Studies On The Inflammation Regulation Mechanism Of Enterococcus Facium HDRsEf1 For IPEC-J2 Cells

Enterococcus faecium has been widely used as lactic acid bacteria of probiotics, it could improve host intestinal health, enhance growth, reduce the rate of diarrhea and enhance the immunity of the host. Presently, few attention is being focused on the beneficial properties of Enterococcus faecium, especially the regulating mechanism of inflammation. So, the study was designed to investigate the effects of Enterococcus faecium HDRsEf1(HDRsEf1) as an inflammation modulatory properties on IPEC-J2 cells. The major content and results are listed as follows:Trial 1 The effect of Enterococcus faecium HDRsEf1 on IPEC-J2 cells.(1) Adhesion test : First, IPEC-J2 cells were cultured 10 days, made it form complete single layer structure. Then, IPEC-J2 cells were co-cultured with Enterococcus faecium HDRsEf1 and(enterotoxigenic e.coli, ETEC) K88 ac, test their adhesion rate respectively. Next, tested the adhesion rate of K88 ac effected by Enterococcus faecium Ef1 and its culture supernatant(S-Ef1). The experiment went like this: IPEC-J2 cells were co-incubated with both HDRsEf1 and K88 ac, or IPEC-J2 cells were pre-cultured with HDRsEf1 and then following incubated with K88 ac, or IPEC-J2 cells were co-cultured with K88 ac firstly, and then incubated with HDRsEf1, the adhesion rate of K88 ac was examined, respectively. The operation of S-Ef1 effect the adhesion rate of K88 ac similar to HDRsEf1. Results showed that HDRsEf1 had a higher adhesion than K88 ac and HDRsEf1 could inhibit the adhesion of K88 ac by exclusion, displacement and competition, and the adherence inhibition rate was 46%(P<0.05),11%(P<0.05),61%(P<0.05) respectively. the results indicate that displacement was the better effect of HDRsEf1. But, S- Ef1 had no effect on the adhesion rate of K88 ac.(2)The effects of Enterococcus faecium HDRsEf1 on transepithelial electrical resistance(TEER) of IPEC-J2 cells. IPEC-J2 cells being cultured for 10 days could fully differentiated and form a complete single layer. Then, the test could be started. First, IPEC-J2 cells were pre-cultured with HDRsEf1 and S-Ef1 for 2 hours and then following co-incubation with K88 ac, the transepithelial electrical resistance was measured after 3,6,12 hours. The results showed that HDRsEf1 and S-Ef1 could protect the membrane integrity of the IPEC-J2 cells, and relieve the membrane structure destruction of IPEC-J2 cells caused by K88 ac.(3) The regulation effects of Enterococcus faecium HDRsEf1 on IL-8 released by IPEC-J2 cells. First, IPEC-J2 cells were pre-cultured with HDRsEf1 and S-Ef1 for 2 hours and then following co-incubation with K88 ac, IL-1β, TNF-ɑ, then tested the expression of IL-8. The results showed that HDRsEf1 and S-Ef1 could relieve the increase of IL-8 caused by ETEC, IL-1β,TNF-ɑ. In order to understand components of HDRsEf1 which could exert inflammatory modulatory effect, the HDRsEf1 and S-Ef1 were treated with heat, and the protein and exopolysaccharides(EPS) were extracted from S-Ef1, anti-inflammatory experiments were carried out respectively. First, IPEC-J2 cells were pre-cultured with heat-treated HDRsEf1, S-Ef1, protein, EPS and then following co-incubating with K88 ac for 2h. The result showed that the heat-treatment had no effects on the anti-inflammatory activities of HDRsEf1 and S-Ef1. The EPS had obvious anti-inflammatory effects, but the protein had no anti-inflammatory effects.Trial 2 The separation and purification of exopolysaccharides(EPS) from Enterococcus faecium HDRsEf1.The experiment revealed HDRsEf1 has the property with high yield EPS, HDRsEf1 were cultured in skim milk culture medium, cultured 30 hours at 30oC, the production of EPS could reach around 320mg/mL. The crude EPS were separated into two polysaccharides by DEAE-Toyopearl 650 M ion-exchange column chromatography, neutral polysaccharide(NPS) and an acidic polysaccharide(APS). Two pure polysaccharides were obtained by Sephadex G-100, Sepharose Cl-6B gelfiltration chromatography, respectively.Trial 3 The study of the inflammatory regulative pathways of Enterococcus faecium HDRsEf1 and its extracellular polysaccharides on IPEC-J2 cells.Objective to revealed the inflammation regulation mechanism of HDRs Ef1 and its extracellular polysaccharides, first, we detected the cytotoxicity test on IPEC-J2 cells. The result showed that the heat-inactivated of HDRsEf1 and its two polysaccharides(neutral polysaccharide and acidic Polysaccharides) had no toxic effects on IPEC-J2 cells, and could improve the activity of cells. Then, the test was carried out to verify the inflammatory regulation properties of HDRsEf1 and its two polysaccharides, the test showed HDRsEf1 and its extracellular polysaccharides could weaken the inflammation of IPEC-J2 cells against lipopolysaccharides(LPS). Next, HDRsEf1 and its two polysaccharides whether could affect the receptors of TLR2 and TLR4 exert the anti-inflammatory effect were studied. The inflammatory regulation properties of HDRsEf1 and its two polysaccharides were tested using the laboratory technology of antibody blocking of these receptors and LPS as the inflammation model. The results showed that TLR2 plays an important role in the inflammatory regulation action of HDRsEf1,While the two polysaccharides suppress inflammatory depended on both TLR4 and TLR2. To further revealed the inflammatory regulation properties of HDRsEf1 and its two polysaccharides, we tested the expression of TLR negative regulators influenced by HDRsEf1 and its two polysaccharides on IPEC-J2 cells stimulated by LPS. The results showed that the TLR negative regulators of Tolliop, IRAKM, A20, Bcl-3 play the important role in the inflammatory regulation action of HDRsEf1, the TLR negative regulators of SIGIRR, Tolliop, IRAKM, A20, Bcl-3play the important role in the inflammatory regulation action of neutral polysaccharide, while the inflammatory regulation action of acidic Polysaccharides depended on Tolliop, A20 and Mkp-1. At last we tested the influence of NF-κB and AP-1 caused by HDRsEf1 and its two polysaccharides using Western Blot. Results showed that the anti-inflammatory effect of HDRsEf1 and its two polysaccharides was exerted through regulate the activity of NF-κB and AP-1.