Neferine inhibits vascular smooth muscle cell proliferation and migration in atherosclerosis by targeting CYS130 and PHE252 in PTEN

Introduction The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are significant pathological factors contributing to atherosclerosis (AS). Neferine (Nef) is a dibenzylisoquinoline alkaloid isolated from the Nelumbo nucifera . Our previous studies revealed that Nef has a good pharmacological effect on AS by inhibiting macrophage glycolytic reprogramming. However, the regulatory mechanism of Nef on abnormal VSMC proliferation and migration, specifically the direct target, has not been clarified. Method High-fat diet (HFD) induces AS model and uses different concentrations of Nef treatment, oil red staining, EdU and scratch experiments to evaluate the pharmacological effects of Nef, molecular docking, molecular dynamics simulation, CETSA, DART experiments to elucidate the direct target of Nef, Western Blot, immunofluorescence and immunohistochemistry to detect the effects of Nef on PTEN/AKT pathways and complexes. Result Nef exhibited significant pharmacological effects against AS, markedly inhibited the abnormal proliferation and migration of VSMCs, reduced the expression levels of PCNA and p-AKT, and promoted PTEN expression. Furthermore, competitive blockade of PTEN by Bpv (HOpic) substantially diminished the regulatory effects of Nef on VSMC proliferation and migration. Mechanistically, we identified PTEN as a direct target of Nef and CYS130 and PHE252 were identified as the binding sites of the Nef-PTEN complex. Molecular dynamics simulations, co-immunoprecipitation (Co-IP) experiments demonstrated that Nef targeted PTEN to promote the formation of the PTEN/AKT complex and inhibit AKT phosphorylation. Competitive PTEN inhibition markedly reduced the regulatory effect of Nef on lipid levels and formation. Conclusion This study presents a novel therapeutic drug of Nef for AS by targeting the PTEN-AKT complex and PTEN.