Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway

**Background:** Diabetic retinopathy (DR) is a common microvascular complication of diabetes and a leading cause of blindness. Advanced glycation end products (AGEs) formed under hyperglycemia contribute to endothelial dysfunction and retinal damage. Genipin, an active compound from Gardenia jasminoides Ellis fruit, has anti-inflammatory and antioxidant properties but its role in DR via AGEs inhibition is unclear. This study aimed to investigate genipin's effects on human retinal microvascular endothelial cells (hRMECs) and in a mouse model of DR, focusing on the CHGA/UCP2/GLUT1 signaling pathway.

**Methods:** In vitro, hRMECs were cultured in high glucose (30 mmol/L D-glucose) with or without genipin (0.4 μmol/L) for 7 days. Cell viability was assessed by CCK-8 and colony formation assays. Apoptosis was measured by flow cytometry. Angiogenesis was evaluated by wound healing, transwell migration, and tube formation assays. Energy metabolism was assessed by ATP levels, mitochondrial membrane potential (MMP), and glucose uptake (2-NBDG). Oxidative stress was measured by ROS levels. Inflammatory cytokines (TNF-α, IL-1β, IL-18, NLRP3) and DR biomarkers (VEGF, SCG3) were quantified by Western blot and immunofluorescence. In vivo, STZ-induced diabetic C57/BL6 mice received intraocular injection (IOI) of genipin (10 mmol/L, 0.4 μL/eye) in the right eye, while the left eye received PBS. Retinal structure was examined by H&E staining. ROS, glucose uptake, and protein expression of CHGA, UCP2, GLUT1, RAGE, and inflammatory markers were measured.

**Key Results:** In vitro, high glucose reduced cell viability by approximately 30% and increased apoptosis (from 30.30% to 61.05%). Genipin (0.4 μmol/L) significantly improved cell viability and reduced early apoptosis from 33.98% to 11.07%. High glucose increased ATP levels by 60%, and genipin reduced them by approximately 20%. ROS, MMP, and glucose uptake were elevated under high glucose and reduced by genipin. High glucose upregulated TNF-α, IL-1β, IL-18, NLRP3, VEGF, and SCG3; genipin downregulated these proteins. In vivo, genipin-treated eyes had reduced retinal thickness (190.70 ± 58.32 μm) compared to untreated eyes (262.10 ± 55.52 μm, P = 0.045). Genipin decreased ROS, glucose uptake, and expression of inflammatory cytokines and CHGA, UCP2, GLUT1, and RAGE in the retina. SCG3 showed higher sensitivity than VEGF as a DR biomarker.

**Clinical Implications:** This study demonstrates that genipin protects retinal endothelial cells from AGEs-induced damage by modulating the CHGA/UCP2/GLUT1 pathway, reducing oxidative stress, inflammation, and abnormal energy metabolism. Intraocular injection of genipin may offer a novel therapeutic strategy for preventing severe retinopathy and vision loss in diabetic patients. The identification of SCG3 as a sensitive biomarker could improve early diagnosis of DR.