Immunoregulatory Effect And Mechanism Of Cell Wall Peptidoglycans Of Lactobacillus Acidophilus

Lactic acid bacteria (LAB) harbors in the human gastrointestinal tract (GIT), which play an important role in maintaining intestinal homeostasis. In addition, LAB have long been used in the manufacture of yogurt, cheese and pickled vegetables. Much attention has been focused on the LAB in relation to host health, and abundant literature shows that LAB have the ability to prevent numerous intestinal disorders.The major structural polymer in the cell walls of most bacteria is PGN. The basic structure of PGN is polymers of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in (0-1,4) linkage, cross-linked by short peptide stems composed of alternating L- and D-amino acids. The most prominent PGN is the substitution of about 50% of the disaccharides (N-acetyl glucan) with muramic acid 8-lactam residue, which occurs predominantly at every alternate disaccharide. An extensive literature shows that murine innate immune cells respond vigorously to various fragments of PGN. PGN has the capacity to induce the Nuclear factor κB (NF-κB) pathway, resulting in the production of a broad range of proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a), a cytokine produced primarily in monocytes and macrophages. Although there is a growing consumption of such probiotics for their health benefits, the precise mechanisms by which they achieve their effects have remained elusive. Researchers have yet to determine how PGN is synthesized, modified and hydrolyzed, and provide a potential candidate for pharmaceutical application of anti-inflammatory drugs.Researchers have found that the PGN of Bifidobacterium can, via mouse peritoneal macrophages, activate nucleotide-binding oligomerization domains (NODs) and interleukin 1β (IL-1β), interleukin 6 (IL-6) and TNF-a secretion. Meanwhile, some researchers found the intact PGN of Bifidobacterium has a potential probiotic based on their tumor-inhibiting capacity through activating the macrophages. Lactobacillus acidophilus (L. acidophilus), a potential probiotic in GIT, they upregulate anti-inflammatory factors and thereby enhance immunity. In this study, L. acidophilus was isolated from the gastrointestinal tract of ribbon fish and identified by 16S rRNA analysis, which had a high DPPH-radical scavenging activity (24.42%) and resistance of neomycin, ceftriaxone, amikacin, gentamicin.The present study sought to evaluate the structure of PGN isolated from L. acidophilus. Despite possible problems related to PGN insolubility, various approaches were used to gain insight into the composition of PGN. Glycoside hydrolase HPLC, NMR, FT-RI discovery, MALDI-TOF/TOP MS and the amino acid analysis provided strong evidence about the presumed PGN structure. The molecular mass of PGN from L. acidophilus is consistent with NAG-(β-1,4)-NAM-L-Ala-D-Glu-L-Lys-D-Ala.The molecular pharmacology of PGN derived from L. acidophilus was analyzed in LPS-stimulated macrophage cells (RAW 264.7), in order to study the molecular mechanisms involved in the induction and repression of intestinal inflammation. With the help of Western Blotting analysis, we presented evidence supporting the hypothesis that the PGN, PGN hydrolysate and monomeric PGN molecule, especially PGN fragment NAM, which showed a significant influence on the anti-inflammatory capacity in relation to LPS-induced inflammation in RAW 264.7 cells. Further research found that the activation of NF-κB is regulated through the cross-talk between TLR4 and NF-Kb pathway. And influenced by cellular kinases p38 in MAPK upon NAM treatment. Meanwhile, iTRAQ was used to evaluate the proteome of LPS-induced RAW 264.7 cells treated with NAM from L. acidophilus. Bioinformatics analysis indicated that this capacity may mediate an anti-inflammatory process via a Ca2+-dependent NF-κB pathway. Attenuation of [Ca2+]i and the up-regulation of SERCA in the NAM-treated group are most consistent with a model in which LPS itself influences SERCA activity through the inhibition of SERCA expression in macrophages, which thereby limits SERCA-dependent ER calcium stores. Lastly, according to the result of ICR mice experiment, the villi changes in E.coli stimulated groups and PGN treated groups did show significant difference.This study shed some light on the functional food, farm livestock and aquatic animal feed fields. PGN and its effector molecules derived from L. acidophilus may provide a new functional material with significant potential in animal feed, such as drug resistance and residues of veterinary drug in food.