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

REN Yaxue; GUO Qianpeng; LI Yuanyuan; XIANG Ming; HUANG Yi

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

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

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

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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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

Cite this article

REN Yaxue , GUO Qianpeng , LI Yuanyuan , XIANG Ming , HUANG Yi . 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[J]. Chinese Journal of Animal Nutrition, 2020 , 32(9) : 4345 -4357 . DOI: 10.3969/j.issn.1006-267x.2020.09.045

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