| Intestinal inflammation disease shows a high incidence recently, how to prevent and cure it became a hot research topic. We usually treat of IBD by drugs to regulate inflammation and immune process, but it can cause side effects. Lactobacillus is a kind of common probiotics, which are believed to have beneficial effects on the anti-inflammatory and anti-cancer. It is important to research on the immunomodulatory capacity of 4 strains selected in laboratory, including L.paracasei subsp.paracasei M5-L, L. casei Q8-L, L.rhamnosus J10-L and L. rhamnosus GG, so that they could be applied widely.To vertify the immune function of 4 Lactobacillus strains on inflammation, we use Lps(1 ug/m L) to stimulate the RAW264.7 cells to establish the inflammatory model, then treat it with 4 heat-killed strains body. After adjust cell concentrations and wash them with PBS, add them to cell model as a medicine. At first, the cytotoxicity of heat-killed body of strains on RAW264.7 cells was detected by MTT accordding to the conditions designed in experimental program. The result shows that they can inhanced the cell viability which may have been lead a low level by Lps, cell viability only account for 61% of the control. When After add M5-L, Q8-L, J10-L and L.GG to the experimental group, the cell viability increased to 68.9%,70.0%,66.3%,101.8% in prevent group(PG);82.12%,65.5%,61.4%,76.7% in co-culture group(CG) and 95.9%,62.2%,64.73%,85.8% in treatment group(TG). When cells stay high viability, measuring the concentration of inflammatory chemokines(NO), proinflammatory cytokines(TNF-α, IL-6, IL-1β) and anti-inflammatory cytokines(IL-10). Studies suggesting that Lactobacillus can inhibit the concentration of NO significantly, L.GG works best. The concentration of NO decreased to 122.99 pg/m L in PG, 248.28 pg/m L in CG, and 218.39 pg/m L in TG from 278.20 pg/mL in Lps group. Meanwhile, all 4 strains can inhibit the concentration of TNF-α obviously, exspecially in Q8. The concentration of TNF-α turns to 7.90 pg/m L in PG,3.90 pg/m L in CG, 8.20 pg/m L in TG, which was 58.95 pg/m L in Lps. For the production of IL-6 and IL-1β, we get inhibit effects indistinctly. Anti-inflammatory cytokine IL-10 increased to over 350 pg/m L compared with control(263.70 pg/m L). Cell cycle results shows that M5 increased the G2/M+S% in PG and TG significantly, Q8 increased it only in TG. J10 and L.GG increased G2/M+S% and decreased S% obviously in all groups, The increase of G0/G1 in Lps means cell was blocked and difficult to proliferation. Lactcobacillus changed the cell cycle to proliferation state.Regulation of 4 strains on inflammation was detected by RT-PCR. Determination of i NOS which was a important upstream regulatory factor in NO production process indicating that Lps inhanced the i NOS in mRNA level, Q8 and J10 have the ability to decreased it significantly. L.GG works best, the effect was not significant in M5; 4 strains could inhibit the expression of TNF-α in mRNA levels effectively; M5 and J 10 can significantly reduce IL-6 in the m RNA expression levels, including the prevention and treatment groups. Inhibitory effect of the treatment group in L.GG was lower than it in M5 and J10 group. Q8 promote the secretion of IL-6. The results show that: M5-L, Q8-L, L.GG and J10-L have effects on regulatory inflammatory factors.Finally, based on the previous experimental results, expression of two proteins in signaling pathway, i NOS and NF-κBp65, were studied by western methods. Different strains affect i NOS expression in different degree. Cells treated with Lps develop to inflammatory status, M5 relieves the inflammation compared with the control group and the positive control group. Q8 has a preventive effect on inflammatory cells. In addition, it can alleviate cellular inflammation in CG; J10 not inhibit the expression of i NOS protein levels; L.GG inhibited only in prevention group. Lps active the NF-κB signaling pathways, futhermore promote the produce of downstream cytokines, accelerated cell inflammation. M5 not inhibit the expression of NF-κBp65; Q8, J10 and L.GG can inhibit it sightly in TG, PG and PG respectively. NF-k Bp65 remains a high levels in the other experimental group. In summary, the 4 heat-killed strains can regulate RAW264.7 macrophage inflammatory response, but the effects of the present strain-specific characteristics. |
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