Dingxin Recipe III Ameliorates Endothelial Cell Senescence in Atherosclerosis by Activating the FOXO3a/Pink1/Parkin Axis.

Ethnopharmacological Relevance: Dingxin Recipe III (DXRIII) is a traditional Chinese medicinal formulation that has been employed in clinics for over two decades. It is utilized in the treatment of cardiovascular diseases associated with atherosclerosis (AS) through mechanisms purported to involve the clearing of heat and detoxification, as well as the promotion of blood circulation and the removal of blood stasis. Despite its widespread application and reported therapeutic benefits, its exact mechanisms remain incompletely elucidated.

Aim Of This Study: This study aimed to confirm the anti-AS efficacy of DXRIII and elucidate its underlying mechanism, specifically focusing on the amelioration of endothelial senescence.

Methods: The mechanisms underlying the anti-AS effects of DXRIII were elucidated through network pharmacology analysis. In vivo and in vitro models were established using high-fat diet (HFD) induced ApoE mice and HO-induced human umbilical vein endothelial cells (HUVECs), respectively. The histopathologic changes of aortic tissues, inflammatory response, oxidative stress, mitochondrial damage, mitophagy, senescence, and FOXO3a/PINK1/Parkin signaling pathway were evaluated through histological staining, transmission electron microscopy, fluorescent probe staining, RT-qPCR, and Western blot. The constituents of DXRIII were identified via liquid chromatography-mass spectrometry, and the binding affinity and stability of its active compounds with target proteins were investigated using molecular docking and cellular thermal shift assay. Foxo3a was silenced in vitro by lentiviral transfection to determine its involvement in DXRIII-induced mitophagy.

Results: DXRIII effectively ameliorated plaque pathology in AS mice and attenuated endothelial cell senescence. Meanwhile, DXRIII suppressed pro-inflammatory factors and adhesion molecules, mitigated oxidative stress and mitochondrial damage, while activating Pink1/Parkin-mediated mitophagy and upregulating FOXO3a expression. Notably, both mitophagy inhibition Mdivi-1 and silencing of FOXO3a in vitro blocked DXRIII's anti-senescence effects. Furthermore, the active ingredients of DXRIII, including berberine, kaempferol, quercetin and luteolin, showed strong binding affinity with FOXO3a and enhanced its protein stability.

Conclusion: Our findings for the first time demonstrated that DXRIII effectively alleviates endothelial senescence and HFD-induced AS, possibly by activating FOXO3a and subsequently enhancing PINK1/Parkin-mediated mitophagy.