Melezitose inhibited glycolytic pathway and enhances anti-Crohn's disease activity via binding to PGK1

Alhagi honey is a light yellow sugar granule formed by concentrating the liquid secreted by Alhagi branches and leaves. It is a traditional Uygur medicine often used to treat abdominal pain, diarrhea, dysentery, and other conditions. Modern research has indicated that the main active components of Alhagi honey are oligosaccharides and polysaccharides. Our previous research had identified that the extract of Alhagi honey exhibits good anti-inflammatory pharmacological activity, however, its efficacy against Crohn's disease (CD) remains to be elucidated. To determine the efficacy of the extract of Alhagi honey in CD and to explore its potential targets and mechanisms. Mel (melitriose) is extracted from dried Alhagi honey. In vivo, 2.5% 2,4,6-trinitrobenzenesulfonic acid (TNBS, At a dosage of 100 mg/kg) is used as an enema to induce CD-like changes in the rat colon. Over the subsequent fortnight, the modeled rats were treated with Mel via gavage. The histopathological alterations and repair ability of colonic injury in the colon tissue were evaluated using hematoxylin and eosin (H&E), Masson's trichrome, and immunofluorescence staining. Additionally, the amelioration of inflammatory responses in the colon was assessed using enzyme-linked immunosorbent assay (ELISA). The reparative capacity of Mel on inflammation was evaluated by inducing inflammation in RAW264.7 cells with lipopolysaccharide (LPS). The Drug Affinity Responsive Target Stability (DARTS) experiment was used to explore the relevant targets of action. Furthermore, network pharmacology was used to investigate the mechanism of action of Mel, to further validate its effects at the cellular level. In the CD rat model, treatment with Mel significantly improved colonic mucosal damage and inflammatory infiltration. It also demonstrated a reduced collagen fiber deposition, thereby ameliorating fibrotic changes in colonic tissue. Furthermore, Mel decreased the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors in colonic tissue and cell supernatants. Further research confirmed that Mel influences the glycolytic pathway by binding to phosphoglycerate kinase 1 (PGK1) and suppressing its activity, leading to reduced production of adenosine triphosphate (ATP) and its metabolites, 2-phosphoglycerate (2-PG), 3-phosphoglycerate (3-PG); thus, playing a role in anti-inflammation and promotion of repair. This mechanism was further validated using the PGK1 inhibitor NG52, which also demonstrated a reduction in the production of ATP, 2-PG, and 3-PG. This study revealed that Mel exerts its anti-inflammatory and reparative capabilities in vitro and in vivo by inhibiting the activity of the key glycolytic enzyme PGK1, leading to reduced production of ATP and its products 2-PG and 3-PG, thereby ameliorating the symptoms of CD. It can emerge as a promising candidate for CD treatment.