6组:正常对照组、模型组、阳性对照组以及黑灵芝多糖低、中、高剂量(25、50、100 mg/(kg·d))组,灌胃期间每天记录小鼠体质量并观察其生活状态,空肠组织进行切片病理学观察,酶联免疫吸附实验测定肠道白细胞介素(interlrukin,IL)-6、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及干扰素-γ(interferon-γ,IFN-γ)的含量,反转录定量聚合酶链式反应分析T-bet、GATA-3 mRNA相对表达水平。结果:与模型组相比,低、中、高剂量黑灵芝多糖有助于体质量回升,改善肠道黏膜形态结构,提高IL-6、IL-10、TNF-α、IFN-γ细胞因子含量,增强T-bet、GATA-3 mRNA相对表达水平,使Th1/Th2平衡向Th1偏移。结论:黑灵芝多糖可改善免疫抑制小鼠的肠道黏膜形态,调节免疫抑制小鼠的肠道黏膜免疫。
Objective: To study the effect of Ganoderma atrum polysaccharide(GAP) on intestinal mucosal morphology and immunity in immunosuppressed mice. Methods: A mouse model of immunosuppression was established by intraperitoneal injection of cyclophosphamide for 3 consecutive days. The mice were randomly divided into 6 groups: normal control, model, low-, medium-and high-dose(25, 50 and 100 mg/(kg·d)) GAP, and positive groups. Body mass was recorded and the status of the mice was observed every day. The mice were sacrificed after administration for 7 days. The mucosal morphology of the jejunum was observed by optical microscopy after hematoxylin-eosin(HE) staining. The enzyme-linked immunosorbent assay(ELISA) was used to determine the levels of interleukin-6(IL-6), interleukin-10(IL-10), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ). Reverse transcription-quantitative polymerase chain reaction(RTqPCR) was used to analyze the relative mRNA expression of T-bet and GATA-3. Results: Compared with the model group, administration of GAP at all three doses increased body mass, improved intestinal mucosal morphology, augmented the levels of IL-6, IL-10, TNF-α and IFN-γ, elevated the relative mRNA expression levels of T-bet and GATA-3, and shifted the Th1/Th2 balance to Th1. Conclusion: GAP can improve intestinal mucosal morphology and regulate intestinal mucosal immunity in immunosuppressed mice.
Objective: To study the effect of Ganoderma atrum polysaccharide(GAP) on intestinal mucosal morphology and immunity in immunosuppressed mice. Methods: A mouse model of immunosuppression was established by intraperitoneal injection of cyclophosphamide for 3 consecutive days. The mice were randomly divided into 6 groups: normal control, model, low-, medium-and high-dose(25, 50 and 100 mg/(kg·d)) GAP, and positive groups. Body mass was recorded and the status of the mice was observed every day. The mice were sacrificed after administration for 7 days. The mucosal morphology of the jejunum was observed by optical microscopy after hematoxylin-eosin(HE) staining. The enzyme-linked immunosorbent assay(ELISA) was used to determine the levels of interleukin-6(IL-6), interleukin-10(IL-10), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ). Reverse transcription-quantitative polymerase chain reaction(RTqPCR) was used to analyze the relative mRNA expression of T-bet and GATA-3. Results: Compared with the model group, administration of GAP at all three doses increased body mass, improved intestinal mucosal morphology, augmented the levels of IL-6, IL-10, TNF-α and IFN-γ, elevated the relative mRNA expression levels of T-bet and GATA-3, and shifted the Th1/Th2 balance to Th1. Conclusion: GAP can improve intestinal mucosal morphology and regulate intestinal mucosal immunity in immunosuppressed mice.
引文
[1]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.DOI:10.7506/spkx1002-6630-201521038.
[2]PETERSON L W,ARTIS D.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nature Reviews Immunology,2014,14(3):141-153.DOI:10.1038/nri3608.
[3]王友湘,陈庆森.益生菌和肠道粘膜免疫关系的研究进展[J].食品科学,2007,28(8):537-542.DOI:10.3321/j.issn:1002-6630.2007.08.136.
[4]李曼,孙学华,周振华,等.慢性乙型肝炎病毒感染者外周血T细胞γ干扰素和白细胞介素4的水平变化[J].细胞与分子免疫学杂志,2016(2):240-244.
[5]郭明,封桂英,郭亚春,等.Th1型细胞因子在胶原蛋白诱导性关节炎小鼠血清中的动态变化[J].细胞与分子免疫学杂志,2014(3):250-253.
[6]严胜泽,马玉芳,廖吕燕,等.太子参多糖对环磷酰胺所致肠道黏膜损伤小鼠SIgA、IL-2、IL-6含量的影响[J].中国畜牧兽医,2015,42(5):1187-1192.DOI:10.16431/j.cnki.1671-7236.2015.05.023.
[7]XU Xiaofei,YANG Jiguo,LUO Zhen,et al.Lentinula edodes-derived polysaccharide enhances systemic and mucosal immunity by spatial modulation of intestinal gene expression in mice[J].Food&Function,2015,6:2068-2080.DOI:10.1039/c4fo01192a.
[8]PATERSON R R M.Ganoderma:A therapeutic fungal biofactory[J].Phyto chemistry,2006,67(18):1985-2001.DOI:10.1016/j.phytochem.2006.07.004
[9]李明春,雷林生,王庆彪,等.灵芝多糖对小鼠T细胞三磷酸肌醇和二酰基甘油体外作用的研究[J].中国药学杂志,2001(8):24-26.
[10]李明春,梁东升,许自明,等.灵芝多糖对小鼠巨噬细胞cAMP含量的影响[J].中国中药杂志,2000(1):43-45.
[11]弓晓峰,谢明勇,陈奕.黑灵芝与赤灵芝提取物的抗氧化作用比较[J].食品科学,2006,27(4):44-47.DOI:10.3321/j.issn:1002-6630.2006.04.006.
[12]张莘莘.黑灵芝多糖的抗肿瘤活性及其分子机制初探[D].南昌:南昌大学,2014:51-72.
[13]LI W J,NIE S P,PENG X P,et al.Ganoderma atrum polysaccharide improves age-related oxidative stress and immune impairment in mice[J].Journal of Agricultural and Food Chemistry,2012,60(6):1413-1418.DOI:10.1021/jf204748a.
[14]朱科学,聂少平,宋丹,等.黑灵芝多糖对Ⅱ型糖尿病大鼠血糖,血脂及肠系膜上动脉病变改善作用[J].食品科学,2013,34(23):300-304.DOI:10.7506/spkx1002-6630-201323060.
[15]李文娟,聂少平,余强,等.黑灵芝多糖对免疫抑制小鼠的免疫调节作用[J].食品科学,2009,30(19):297-299.DOI:10.3321/j.issn:1002-6630.2009.19.070.
[16]王君巧,聂少平,余强,等.黑灵芝多糖对免疫抑制小鼠的免疫调节和抗氧化作用[J].食品科学,2012,33(23):274-277.
[17]CHEN Y,XIE M Y,NIE S P,et al.Purification,composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum[J].Food Chemistry,2008,107(1):231-241.DOI:10.1016/j.foodchem.2007.08.021.
[18]CHEN Changying,TIAN Li,ZHANG Mingzhi,et al.Protective effect of amifostine on high-dose methotrexate-induced small intestinal mucositis in mice[J].Digestive Diseases and Sciences,2013,58(11):3134-3143.DOI:10.1007/s10620-013-2826-3.
[19]廖吕燕.“芪苓”制剂多糖对免疫抑制小鼠肠道黏膜免疫及免疫调节作用的影响[D].福州:福建农林大学,2010:34-44.
[20]PELAEZ B,CAMPILLO J A,LOPEZ-ASENJO J A,et al.Cyclophosphamide induces the development of early myeloid cells suppressing tumor cell growth by a nitric oxide-dependent mechanism[J].The Journal of Immunology,2001,166:6608-6615.DOI:10.4049/jimmunol.166.11.6608.
[21]MADONDO M T,QUINN M,PLEBANSKI M.Low dose cyclophosphamide:mechanisms of T cell modulation[J].Cancer Treatment Reviews,2016,42:3-9.DOI:10.1016/j.ctrv.2015.11.005.
[22]XIE J H,YU Q,NIE S P,et al.Effects of Lactobacillus plantarum NCU116 on intestine mucosal immunity in immunosuppressed mice[J].Journal of Agricultural and Food Chemistry,2015,63(51):10914-10920.DOI:10.1021/acs.jafc.5b04757.
[23]TAKAHASHI I,KIYONO H.Gut as the largest immunologic tissue[J].Journal of Parenteral and Enteral Nutrition,1999,23(Suppl 5):S7-S12.DOI:10.1177/014860719902300503.
[24]ZUO T,CAO L,XUE C,et al.Dietary squid ink polysaccharide induces goblet cells to protect small intestine from chemotherapy induced injury[J].Food&Function,2015,6(3):981-986.DOI:10.1039/c4fo01191k.
[25]石玉祥,闫金坤,王雪敏.枸杞多糖对小鼠肠道上皮内淋巴细胞和杯状细胞数量,分布及对IL-2水平影响[J].食品科学,2011,32(13):318-320.
[26]陈凌锋,蔡旭滨,檀新珠,等.太子参茎叶多糖对断奶仔猪肠道免疫功能,肠黏膜形态结构及盲肠内容物菌群的影响[J].动物营养学报,2017,29(3):1012-1020.
[27]齐育英,林振忠,明德松.IL-6、IL-10、IL-17在慢性乙型病毒性肝炎患者血中水平分析[J].中国免疫学杂志,2013,29(11):1177-1180.
[28]LI W J,NIE S P,CHEN Y,et al.Ganoderma atrum polysaccharide protects cardiomyocytes against anoxia/reoxygenation-induced oxidative stress by mitochondrial pathway[J].Journal of Cellular Biochemistry,2010,110(1):191-200.DOI:10.1002/jcb.22526.
[29]黄璟,周毅.肿瘤坏死因子α及其抑制剂的研究进展[J].国际口腔医学杂志,2015,42(1):63-68.
[30]HEHLGANS T,PFEFFER K.The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily:players,rules and the games[J].Immunology,2005,115(1):1-20.DOI:10.1111/j.1365-2567.2005.02143.x.
[31]LUO Q,CUI H,PENG X,et al.The association between cytokines and intestinal mucosal immunity among broilers fed on diets supplemented with fluorine[J].Biological Trace Element Research,2013,152(2):212-218.DOI:10.1007/s12011-013-9612-0.
[32]孙晓雨,崔子寅,张明亮,等.枸杞多糖和茯苓多糖对免疫抑制小鼠免疫增强及对肠道黏膜的免疫调节作用[J].中国兽医学报,2015(3):450-455.
[33]但刚,刘晨霞,吴丽娟.T淋巴细胞功能及检测方法[J].国际检验医学杂志,2015(3):377-379;380.DOI:10.3969/j.issn.1673-4130.2015.03.039.
[34]CHAKIR H,WANG H,LEFEBVRE D E,et al.T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell populations:predominant role of GATA-3[J].Journal of Immunological Methods,2003,278(1):157-169.DOI:10.1016/S0022-1759(03)00200-X.
[2]PETERSON L W,ARTIS D.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nature Reviews Immunology,2014,14(3):141-153.DOI:10.1038/nri3608.
[3]王友湘,陈庆森.益生菌和肠道粘膜免疫关系的研究进展[J].食品科学,2007,28(8):537-542.DOI:10.3321/j.issn:1002-6630.2007.08.136.
[4]李曼,孙学华,周振华,等.慢性乙型肝炎病毒感染者外周血T细胞γ干扰素和白细胞介素4的水平变化[J].细胞与分子免疫学杂志,2016(2):240-244.
[5]郭明,封桂英,郭亚春,等.Th1型细胞因子在胶原蛋白诱导性关节炎小鼠血清中的动态变化[J].细胞与分子免疫学杂志,2014(3):250-253.
[6]严胜泽,马玉芳,廖吕燕,等.太子参多糖对环磷酰胺所致肠道黏膜损伤小鼠SIgA、IL-2、IL-6含量的影响[J].中国畜牧兽医,2015,42(5):1187-1192.DOI:10.16431/j.cnki.1671-7236.2015.05.023.
[7]XU Xiaofei,YANG Jiguo,LUO Zhen,et al.Lentinula edodes-derived polysaccharide enhances systemic and mucosal immunity by spatial modulation of intestinal gene expression in mice[J].Food&Function,2015,6:2068-2080.DOI:10.1039/c4fo01192a.
[8]PATERSON R R M.Ganoderma:A therapeutic fungal biofactory[J].Phyto chemistry,2006,67(18):1985-2001.DOI:10.1016/j.phytochem.2006.07.004
[9]李明春,雷林生,王庆彪,等.灵芝多糖对小鼠T细胞三磷酸肌醇和二酰基甘油体外作用的研究[J].中国药学杂志,2001(8):24-26.
[10]李明春,梁东升,许自明,等.灵芝多糖对小鼠巨噬细胞cAMP含量的影响[J].中国中药杂志,2000(1):43-45.
[11]弓晓峰,谢明勇,陈奕.黑灵芝与赤灵芝提取物的抗氧化作用比较[J].食品科学,2006,27(4):44-47.DOI:10.3321/j.issn:1002-6630.2006.04.006.
[12]张莘莘.黑灵芝多糖的抗肿瘤活性及其分子机制初探[D].南昌:南昌大学,2014:51-72.
[13]LI W J,NIE S P,PENG X P,et al.Ganoderma atrum polysaccharide improves age-related oxidative stress and immune impairment in mice[J].Journal of Agricultural and Food Chemistry,2012,60(6):1413-1418.DOI:10.1021/jf204748a.
[14]朱科学,聂少平,宋丹,等.黑灵芝多糖对Ⅱ型糖尿病大鼠血糖,血脂及肠系膜上动脉病变改善作用[J].食品科学,2013,34(23):300-304.DOI:10.7506/spkx1002-6630-201323060.
[15]李文娟,聂少平,余强,等.黑灵芝多糖对免疫抑制小鼠的免疫调节作用[J].食品科学,2009,30(19):297-299.DOI:10.3321/j.issn:1002-6630.2009.19.070.
[16]王君巧,聂少平,余强,等.黑灵芝多糖对免疫抑制小鼠的免疫调节和抗氧化作用[J].食品科学,2012,33(23):274-277.
[17]CHEN Y,XIE M Y,NIE S P,et al.Purification,composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum[J].Food Chemistry,2008,107(1):231-241.DOI:10.1016/j.foodchem.2007.08.021.
[18]CHEN Changying,TIAN Li,ZHANG Mingzhi,et al.Protective effect of amifostine on high-dose methotrexate-induced small intestinal mucositis in mice[J].Digestive Diseases and Sciences,2013,58(11):3134-3143.DOI:10.1007/s10620-013-2826-3.
[19]廖吕燕.“芪苓”制剂多糖对免疫抑制小鼠肠道黏膜免疫及免疫调节作用的影响[D].福州:福建农林大学,2010:34-44.
[20]PELAEZ B,CAMPILLO J A,LOPEZ-ASENJO J A,et al.Cyclophosphamide induces the development of early myeloid cells suppressing tumor cell growth by a nitric oxide-dependent mechanism[J].The Journal of Immunology,2001,166:6608-6615.DOI:10.4049/jimmunol.166.11.6608.
[21]MADONDO M T,QUINN M,PLEBANSKI M.Low dose cyclophosphamide:mechanisms of T cell modulation[J].Cancer Treatment Reviews,2016,42:3-9.DOI:10.1016/j.ctrv.2015.11.005.
[22]XIE J H,YU Q,NIE S P,et al.Effects of Lactobacillus plantarum NCU116 on intestine mucosal immunity in immunosuppressed mice[J].Journal of Agricultural and Food Chemistry,2015,63(51):10914-10920.DOI:10.1021/acs.jafc.5b04757.
[23]TAKAHASHI I,KIYONO H.Gut as the largest immunologic tissue[J].Journal of Parenteral and Enteral Nutrition,1999,23(Suppl 5):S7-S12.DOI:10.1177/014860719902300503.
[24]ZUO T,CAO L,XUE C,et al.Dietary squid ink polysaccharide induces goblet cells to protect small intestine from chemotherapy induced injury[J].Food&Function,2015,6(3):981-986.DOI:10.1039/c4fo01191k.
[25]石玉祥,闫金坤,王雪敏.枸杞多糖对小鼠肠道上皮内淋巴细胞和杯状细胞数量,分布及对IL-2水平影响[J].食品科学,2011,32(13):318-320.
[26]陈凌锋,蔡旭滨,檀新珠,等.太子参茎叶多糖对断奶仔猪肠道免疫功能,肠黏膜形态结构及盲肠内容物菌群的影响[J].动物营养学报,2017,29(3):1012-1020.
[27]齐育英,林振忠,明德松.IL-6、IL-10、IL-17在慢性乙型病毒性肝炎患者血中水平分析[J].中国免疫学杂志,2013,29(11):1177-1180.
[28]LI W J,NIE S P,CHEN Y,et al.Ganoderma atrum polysaccharide protects cardiomyocytes against anoxia/reoxygenation-induced oxidative stress by mitochondrial pathway[J].Journal of Cellular Biochemistry,2010,110(1):191-200.DOI:10.1002/jcb.22526.
[29]黄璟,周毅.肿瘤坏死因子α及其抑制剂的研究进展[J].国际口腔医学杂志,2015,42(1):63-68.
[30]HEHLGANS T,PFEFFER K.The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily:players,rules and the games[J].Immunology,2005,115(1):1-20.DOI:10.1111/j.1365-2567.2005.02143.x.
[31]LUO Q,CUI H,PENG X,et al.The association between cytokines and intestinal mucosal immunity among broilers fed on diets supplemented with fluorine[J].Biological Trace Element Research,2013,152(2):212-218.DOI:10.1007/s12011-013-9612-0.
[32]孙晓雨,崔子寅,张明亮,等.枸杞多糖和茯苓多糖对免疫抑制小鼠免疫增强及对肠道黏膜的免疫调节作用[J].中国兽医学报,2015(3):450-455.
[33]但刚,刘晨霞,吴丽娟.T淋巴细胞功能及检测方法[J].国际检验医学杂志,2015(3):377-379;380.DOI:10.3969/j.issn.1673-4130.2015.03.039.
[34]CHAKIR H,WANG H,LEFEBVRE D E,et al.T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell populations:predominant role of GATA-3[J].Journal of Immunological Methods,2003,278(1):157-169.DOI:10.1016/S0022-1759(03)00200-X.