动物营养学报 >

2020 , Vol. 32 >Issue 9: 4345 - 4357

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.09.045

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

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

  • 任亚雪 ,
  • 郭乾鹏 ,
  • 李媛媛 ,
  • 向明 ,
  • 黄怡
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  • 广西大学动物科学技术学院, 南宁 530004
任亚雪(1994-),女,山东菏泽人,硕士研究生,动物营养与饲料科学专业。E-mail:343704642@qq.com

收稿日期: 2020-03-17

  网络出版日期: 2020-09-17

基金资助

国家自然科学基金项目（31460607）

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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

  • REN Yaxue ,
  • GUO Qianpeng ,
  • LI Yuanyuan ,
  • XIANG Ming ,
  • HUANG Yi
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  • College of Animal Science and Technology, Guangxi University, Nanning 530004, China

Received date: 2020-03-17

  Online published: 2020-09-17

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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），2 μg/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（2 μg/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。

关键词： 屎肠球菌; 活菌; 热灭活菌; 脂多糖; 肿瘤坏死因子-&alpha; 白细胞介素-10; MAPK信号通路; RAW264.7细胞

本文引用格式

任亚雪 , 郭乾鹏 , 李媛媛 , 向明 , 黄怡 . 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[J]. 动物营养学报, 2020 , 32(9) : 4345 -4357 . DOI: 10.3969/j.issn.1006-267x.2020.09.045

Abstract

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), 2 μg/mL] were set at 3 and 6 h, respectively. The contents of TNF-α and 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 (2 μg/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-α and 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-α or 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-α secretion 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 (106 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.

Key words： Enterococcus faecium; viable bacteria; heat-inactivated bacteria; LPS; TNF-α; IL-10; MAPK pathway; RAW264.7 cells

参考文献

[1] BOYLE R J,ROBINS-BROWNE R M,TANG M L K.Probiotic use in clinical practice:what are the risks[J].The American Journal of Clinical Nutrition,2006,83(6):1256-1264.  
[2] BACCOURI O,BOUKERB A M,BEN FARHAT L,et al.Probiotic potential and safety evaluation of Enterococcus faecalis OB14 and OB15,isolated from traditional tunisian testouri cheese and rigouta,using physiological and genomic analysis[J].Frontiers Microbiology,2019,10:881,doi:10.3389/fmicb.2019.00881.
[3] FU S Z,YANG Q,HE F L,et al.National safety survey of animal-use commercial probiotics and their spillover effects from farm to humans:an emerging threat to public health[J].Clinical Infectious Diseases,2019,70(11):2386-2395,doi:10.1093/cid/ciz642.
[4] MARKOWICZ C,KUBIAK P,GRAJEK W,et al.Inactivation of Lactobacillus rhamnosus GG by fixation modifies its probiotic properties[J].Canadian Journal of Microbiology,2016,62(1):72-82.  
[5] GIAHI L,ELMADFA I,HOSEINI M,et al.Heat-inactivated Lactobacillus rhamnosus and Lactobacillus delbrueckii induce efficient maturation and differential cytokine production in human monocyte derived dendritic cells[J].Food and Agricultural Immunology,2013,24(1):95-109.  
[6] UCHINAKA A,AZUMA N,MIZUMOTO H,et al.Anti-inflammatory effects of heat-killed Lactobacillus plantarum L-137 on cardiac and adipose tissue in rats with metabolic syndrome[J].Scientific Reports,2018,8:8156.
[7] MAKIOKA Y,TSUKAHARA T,IJICHI T,et al.Oral supplementation of Bifidobacterium longum strain BR-108 alters cecal microbiota by stimulating gut immune system in mice irrespectively of viability[J].Bioscience,Biotechnology,and Biochemistry,2018,82(7):1180-1187.  
[8] KIM J E,SHARMA A,SHARMA G,et al.Lactobacillus pentosus modulates immune response by inducing IL-10 producing TRL cells[J].Immune Network,2019,19(6):e39.
[9] CLARK I A.How TNF was recognized as a key mechanism of disease[J].Cytokine & Growth Factor Reviews,2007,18(3/4):335-343.
[10] MOORE K W,DE WAAL MALEFYT R,COFFMAN R L,et al.Interleukin-10 and the interleukin-10 receptor[J].Annual Review of Immunology,2001,19:683-765.
[11] IYER S S,CHENG G H.Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease[J].Critical ReviewsTM in Immunology,2012,32(1):23-63.  
[12] CROSS M L,GANNER A,TEILAB D,et al.Patterns of cytokine induction by Gram-positive and gram-negative probiotic bacteria[J].FEMS Immunology & Medical Microbiology,2004,42(2):173-180.  
[13] WANG Y Z,XIE J M,WANG N,et al.Lactobacillus casei Zhang modulate cytokine and Toll-like receptor expression and beneficially regulate poly Ⅰ:C-induced immune responses in RAW264.7 macrophages[J].Microbiology and Immunology,2013,57(1):54-62.  
[14] SHIN R,ITOH Y,KATAOKA M,et al.Anti-tumor activity of heat-killed Lactobacillus plantarum BF-LP284 on Meth-A tumor cells in BALB/c mice[J].International Journal of Food Sciences and Nutrition,2016,67(6):641-649.  
[15] OH N S,JOUNG J Y,LEE J Y,et al.Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant feces[J].PLoS One,2018,13(2):e0192021,doi:10.1371/journal.pone.0192021.
[16] LI N,RUSSELL W M,DOUGLAS-ESCOBAR M,et al.Live and heat-killed Lactobacillus rhamnosus GG:effects on proinflammatory and anti-inflammatory cytokines/chemokines in gastrostomy-fed infant rats[J].Pediatric Research,2009,66(2):203-207.  
[17] DONG C,DAVIS R J,FLAVELL R A.Map kinases in the immune response[J].Annual Review of Immunology,2002,20:55-72.
[18] KIM S O,SHEIKH H I,HA S D,et al.G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages[J].Cellular Microbiology,2006,8(12):1958-1971.  
[19] LIN Y P,THIBODEAUX C H,PEÑA J A,et al.Probiotic Lactobacillus reuteri suppress proinflammatory cytokines via c-Jun[J].Inflammatory Bowel Diseases,2008,14(8):1068-1083.  
[20] LATVALA S,MIETTINEN M,KEKKONEN R A,et al.Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3(SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages[J].Clinical Experimental Immunology,2011,165(1):94-103.  
[21] KAJI R,KIYOSHIMA-SHIBATA J,NAGAOKA M,et al.Bacterial teichoic acids reverse predominant IL-12 production induced by certain Lactobacillus strains into predominant IL-10 production via TLR2-dependent ERK activation in macrophages[J].The Journal of Immunology,2010,184(7):3505-3513.  
[22] UENO N,FUJIYA M,SEGAWA S,et al.Heat-killed body of Lactobacillus brevis sbc8803 ameliorates intestinal injury in a murine model of colitis by enhancing the intestinal barrier function[J].Inflammatory Bowel Diseases,2011,17(11):2235-2250.  
[23] POPOVI? N,DJOKI? J,BRDARI? E,et al.The Influence of heat-killed Enterococcus faecium BGPAS1-3 on the tight junction protein expression and immune function in differentiated Caco-2 cells infected with Listeria monocytogenes ATCC 19111[J].Frontiers Microbiology,2019,10:412.
[24] CHUNG I C,OUYANG C N,YUAN S N,et al.Pretreatment with a heat-killed probiotic modulates the NLRP3 inflammasome and attenuates colitis-associated colorectal cancer in mice[J].Nutrients,2019,11(3):516-531.  
[25] CHEN M F,WENG K F,HUANG S Y,et al.Pretreatment with a heat-killed probiotic modulates monocyte chemoattractant protein-1 and reduces the pathogenicity of influenza and enterovirus 71 infections[J].Mucosal Immunology,2017,10(1):215-227.  
[26] 戴正浩,刘虎传,江科,等.屎肠球菌的功能特性及其在养猪业中的应用[J].山东畜牧兽医,2018,39(5):78-79.
[27] HUANG Y,LI Y L,HUANG Q,et al.Effect of orally administered Enterococcus faecium EF1 on intestinal cytokines and chemokines production of suckling piglets[J].Pakistan Veterinary Journal,2012,32(1):81-84.
[28] 向明,李媛媛,郭乾鹏,等.屎肠球菌对哺乳期仔猪结肠微生物群落优势门属的影响[J].南方农业学报,2019,50(3):477-484.
[29] 李媛媛,郭乾鹏,梁世忠,等.热灭活屎肠球菌及其活菌对仔猪腹泻率、消化酶活性和脏器指数的影响[J].黑龙江畜牧兽医,2018(15):1-5,10.
[30] CHON H,CHOI B,LEE E,et al.Immunomodulatory effects of specific bacterial components of Lactobacillus plantarum KFCC11389P on the murine macrophage cell line RAW 264.7[J].Journal of Applied Microbiology,2009,107(5):1588-1597.  
[31] KIM J Y,KIM H,JUNG B J,et al.Lipoteichoic acid isolated from Lactobacillus plantarum suppresses LPS-mediated atherosclerotic plaque inflammation[J].Molecules and Cells,2013,35(2):115-124.  
[32] AZAD A K,SARKER M,WAN D.Immunomodulatory effects of probiotics on cytokine profiles[J].BioMed Research International,2018,2018:8063647,doi:10.1155/2018/8063647.
[33] HE F,MORITA H,OUWEH A C,et al.Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains[J].Microbiology and Immunology,2002,46(11):781-785.  
[34] PERCY M G,GRVNDLING A.Lipoteichoic acid synthesis and function in Gram-positive bacteria[J].Annual Review of Microbiology,2014,68:81-100.
[35] NUSSENBLATT V,MUKASA G,METZGER A,et al.Anemia and interleukin-10,tumor necrosis factor alpha,and erythropoietin levels among children with acute,uncomplicated Plasmodium falciparum malaria[J].Clinical and Diagnostic Laboratory Immunology,2001,8(6):1164-1170.  
[36] ISMAIL N,STEVENSON H L,WALKER D H.Role of tumor necrosis factor alpha (TNF-α) and interleukin-10 in the pathogenesis of severe murine monocytotropic ehrlichiosis:increased resistance of TNF receptor p55- and p75-deficient mice to fatal ehrlichial infection[J].Infection and Immunity,2006,74(3):1846-1856.  
[37] LECH-MARANDA E,GRZYBOWSKA-IZYDORCZYK O,WYKA K,et al.Serum tumor necrosis factor-α and interleukin-10 levels as markers to predict outcome of patients with chronic lymphocytic leukemia in different risk groups defined by the IGHV mutation status[J].Archivum Immunologiae et Therapiae Experimentalis,2012,60(6):477-486.  
[38] KOZICKY L K,MENZIES S C,ZHAO Z Y,et al.IVIg and LPS co-stimulation induces IL-10 production by human monocytes,which is compromised by an FcγRIIA disease-associated Ge[J/OL].Frontiers in Immunology,2018,9:2676,doi:10.3389/fimmu.2018.02676.
[39] PEÑA J A,VERSALOVIC J.Lactobacillus rhamnosus GG decreases TNF-α production in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism[J].Cellular Microbiology,2003,5(4):277-285.  
[40] MARTIN I,CABÁN-HERNÁNDEZ K,FIGUEROA-SANTIAGO O,et al.Fasciola hepatica fatty acid binding protein inhibits TLR4 activation and suppresses the inflammatory cytokines induced by lipopolysaccharide in vitro and in vivo[J].The Journal of Immunology,2015,194(8):3924-3936.  
[41] IYER S S,AIL GHAFFARI A,CHENG G H.Lipopolysaccharide-mediated IL-10 transcriptional regulation requires sequential induction of type Ⅰ IFNs and IL-27 in macrophages[J].The Journal of Immunology,2010,185(11):6599-6607.  
[42] KIM H G,KIM N R,GIM M G,et al.Lipoteichoic acid isolated from Lactobacillus plantarum inhibits lipopolysaccharide-induced TNFα production in THP-1 cells and endotoxin shock in mice[J].Journal of Immunology,2008,180(4):2553-2561.  
[43] MUNIANDY K,GOTHAI S,BADRAN K M H,et al.Suppression of proinflammatory cytokines and mediators in LPS-induced RAW 264.7 macrophages by stem extract of Alternanthera sessilis via the inhibition of the NF-κB pathway[J].Journal of Immunology Research,2018,2018:3430684,doi:10.1155/2018/3430684.
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