Deciphering the therapeutic mechanism of kaempferol in diabetic retinopathy via the P21/Thioredoxin axis

Diabetic retinopathy (DR) is an irreversible microvascular complication in individuals with diabetes. Kaempferol, a flavonoid with anti-inflammatory, antioxidant, and hypoglycemic activities, has exhibited therapeutic potential in previous investigations for treating DR. However, its accurate molecular mechanisms remain elusive. This study aimed to elucidate similarity underlying the progression of DR from early to late stages, along with exploring the key targets of kaempferol for DR therapy. Combined with weighted gene co-expression network analysis (WGCNA) and single-cell RNA sequencing (scRNA-seq) analysis, we elucidated hub regulatory genes and cell subpopulations. Molecular docking was conducted to analyze molecular interactions. Evans Blue (EB) leakage assay, Hematoxylin & Eosin (H&E) and Periodic Acid-Schiff (PAS) staining was utilized to assess retinal structural and vascular damage. Additionally, TUNEL staining was applied to evaluate retinal apoptosis. Comprehensive analyses, including enzyme-linked immunosorbent assays (ELISA), immunofluorescence, Western blotting, and real-time PCR were employed to monitor cytokine levels and protein expression. Our findings preliminarily unveiled that kaempferol could modulate the P21/Thioredoxin pathway, and exerted protective effects on DR by regulating metabolism disorder and cellular dysregulation. Moreover, a novel mechanistic connection was established between fibroblasts activity and DR fibrosis progression, underscoring the pivotal role of the VCAM signaling pathway in vascular cell regulation and its contribution to disease pathogenesis. This study provides new perspectives on the therapeutic potential of kaempferol in DR, particularly regulating vascular injury and cellular senescence via the P21/Thioredoxin axis, which expand the horizon of natural compounds in addressing the vision-threatening complications associated with diabetes.