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

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

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)屎肠球菌活菌和热灭活菌分别通过促进IL-10分泌(P<0.01)和抑制TNF-α释放(P<0.01)来抵抗LPS对TNF-α/IL-10平衡的破坏作用,经热灭活菌预处理的细胞能够保持TNF-α/IL-10平衡不受LPS干扰。此外,屎肠球菌活菌和热灭活菌预处理细胞对LPS活化细胞外信号调节激酶(ERK)信号通路没有产生显著影响(P>0.05),但热灭活菌预处理细胞极显著促进了LPS活化p38丝裂原活化蛋白激酶(p38 MAPK)信号通路(P<0.01)。综上可知,屎肠球菌的类型和剂量是影响TNF-α/IL-10平衡的主要因素,而且屎肠球菌的类型与剂量、剂量与作用时间之间都有交互效应,低剂量热灭活菌且作用时间短才有利于维持TNF-α/IL-10平衡。活菌与热灭活菌以不同方式减轻LPS引起的TNF-α/IL-10失衡,但它们对MAPK信号通路的确切影响还有待进一步研究确定。

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),phosphorylated p38(p-p38),and phosphorylated c-Jun amino-terminal kinases(p-JNK)were determined by Western blot method.The results showed as follows:1)one or more factors in Enterococcus faecium type,dose and duration time significantly affected TNF-αor IL-10 contents(P<0.01),or TNF-α/IL-10 ratio(P<0.01),and the interactions between two or three of these three factors varied depending on the cytokines.The induction effect of heat-inactivated Enterococcus faecium on TNF-αsecretion was significantly weaker than that of viable Enterococcus faecium(P<0.01),the effect on IL-10 content was similar to that of viable Enterococcus faecium(P>0.05),and the effect on TNF-α/IL-10 ratio was smaller than that of viable Enterococcus faecium(P<0.05 or P<0.01).In addition,the low dose(10^6 CFU/mL)of heat-inactivated Enterococcus faecium could maintain TNF-α/IL-10 balance in a short duration time(3 h).2)The viable and the heat-inactivated Enterococcus faecium resisted the damage of LPS to the TNF-α/IL-10 balance by promoting the secretion of IL-10 and inhibiting the release of TNF-α,respectively.The cells pretreated by heat-inactivated bacteria could maintain TNF-α/IL-10 balance without interference by LPS.In addition,viable and heat-inactivated Enterococcus faecium pretreated macrophages had no significant effect on the activation of extracellular signal-regulated protein kinases(ERK)signaling pathway by LPS(P>0.05),but heat-inactivated bacteria pretreated cells significantly promoted the activation of p38 mitogen-activated protein kinases(p38 MAPK)signaling pathway by LPS(P<0.01).In summary,the type and dose of Enterococcus faecium are the main factors affecting TNF-α/IL-10 balance.And there are interactions between type and dose,as well as dose and duration time of Enterococcus faecium.The low dose of heat-inactivated bacteria with short duration time is conducive to maintaining TNF-α/IL-10 balance.The TNF-α/IL-10 imbalance caused by LPS is reduced in different ways by viable and heat-inactivated bacteria.However,the exact effects of viable or heat-inactivated Enterococcus faecium on the MAPK signaling pathway have yet to be determined.