Autophagy-Mediated Inhibition of Apoptosis Underlies the Ameliorative Effect of  Citri Reticulatae  Semen on Diabetic Retinopathy

This study aimed to investigate the protective effects of the active fraction of Citri Reticulatae Semen (CRSE4#) in DR models and to elucidate its underlying molecular mechanisms, with a focus on autophagy‑mediated regulation of apoptosis and BRB function.Methods: In vitro, DR models were established in high glucose–treated HRECs and MIO-M1 cells in the presence of autophagy inhibitors to assess the effects of CRSE4# on autophagy, apoptosis, and BRB function. In vivo, db/db mice were orally administered CRSE4# for 10 consecutive weeks. Electroretinography was performed, along with morphological and molecular analyses, to assess the retinal protective effects of CRSE4#. Bioactive components were identified by serum LC–MS/MS. RNA sequencing and Gene Set Enrichment Analysis were performed, and pharmacological inhibitors were used to validate the mechanisms underlying the effects of CRSE4#.Results: In vitro, CRSE4# restored autophagic flux, reduced REC apoptosis, and improved BRB function. In db/db mice, CRSE4# improved visual function and retinal structure, restored autophagy, suppressed REC apoptosis, and preserved BRB integrity. RNA sequencing and pathway analyses indicated that CRSE4# exerts anti-DR effects by regulating autophagy via the IRS1/PI3K/AKT signaling pathway. Serum pharmacochemical analysis identified 33 absorbed components, including 10 high‑confidence compounds: kaempferol, ardisiaquinone E, 26-deoxyactein, cimifugin, arabinose, cimigenoside, nicotinic acid, madecassic acid, 5′-O-β-D-glucosylpyridoxine, and hesperidin. Conclusion: CRSE4# exerts protective effects against DR by restoring autophagic activity via the IRS1/PI3K/AKT pathway, thereby inhibiting REC apoptosis and preserving BRB function. Serum pharmacochemical analysis further identified 10 candidate compounds that may contribute to its biological activity. Collectively, these findings underline the pharmacological effects of CRSE4# in regulating autophagy, inhibiting apoptosis, and protecting the BRB, highlighting its potential as a natural protective agent that warrants further preclinical investigation.