Asiaticoside attenuates endothelial cells senescence in diabetic retinopathy via CCND1-mediated mitophagy through the PI3K/AKT/mTOR pathway

Diabetic retinopathy (DR), a predominant microvascular complication of diabetes mellitus, is pathologically characterized by endothelial dysfunction and blood-retinal barrier (BRB) disruption. Asiaticoside (AC), a bioactive phytochemical constituent derived from traditional Chinese medicinal herbs, has shown promising therapeutic potential against a spectrum of diabetic complications. Nevertheless, the explicit molecular targets and the detailed pharmacological mechanisms underlying the protective effects of this herbal ingredient against DR have not yet been fully elucidated.

This study aimed to investigate the therapeutic effects and underlying mechanisms of AC in attenuating the progression of DR by targeting Cyclin D1 (CCND1) to negatively regulate the PI3K-AKT-mTOR pathway and activating mitophagy, thereby providing a promising therapeutic strategy for DR treatment.

C57BL/6J mice were utilized to establish diabetic mouse model via intraperitoneal injection of streptozotocin (STZ) then treated with AC. Human microvascular endothelial cells (HMECs) were exposed to high glucose (HG; 35 mM) to construct high-glucose model and treated with AC, mitophagy inhibitor 3-MA, mitophagy agonist CCCP and PI3K agonist 740-YP. Genetic knockdown via siRNA was performed to validate CCND1 effect on cellular senescence and mitophagy. The senescence level of retina, mice visual contrast sensitivity and retinal vascular leakage were evaluated by western blotting, immunofluorescence, OptoDrum eyes tracker system and evans blue staining respectively. Cell viability, cellular senescence, cell migratory capacity, apoptosis, mitophagy, mitochondrial morphology, mitochondrial function, and ultrastructural changes were assessed by CCK8 assays, wound-healing assay, western blotting, immunofluorescence, and transmission electron microscopy (TEM) respectively.

AC treatment significantly lowered the visual contrast threshold by approximately 18% and reduced retinal vascular leakage in diabetic mice. In vitro, AC ameliorated high glucose-induced endothelial cell senescence, attenuated cellular and mitochondrial dysfunction as well as reactive oxygen species (ROS) production, and induced mitophagy. Bioinformatics analysis identified CCND1 as one of the key senescence-related genes upregulated in DR, AC intervention significantly inhibited its overexpression. Furthermore, knockdown of CCND1 recapitulated the protective effects of AC, enhancing mitophagy and suppressing senescence phenotypes. Mechanistically, AC treatment or CCND1 knockdown inhibited high glucose-induced activation of the PI3K-AKT-mTOR signaling pathway, while the PI3K agonist 740 YP completely reversed these beneficial effects of AC.

We firstly revealed that AC attenuates DR progression by alleviating cellular senescence in HMECs by modulating CCND1. Notably, the present study is the first to explore the pathological role of CCND1 in DR beyond its classical cell-cycle regulatory function, which provides a novel and promising therapeutic target and intervention strategy for DR treatment.