Mechanism of XiJiaQi in the treatment of chronic heart failure: Integrated analysis by pharmacoinformatics, molecular dynamics simulation, and SPR validation.

OBJECTIVE: Chronic heart failure (CHF) is a complicated clinical syndrome with a high mortality rate. XiJiaQi (XJQ) is a traditional Chinese medicine used in the clinical treatment of CHF, but its bioactive components and their modes of action remain unknown. This study was designed to unravel the molecular mechanism of XJQ in the treatment of CHF using multiple computer-assisted and experimental methods. METHODS: Pharmacoinformatics-based methods were used to explore the active components and targets of XJQ in the treatment of CHF. ADMETlab was then utilized to evaluate the pharmacokinetic and toxicological properties of core components. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were to explore the underlying mechanism of XJQ treatment. Molecular docking, surface plasmon resonance (SPR), and molecular dynamics (MD) were employed to evaluate the binding of active components to putative targets. RESULTS: Astragaloside IV, formononetin, kirenol, darutoside, periplocin and periplocymarin were identified as core XJQ-related components, and IL6 and STAT3 were identified as core XJQ targets. ADME/T results indicated that periplocin and periplocymarin may have potential toxicity. GO and KEGG pathway analyses revealed that XJQ mainly intervenes in inflammation, apoptosis, diabetes, and atherosclerosis-related biological pathways. Molecular docking and SPR revealed that formononetin had a high affinity with IL6 and STAT3. Furthermore, MD simulation confirmed that formononetin could firmly bind to the site 2 region of IL6 and the DNA binding domain of STAT3. CONCLUSION: This study provides a mechanistic rationale for the clinical application of XJQ. Modulation of STAT3 and IL-6 by XJQ can impact CHF, further guiding research efforts into the molecular underpinnings of CHF.

Macromolecular assemblies of complex polysaccharides with galectin-3 and their synergistic effects on function.

Although pectin-derived polysaccharides can antagonize galectin function in various pathological disorders, the nature of their binding interactions needs to be better defined for developing them as drugs. Moreover, given their relatively large size and complexity, pectin-derived polysaccharides are also useful as model systems to assess inter-polysaccharide and protein-polysaccharide interactions. Here, we investigated interactions between galectin-3 (Gal-3) and pectin-derived polysaccharides: a rhamnogalacturonan (RG) and two homogalacturonans (HGs). BioLayer Interferometry and fluorescence-linked immunosorbent assays indicate that these polysaccharides bind Gal-3 with macroscopic or apparent KD values of 49 nM, 46 µM, and 138 µM, respectively. 15N-1H heteronuclear single quantum coherence (HSQC) NMR studies reveal that these polysaccharides interact primarily with the F-face of the Gal-3 carbohydrate recognition domain. Even though their binding to Gal-3 does not inhibit Gal-3-mediated T-cell apoptosis and only weakly attenuates hemagglutination, their combination in specific proportions increases activity synergistically along with avidity for Gal-3. This suggests that RG and HG polysaccharides act in concert, a proposal supported by polysaccharide particle size measurements and 13C-1H HSQC data. Our model has HG interacting with RG to promote increased avidity of RG for Gal-3, likely by exposing additional lectin-binding sites on the RG. Overall, the present study contributes to our understanding of how complex HG and RG polysaccharides interact with Gal-3.

Multiple approaches to assess pectin binding to galectin-3.

Although several approaches have been used to evaluate binding of carbohydrates to lectins, results are not always comparable, especially with larger polysaccharides. Here, we quantitatively assessed and compared binding of pectin-derived polysaccharides to galectin-3 (Gal-3) using five methods: surface plasmon resonance (SPR), bio-layer interferometry (BLI), fluorescence polarization (FP), competitive fluorescence-linked immunosorbance (cFLISA), and the well-known cell-based hemagglutination assay (G3H). Our studies revealed that whereas Gal-3-pectin binding parameters determined by SPR and BLI were comparable and correlated with inhibitory potencies from the G3H assay, results using FP and cFLISA assays were highly variable and depended greatly on the probe and mass of the polysaccharide. In the cFLISA assay, for example, pectins showed no inhibition when using the DTAF-labeled asialofetuin probe, but did when using a DTAF-labeled pectin probe. And the FP approach with the DTAF-lactose probe did not work on polysaccharides and large galactan chains, although it did work well with smaller galactans. Nevertheless, even though results derived from all of these methods are in general agreement, derived KD, IC50, and MIC values do differ. Our results reflect the variability using various techniques and therefore will be useful to investigators who are developing pectin-derived Gal-3 antagonists as anti-cancer agents.