Molecular insight into the role of isoflavones targeting estrogen β receptor in diabetic wound healing pathway: A computational and in-vitro studies

This study aims to investigate the modulation of ER-beta by phytoestrogens (isoflavones) in target diabetic wound healing using computational tools and in vitro studies. One of the primary limitations associated with delayed wound healing is inhibiting antioxidant pathways mediated by estrogen receptor-beta (ER-beta). In this study, we screened 50 isoflavones against the active pocket of ER-beta (5TOA.pdb). Through the XP docking score, we evaluated the binding pattern and dynamic changes of the top 5 isoflavones having the highest docking scores, binding energy, and interactions with ER-beta. Further, in vitro, MTT assay was performed on the L929 cell line, followed by scratch assay, DPPH assay and glucose uptake assay. The XP (Extra precision) docking score of the top 5 isoflavones were S-Equol -9.90, Genistein -9.71, Icaritin (ICT) -9.92, Biochanin A -8.97, and Formononetin -8.87 respectively when compared with standard estradiol -10.61 when docked with ER-beta. The binding free energy values (Delta G bind) of the top 5 isoflavones were S-Equol (-27.15 kcal/mol), Genistein (-41.49 kcal/mol), ICT (-46.56 kcal/mol) Biochanin A (-37.01 kcal/Mol) and Formononetin (-42.96 kcal/ Mol) respectively when compared with standard estradiol (-97.15 kcal/mol). Based on the XP glide score and free binding energy calculations, ICT was subjected to 100 ns MD simulation, confirming the stability of ICT with ER-beta. The in-vitro results showed no cytotoxicity found up to 125 mu M concentration, revealing normal proliferation of fibroblasts reduced total ROS production and prevented cell glucose uptake. These results highlighted the interaction potential of ICT for targeting ER-beta, antioxidant, and anti-diabetic activity for diabetic wound healing.