摘要
本试验旨在探究芜菁多糖对黄羽鹌鹑外周血淋巴细胞免疫和抗氧化功能的影响。将经过粉碎机粉碎的芜菁块根,通过热水浸提、大孔树脂吸附和DEAE-52纤维素柱纯化,获得纯度为99.85%、分子质量为118 ku的芜菁多糖。静脉采血收集黄羽鹌鹑的静脉血液样品,通过PBLs提取试剂盒收集黄羽鹌鹑淋巴细胞,悬于RPMI-1640培养基(含10%胎牛血清+1%双抗)中,然后使用台盼蓝染色计数,调整外周血淋巴细胞悬浮液中细胞浓度为1×10^(7)个/mL,用于外周血淋巴细胞体外培养试验。外周血淋巴细胞体外培养试验中设置5个芜菁多糖浓度(0、25、50、100和200μg/mL),每个浓度设6个重复孔,外周血淋巴细胞体外培养24 h后收集上清液,测定免疫指标[肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)、白细胞介素-10(IL-10)、免疫球蛋白A(IgA)、免疫球蛋白G(IgG)和免疫球蛋白M(IgM)含量]与抗氧化指标[谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量];同时,采用实时荧光定量PCR(RT-qPCR)方法测定免疫相关基因IL-1β、IL-8、IL-10、TNF-α、Toll样受体4(TLR4)、核因子-κB(NF-κB)、髓样分化因子88(MyD88)与抗氧化相关基因GSH-Px、CAT、SOD的mRNA相对表达量。结果显示:1)在免疫功能方面,与空白对照组(0μg/mL芜菁多糖)相比,50、100和200μg/mL芜菁多糖可极显著提高上清液中IgA、IgM含量(P<0.01),极显著降低上清液中IL-1β、IL-8、TNF-α含量(P<0.01);此外,50和100μg/mL芜菁多糖还可显著提高上清液中IgG含量(P<0.05)。2)在抗氧化功能方面,与空白对照组相比,50、100和200μg/mL芜菁多糖可极显著提高上清液中CAT活性(P<0.01);同时,上清液中TLR4、NF-κB、IL-8、TNF-α的mRNA相对表达量在芜菁多糖浓度为100和200μg/mL时极显著降低(P<0.01),SOD、GSH-Px和CAT的mRNA相对表达量在芜菁多糖浓度为100和200μg/mL时显著或极显著升高(P<0.05或P<0.01),MyD88的mRNA相对表达量在芜菁多糖浓度为25和50μg/mL时极显著升高(P<0.01)。综上所述,芜菁多糖可通过调节淋巴细胞响应,提高黄羽鹌鹑外周血淋巴细胞的免疫和抗氧化功能,本试验条件下的最佳作用浓度为100μg/mL。
This study aimed to evaluate the effects of Brassica rapa L.polysaccharides(BRP)on the immune and antioxidant functions of peripheral blood lymphocytes(PBLs)in yellow-feathered quails.The Brassica rapa L.roots were crushed,and BRP was extracted using hot water extraction,macroporous resin adsorption,and DEAE-52 cellulose column purification,resulting in a purity of 99.85% and a molecular weight of 118 ku.Blood samples were taken from the veins of yellow-feathered quails,and lymphocytes were isolated with a PBLs extraction kit.Lymphocytes were suspended in RPMI-1640 medium(with 10% fetal bovine serum and 1%double antibiotics),and the cell concentration was adjusted to 1×10^(7) cells/mL using Trypan blue staining and counting for PBLs culture in vitro test.The PBLs culture in vitro test involved five concentrations(0,25,50,100 and 200μg/mL)of BRP,with six replicates for each concentration.After 24 hours of PBLs culture,and the supernatant was collected to measure immune indexes[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-8(IL-8),interleukin-10(IL-10),immunoglobulin A(IgA),immunoglobulin G(IgG)and immunoglobulin M(IgM)contents]and antioxidant indexes[glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),catalase(CAT)activities and malondialdehyde(MDA)content].Real-time quantitative PCR(RT-qPCR)method was also performed to assess the mRNA relative expression levels of immune-related genes,including IL-1β,IL-8,IL-10,TNF-α,Toll-like receptor 4(TLR4),nuclear factor-κB(NF-κB),myeloid differentiation factor 88(MyD88)and antioxidant-related genes,including GSH-Px,CAT,SOD.The results showed as follows:1)in terms of immune function,compared with the blank control group(0μg/mL BRP),BRP at the concentrations of 50,100 and 200μg/mL extremely significantly increased IgA and IgM contents in the supernatant(P<0.01),while extremely significantly reduced IL-1β,IL-8 and TNF-α contents in the supernatant(P<0.01);moreover,BRP at the concentrations of 50 and 100μg/mL also significantly increased IgG content in the supernatant(P<0.05).2)In terms of antioxidant function,compared with the blank control group,BRP extremely significantly increased the supernatant CAT activity at the concentrations of 50,100 and 200μg/mL(P<0.01);furthermore,the mRNA relative expression levels of TLR4,NF-κB,IL-8 and TNF-α in the supernatant were extremely significantly reduced by BRP at the concentrations of 50,100 and 200μg/mL(P<0.01),whereas the mRNA relative expression levels of SOD,GSH-Px and CAT were significantly or extremely significantly elevated by BRP at the concentrations of 100 and 200μg/mL(P<0.05 or P<0.01),and the mRNA relative expression level of MyD88 was extremely significantly elevated by BRP at the concentrations of 25 and 50μg/mL(P<0.01).In conclusion,BRP enhances the immune and antioxidant functions of PBLs in yellow-feathered quails by modulating lymphocyte responses,with 100μg/mL being the optimal concentration under this experimental condition.
作者
朱建军
柳婷婷
王政力
马艳
申红
王俊刚
ZHU Jianjun;LIU Tingting;WANG Zhengli;MA Yan;SHEN Hong;WANG Jungang(College of Animal Science and Technology,Shihezi University,Shihezi 832061,China)
出处
《动物营养学报》
北大核心
2025年第2期1234-1244,共11页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(31660703)。
关键词
芜菁多糖
黄羽鹌鹑
外周血淋巴细胞
免疫
抗氧化
NF-κB信号通路
Brassica rapa L.polysaccharides
yellow-feathered quails
peripheral blood lymphocytes
immune
antioxidant
NF-κB signal pathway
