In Vitro Study On The Immunomodulatory Properties Of Probiotic Enterococcus Faecium

The aim of the study was to characterize the immunomodulatory activity of viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant of the strain Enterococcus faecium in vitro using macrophage-RAW264.7 cell line and to study its anti-inflammatory potential in lipopolysaccharide?LPS?-induced RAW264.7 cells.Thus provides a scientific reference for fully understanding the immunoregulation effect of Enterococcus faecium and clarifying its mechanism beneficial to host health.Methods:1)RAW264.7 cells were co-cultured with low dose?10⁶CFU/m L?,medium dose?10⁸CFU/m L?or high dose(10¹⁰CFU/m L)Enterococcus faecium for 3 and 6 hours,respectively,and cell proliferation rate was detected by Cell Counting Kit-8?CCK-8?method.The concentration of Enterococcus faecium used in subsequent experiments was determined based on cell proliferation.2)RAW264.7 cells were incubated with serum free,antibiotics free DMEM culture medium?negative control?,LPS?positive control?,viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant of Enterococcus faecium for 3 and 6 hours respectively.The concentration of TNF?and IL-10 in the culture supernatant of each treatment were detected by ELISA.3)RAW264.7 cells were treated with ERK,p38 or JNK inhibitor for 30 min respectively,and then incubated with serum free,antibiotics free DMEM,LPS,viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant of Enterococcus faecium for 3 hours respectively.The concentration of TNF?in the culture supernatant of each treatment was detected by ELISA.4)RAW264.7 cells were treated with serum free,antibiotics free DMEM culture solution or LPS respectively,or cells were pretreated with viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant of Enterococcus faecium,and then stimulated with LPS;or cells were treated with LPS first,and then incubating with viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant respectively;or cells were treated with LPS and viable bacteria,heat inactivated bacteria,broken cells or cell-free culture supernatant simultaneously.The concentration of TNF?and IL-10 in the culture supernatant of each treatment was detected by ELISA,and the relative expression of phosphorylated ERK,p38 and JNK protein was detected by Western blot.The main results were as follows:1)Cell proliferation was not affected at low dose by four forms of Enterococcus faecium,while cell proliferation was not affected or promoted at medium dose,high dose inhibits cell proliferation.Low or medium dose was suitable for subsequent experiments.2)The secretion of TNF?could be induced by all treatment groups.The order of their actions was:viable bacteria,broken cells,cell-free culture supernatant,heat inactivated bacteria.In addition to the supernatant treatment group,the ability of other groups to induce TNF?secretion increased with increasing concentration,and the viable bacteria treatment group and the broken cell treatment group were equivalent to the lipopolysaccharide treatment group.The induction effect of IL-10 secretion by all treatment groups only appeared in a short time?3h?.The effect of each treatment group was in order of culture supernatant,broken cells,heat inactivated bacteria and viable bacteria,and the ability of all treatment groups to induce IL-10 secretion was weakened by the increase of concentration.3)The secretion of TNF?by macrophages was completely blocked by ERK protein inhibitor,p38 protein inhibitor or JNK protein inhibitor in all treatment groups.4)The production of a large amount of TNF?was stimulated by Lipopolysaccharide,but at the same time did not affect the secretion of IL-10,thus causing a serious pro-inflammatory response.TNF?production could be significantly reduced by pretreatment of macrophages with heat-inactivated bacteria or co-treatment of cells with heat-inactivated bacteria and lipopolysaccharid.In addition,the phosphorylation of ERK,p38 and JNK proteins of RAW264.7 cells by lipopolysaccharide was not affected of macrophages pretreated with viable bacteria,but the expression of phosphorylated p38 protein of macrophages pretreated with heat inactivated bacteria was significantly increased,and the expression of phosphorylated ERK,p38 and JNK proteins of macrophages pretreated with broken cells were significantly increased.Conclusion:1)The viable bacteria,heat inactivated bacteria,broken cells and cell-free culture supernatant of Enterococcus faecium induce macrophages to secrete TNF?and IL-10 to varying degrees,and MAPK pathway participates in the regulation of TNF?secretion.2)Different forms of Enterococcus faecium can reduce the inflammatory response induced by lipopolysaccharide,but the mode of action of heat inactivated bacteria is different from the other three forms.The anti-inflammatory effects of heat-inactivated bacteria and broken cells of Enterococcus faecium may be related to the MAPKs pathway,but the signaling pathways related to the anti-inflammatory effects of viable bacteria need further study.