Structural characterization of an apple polysaccharide and its anti-inflammatory effect through suppressing TLR4/NF-κB signaling

The current study isolated a homogeneous polysaccharide (AP) with a molecular weight of 7.9 kDa from the pomace of Fuji apples. AP was found to consists of rhamnose, galactose, arabinose, glucose, and galacturonic acid in a ratio of 4.3:5.2:2.6:1.0:11.9. Ten sugar residues in AP, including T-Araf, 1,5-Araf, 1,2-Rhap, 1,3-Rhap, TGalp, 1,3,5-Araf, 1,4-Galp, 1,4-GalpA, 1,6-Glcp, and 1,3,6-Glcp were identified using methylation and GC-MS. Combined with 1D and 2D NMR, it was further revealed that AP possesses a backbone of alpha-Galp-(1 -> [3)-alpha-Rhap- (1 -> 2)-alpha-Rhap-(1]2 -> [4)-alpha-GalpA-(1]10 -> 3,6)-beta-Glcp-(1 -> 6)-beta-Glcp-(1 -> 4)-beta-Galp-(1 -> 4)-beta-Galp-(1 ->, with two branches: alpha-Araf-(1 -> 5)-alpha-Araf-(1 -> 5)-alpha-Araf-(1 -> 3,5)-alpha-Araf-(1 -> 6)-beta-Glcp-(1 -> and -> 3)-alpha-Rha p-(1 -> 5)-alpha-Araf-(1 -> 3,6)-beta-Glcp-(1 -> bonded to the C-3 of beta-1,3,6-Glc p . AP significantly inhibited the release of cytokines and inflammatory mediators, such as TNF-alpha, IL-1 beta, IL-6, reactive oxygen species (ROS) and nitric oxide (NO). Western blotting results indicated that AP treatment markedly downregulated iNOS and NF-kappa B protein expression in LPS-induced RAW264.7 cells, leading to decreased levels of phosphorylated proteins (p-NF-kappa B and p-& Iukcy;kappa & Vcy;alpha) and preventing the degradation of & Iukcy;kappa & Vcy; alpha. Furthermore, in LPS-induced RAW264.7 macrophages, AP inhibited the expression of TLR4 protein, which in turn inhibited the activity of the NF-kappa B pathway. The findings demonstrated that AP exhibits anti-inflammatory properties in vitro by targeting the TLR4/NF-kappa B signaling pathway, thus impeding the nuclear translocation of NF-kappa Bp65, suppressing the expression of related pro- inflammatory factors.