屎肠球菌活菌和热灭活菌对RAW264.7细胞肿瘤坏死因子-α/白细胞介素-10平衡以及丝裂原活化蛋白激酶信号通路的影响  被引量:5

Effects of Viable and Heat-Inactivated Enterocuccus faecium on Tumor Necrosis Factor-α/Interleukin-10 Balance and Mitogen-Activated Protein Kinase Signaling Pathway in RAW264.7 Cells

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作  者:任亚雪 郭乾鹏 李媛媛[1] 向明 黄怡[1] REN Yaxue;GUO Qianpeng;LI Yuanyuan;XIANG Ming;HUANG Yi(College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区:[1]广西大学动物科学技术学院,南宁530004

出  处:《动物营养学报》2020年第9期4345-4357,共13页CHINESE JOURNAL OF ANIMAL NUTRITION

基  金:国家自然科学基金项目(31460607)。

摘  要:本试验旨在通过体外试验研究益生菌屎肠球菌活菌和热灭活菌免疫调节作用的差异。试验分为2部分:1)研究屎肠球菌的类型、剂量和作用时间对RAW264.7细胞的肿瘤坏死因子-α(TNF-α)/白细胞介素-10(IL-10)平衡的影响。试验共设12个组,每个组6个重复。采用3因素2水平设计试验方案。因素1及水平:屎肠球菌类型,活菌或热灭活菌;因素2及水平:剂量,106或108 CFU/mL;因素3及水平:作用时间,3或6 h。在3和6 h作用时间下分别设置阴性对照(DMEM细胞基础培养液)和阳性对照[脂多糖(LPS),2μg/mL]。采用酶联免疫吸附测定(ELISA)法检测各组细胞培养上清中TNF-α和IL-10含量,据此计算TNF-α/IL-10的比值。2)研究屎肠球菌活菌或热灭活菌对由LPS引起RAW264.7细胞的TNF-α/IL-10失衡的调节作用。试验共设8个组,每个组6个重复。对照组用DMEM细胞基础培养液处理细胞3 h,LPS组用LPS(2μg/mL)处理细胞3 h;LB-LPS或HB-LPS组先用屎肠球菌活菌或热灭活菌处理细胞3 h,再用LPS处理3 h;LPS-LB或LPS-HB组先用LPS处理细胞3 h,再用屎肠球菌活菌或热灭活菌处理3 h;LPS+LB或LPS+HB组用屎肠球菌活菌或热灭活菌与LPS同时共处理细胞3 h。采用ELISA法检测各组细胞培养上清中TNF-α和IL-10含量,然后计算TNF-α/IL-10的比值,用Western blot法检测磷酸化细胞外信号调节激酶(p-ERK)、磷酸化p38(p-p38)以及磷酸化c-Jun氨基末端激酶(p-JNK)的蛋白相对表达量。结果表明:1)屎肠球菌类型、剂量、作用时间中的1个或多个因素极显著影响TNF-α或IL-10含量(P<0.01),或者TNF-α/IL-10比值(P<0.01),3个因素的两两或三者之间的交互效应因细胞因子而异。屎肠球菌热灭活菌对TNF-α分泌的诱导作用极显著弱于活菌(P<0.01),对IL-10含量的影响与活菌相似(P>0.05),对TNF-α/IL-10比值的影响小于活菌(P<0.05或P<0.01),且低剂量(106 CFU/mL)热灭活菌在作用时间较短(3 h)时能维持TNF-α/IL-10平衡。2)屎�The purpose of this study was to investigate the differences in the immunomodulatory effects of viable and heat-inactivated probiotic Enterococcus faecium in vitro.The experiment included two parts:1)to investigate the effect of type,dose and duration time of Enterococcus faecium on tumor necrosis factor-α(TNF-α)/interleukin-10(IL-10)balance in RAW264.7 cells.Twelve groups were set up in the experiment,with 6 replicates for each.A two-factor three-level test scheme was used in this experiment.Factor 1 and levels:Enterococcus faecium type,viable bacteria or heat-inactivated bacteria;factor 2 and levels:dose,106 or 108 CFU/mL;factor 3 and levels:duration time,3 or 6 h.Negative control(DMEM cell base culture medium)and positive control[lipopolysaccharide(LPS),2μg/mL]were set at 3 and 6 h,respectively.The contents of TNF-αand IL-10 in the cell culture supernatant of each group were detected by enzyme-linked immunosorbent assay(ELISA)method,and the ratio of TNF-α/IL-10 was calculated accordingly.2)To investigate the regulation effect of viable or heat-inactivated Enterococcus faecium on the TNF-α/IL-10 imbalance in LPS-induced RAW264.7 cells.Eight groups were set up in the experiment,with 6 replicates for each.The control group was treated with DMEM cell base culture medium for 3 h,while the LPS group was treated with LPS(2μg/mL)for 3 h.The LB-LPS or HB-LPS groups were firstly treated with viable or heat-inactivated Enterococcus faecium for 3 h,and then treated with LPS for 3 h.The LPS-LB or LPS-HB groups were firstly treated with LPS for 3 h,and then treated with viable or heat-inactivated Enterococcus faecium for 3 h.The LPS+LB or LPS+HB groups were treated with viable or heat-inactivated Enterococcus faecium and LPS for 3 h.ELISA method was used to examine the contents of TNF-αand IL-10 in the cell culture supernatant of each group,then the ratio of TNF-α/IL-10 was calculated,and the relative expression levels of proteins of phosphorylated extracellular signal-regulated protein kinases(p-ERK),phosphorylate

关 键 词:屎肠球菌 活菌 热灭活菌 脂多糖 肿瘤坏死因子-Α 白细胞介素-10 MAPK信号通路 RAW264.7细胞 

分 类 号:S816.7[农业科学—饲料科学]

 

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