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, T-Galp, 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 α-Galp-(1 → [3)-α-Rhap-(1 → 2)-α-Rhap-(1]₂ → [4)-α-GalpA-(1]₁₀ → 3,6)-β-Glcp-(1 → 6)-β-Glcp-(1 → 4)-β-Galp-(1 → 4)-β-Galp-(1→, with two branches: α-Araf-(1 → 5)-α-Araf-(1 → 5)-α-Araf-(1 → 3,5)-α-Araf-(1 → 6)-β-Glcp-(1→ and →3)-α-Rhap-(1 → 5)-α-Araf-(1 → 3,6)-β-Glcp-(1→ bonded to the C-3 of β-1,3,6-Glcp. AP significantly inhibited the release of cytokines and inflammatory mediators, such as TNF-α, IL-1β, IL-6, reactive oxygen species (ROS) and nitric oxide (NO). Western blotting results indicated that AP treatment markedly downregulated iNOS and NF-κB protein expression in LPS-induced RAW264.7 cells, leading to decreased levels of phosphorylated proteins (p-NF-κB and p-ΙκΒα) and preventing the degradation of ΙκΒα. Furthermore, in LPS-induced RAW264.7 macrophages, AP inhibited the expression of TLR4 protein, which in turn inhibited the activity of the NF-κB pathway. The findings demonstrated that AP exhibits anti-inflammatory properties in vitro by targeting the TLR4/NF-κB signaling pathway, thus impeding the nuclear translocation of NF-κBp65, suppressing the expression of related pro-inflammatory factors.