Lactobacillus Regulation Of Lps-induced Thp-1 Cells In Inflammatory Cytokine Release And The Possible Mechanism

Background: Lactic acid bacteria, belonging to the normal flora, contribute to regulate the immunological responses to maintain the balance of intestinal microenvironment. More and more researches have revealed lactic acid bacteria exhibit kinds of immunological activities, enhancing the activity of NK cells, promoting the secretion of IgA, maturation of dendric cell differentiation of regulatory T cells and releasing inflammation included. Besides, LTA,PGN and LP of lactic acid bacteria can binding immune cells by recognizing the membrane receptor TLR2. TLR2 can be connected to TLR1 and TLR6 respectively to recognize propionyl- and acetyl-LP subsequently, which may participate in the complicated signal transduction of THP-1 when stimulated by lactic acid bacteria. However, the particular immunological molecular mechanism of lactic acid bacteria is still not very clear.Objective: We aim to explore the regulatory effects of Lactobacillus paracasei and Lactobacillus acidophilus on the secretion of LPS-induced TNF-α, IL-12P40 and TGF-βin THP-1 cells on behalf of studying the molecular mechanism primarily.Methods: (1) THP-1 cells were firstly stimulated by PMA to differentiate into macrophage-like cells, and were secondly treated by LPS to induce inflammatory cytokines. Six groups were designed, including blank control group, LPS-treated group, L.para-treated alone group, L.para plus LPS-treated group, L.acid-treated alone group and L.acid plus LPS-treated group. mRNA levels of TNF-αin THP-1 cells were determined by RT-PCR. TNF-α, IL-12P40 and TGF-βin the supernatant of THP-1 cells were assayed by ELISA. (2) The whole proteins of the blank control group, LPS-treated group, L.para-treated alone group, L.para plus LPS-treated group, L.acid-treated alone group and L.acid plus LPS-treated group cells were extracted for the quantitation of phosphorylation of IκBα. (3) The subcellular localization of NF-κB/P65 was observed by immunofluorescence, while the quantitation of NF-κB(P65/P50)in THP-1 nucleus was detected by TransAMTM NF-κB Kit. Results: (1)The secretions of TNF-α, IL-12P40 and TGF-βin PMA-induced macrophage-like THP-1 cells were remarkably increased when treated by LPS, the same as L.para or L.acid treated alone respectively. However, TNF-αand IL-12P40 in L.para-treated alone group were lower than that in LPS-treated group, whereas TNF-αand IL-12P40 in L.acid-treated alone group were similar to the LPS-treated group. TGF-βinduced by L.para and L.acid was strikingly higher than that of LPS. Interestingly, TNF-αand IL-12P40 in PMA-induced macrophage-like THP-1 cells stimulated by LPS were notably inhibited when cells were treated with L.para plus LPS or L.acid plus LPS. TNF-αand IL-12P40 in L.para plus LPS-treated group were much lower than in LPS-treated group. No inhibition of TGF-βwere observed in PMA-induced macrophage-like THP-1 cells when treated with both lactobacilli. (2) Phosphorylation of IκBαin PMA-induced macrophage-like THP-1 cells increased in LPS-treated group, L.para-treated alone group and L.acid-treated alone group, while non-phosphorylation of IκBαaccordingly decreased .The regulatory effect of L.para was a little weaker than L.acid. Phosphorylation of IκBαwere significantly reduced in PMA-induced macrophage-like THP-1 cells when treated with L.para plus LPS or L.acid plus LPS. (3) Similar to the above results, nuclear NF-κB(P65/P50)in PMA-induced macrophage-like THP-1 cells increased in LPS-treated group, L.para-treated alone group and L.acid-treated alone group.The regulatory effect of L.para was a little weaker than L.acid. Nuclear translocation of NF-κB were significantly reduced in PMA-induced macrophage-like THP-1 cells when treated with L.para plus LPS or L.acid plus LPS.Conclusion: L.para and L.acid can inhibit the secretion of LPS-induced inflammatory cytokines TNF-αand IL-12P40 when they interacted with LPS respectively. The negative regulation of lactic acid bacteria was related to inhibition of NF-κB signaling. Besides, the regulatory effects of lactic acid bacteria were evidently different among species. As a result, clinical applications of L.para and L.acid to prevent the development and release of ulcerative colitis and Crohn's disease are reasonable.