Evaluation of Kusuantongtiao Formula as adjunctive therapy for Insulin Resistance: An integrated in silico exploration and experimental validation

Abstract

Abstract Background: Kusuantongtiao Formula (KSTTF) have been shown to be effective in treating insulin resistance (IR) of type 2 diabetes (T2DM), but the mechanism by which KSTTF remains unclear. Objective This study revealed the pharmacological mechanisms of KSTTF acting on IR through network pharmacology, molecular docking, dynamics and vitro experiments. Materials and methods In this study, the ingredients and potential targets of KSTTF were obtained from the TCMSP and HERB databases, and the potential targets of IR were obtained from GeneCard and OMIM database. Cytoscape 3.9.1 constructed network and predicted potential active ingredients and targets. Protein–protein interaction network was constructed using the STRING. AutoDock Vina was used for molecular docking between active ingredients and targets. Subsequently, molecular dynamics simulation was used to analyze the conformational stability of the protein-ligand complexes by GROMACS. Furthermore, the effect and mechanism of KSTTF on insulin resistance were verified by cell viability assay, glucose uptake and western blotting. Results In the formula, we found 172 potential active compounds, such as berberine, baicalin, rhein. A total of 270 intersection targets of KSTTF and IR were obtained, including peroxisome proliferator-activated receptor-gamma (PPARG), glycogen synthase kinase 3β (GSK3B), nitric-oxide synthase (NOS2), and dipeptidyl peptidase 4 (DPP4). Gene ontology and KEGG pathway enrichment revealed that KSTTF could regulate PI3K/Akt signaling pathway. The results of molecular docking showed that GSK3B-1,2,5,6-tetrahydrotanshinone, NOS2-1,2,5,6-tetrahydrotanshinone, PPARG-1,2,5,6-tetrahydrotanshinone, and DPP4-cryptotanshinone had higher binding energy. Molecular dynamics simulation verified the stability of PPARG-1,2,5,6- tetrahydrotanshinone. Binding free energy analysis showed that van der Waals interaction was the main binding form of protein and ligand. Finally, the result from vitro experiments showed that KSTTF improved glucose consumption and increased the phosphorylation levels of IRS1, PI3K, and Akt in palmitic acid-induced C2C12. Conclusions This study predicted the active components and potential targets of KSTTF through network pharmacology, and analyzed its possible mechanism of action in the treatment of insulin resistance. The key targets were validated by molecular docking and molecular dynamics simulation. The vitro experiments confirmed that KSTTF may treat IR by promoting IRS1/PI3K/Akt conduction and glucose uptake consumption. We possess a reason to believe that their mechanisms for insulin resistance are synergy between several different signaling pathways and targets. However, further experiment is crucial to reveal the effect of KSTTF on insulin resistance.