Immunoregulatory effect of Lactobacillus paracasei VL8 exopolysaccharide on RAW264.7 cells by NF-κB and MAPK pathways

[Display omitted] •VL8-EPS promoted the cell proliferation and upregulated the mRNA expression of proinflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-10.•RAW264.7 macrophages mainly recognized polysaccharides through TLR4.•VL8-EPS promoted nuclear translocation of the p65 subunit of NF-κB.•VL8-EPS stimulated RAW264.7 macrophages through the upregulation of the NF-κB and MAPK signaling pathways. This study explored the immunoregulatory mechanism of VL8-EPS, obtained from Viili. An RAW264.7 macrophage cell-line was used along with real-time quantitative PCR to evaluate the impact of VL8-EPS on mRNA expression of IL-1β, TNF-α, IL-6, IL-10, and TLR4. Moreover, western blot assay was conducted to study the signal pathway of NF-κB and MAPK. The VL8-EPS was found to upregulate the mRNA expression of these cytokines and TLR4, while it promoted protein phosphorylation of p65, IκBα, ERK1/2, and JNK. The immunofluorescence staining and inhibitor blocking experiments proved that TLR4, NF-κB, and MAPK signal pathway were involved to secrete immune-enhancing substances. In conclusion, VL8-EPS was involved to activate downstream signal pathways of NF-κB and MAPK through TLR4 identification, which caused an increase in mRNA expression and cytokines release, ultimately exerting immune regulation.