Protocatechualdehyde attenuates oxidative stress in diabetic cataract via GLO1-mediated inhibition of AGE/RAGE glycosylation

Abstract

Background: Protocatechualdehyde (PCA), a phenolic compound derived from Salvia miltiorrhiza, exhibits anti-proliferative and antioxidant properties. However, its molecular mechanisms in reducing oxidative stress in diabetic cataract (DC) remain unclear. This study systematically investigated the role of PCA in modulating glyoxalase-1 (GLO1)-dependent suppression of advanced glycation end product (AGE)-receptor for AGE (RAGE) axis activation and oxidative stress in DC models.

Methods: A galactose-induced DC rat model and high glucose-stimulated human lens epithelial cells (HLECs) were employed. Lens opacity was assessed using slit-lamp microscopy. GLO1, AGE, and RAGE expressions were analyzed through immunohistochemistry (IHC), immunofluorescence (IF), ELISA, and Western blotting. Molecular docking was performed to validate PCA-GLO1 interactions.

Results: PCA administration (25 mg/kg) significantly alleviated lens opacity and epithelial cell disorganization in DC rats (p < 0.01). In vitro, PCA (10 μM) restored HLEC viability under hyperglycemic conditions (p < 0.05). Mechanistically, PCA upregulated GLO1 expression while suppressing AGE accumulation and RAGE activation in both models. Molecular docking revealed strong binding affinities between PCA and GLO1 (-CDOCKER energy: 26.41 kcal/mol).

Conclusion: PCA ameliorates DC progression by enhancing the GLO1-mediated detoxification of AGE precursors, thereby inhibiting AGE/RAGE-driven oxidative stress. These findings provide a foundation for PCA as a therapeutic candidate for DC.

Keywords: AGEs/RAGE; Glo1; Protocatechualdehyde; diabetic cataract; oxidative stress.

FIGURE 1

Effect of PCA (25 mg/kg) on body weight of diabetic cataract rat.

FIGURE 2

Effect of PCA (25 mg/kg) on lens opacity of diabetic cataract rats. (A) At the end of the experiment, the lens of the rat was placed in water, and its opacity was measured by ordinary microscope. (B) On days 3 and 26 of the experiment, the opacity of the rat lens was measured by slit-lamp microscopy. (C) At the end of the experiment, rat lens opacity was assessed according to the scoring criteria.

FIGURE 3

Effect of PCA (25 mg/kg) on the pathological changes of the lens epithelium in diabetic cataract rats by HE staining (original magnification ×20).

FIGURE 4

Effect of PCA (25 mg/kg) on the expression of GLO1 in diabetic cataract rats. (A) Expression of GLO1 in rat lens tissue by IHC staining (original magnification ×40). (B) Expression of GLO1 in rat lens tissue by IF staining (original magnification ×73). (C) Expression of GLO1 in rat lens tissue by ELISA. (D) Expression of GLO1 in rat lens tissue by WB. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 5

Effect of PCA (25 mg/kg) on the expression of AGEs in diabetic cataract rats. (A) Expression of AGEs in rat lens tissue by IHC staining (original magnification ×40). (B) Expression of AGEs in rat lens tissue by IF staining (original magnification ×73). (C) Expression of AGEs in rat lens tissue by WB. Data were represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 6

Effect of PCA (25 mg/kg) on expression of RAGE in diabetic cataract rats. (A) Expression of RAGE in rat lens tissue by IHC staining (original magnification ×40). (B) Expression of RAGE in rat lens tissue by IF staining (original magnification ×73). (C) Expression of RAGE in rat lens tissue by WB. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 7

Effect of PCA (25 mg/kg) on oxidative stress response in diabetic cataract rats. (A) Level of CAT in rat lens tissue by ELISA. (B) Level of GPX in rat lens tissue by ELISA. (C) Level of SOD in rat lens tissue by ELISA. (D) Level of AOPP in rat lens tissue by ELISA. (E) Level of GSSG in rat lens tissue by ELISA. (F) Level of TBARS in rat lens tissue by ELISA. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 8

Effect of PCA (10 μM) on cell viability in human lens epithelial cells. (A) Cell viability of human lens epithelial cells measured by CCK-8 kit. (B) Morphology of human lens epithelial cells photographed with microscope (original magnification ×40). Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 9

Effect of PCA (10 μM) on expression of GLO1 in human lens epithelial cells. (A) Expression of GLO1 in human lens epithelial cells by IF staining (original magnification ×40). (B) Expression of GLO1 in human lens epithelial cells by WB. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 10

Effect of PCA (10 μM) on expression of AGEs in human lens epithelial cells. (A) Expression of AGEs in human lens epithelial cells by IF staining (original magnification ×40). (B) Expression of AGEs in human lens epithelial cells by WB. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 11

Effect of PCA (10 μM) on expression of RAGE in human lens epithelial cells. (A) Expression of RAGE in human lens epithelial cells by IF staining (original magnification ×40). (B) Expression of RAGE in human lens epithelial cells by WB. Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group, and ** for p < 0.01 between the PCA group and model group.

FIGURE 12

Effect of PCA (10 μM) on oxidative stress response in human lens epithelial cells. (A) Expression of 3-NT in human lens epithelial cells by IF staining (original magnification ×40). (B, C) Level of ROS in human lens epithelial cells by ROS staining (original magnification ×40). Data represented as mean ± SD. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. model group. ^# for p < 0.05 between the model group and control group, ^## for p < 0.01 between the model group and control group, * for p < 0.05 between the PCA group and model group and, ** for p < 0.01 between the PCA group and model group.

FIGURE 13

Docking patterns of PCA interacting with 2ZA0 (A), 3VW9 (B) and 7WT0 (C) as produced using CDOCKER.

FIGURE 14

Results of molecular dynamics simulation of PCA and GLO1. (A) Result of RMSD between PCA and GLO1. (B) Result of RMSF between PCA and GLO1. (C) Result of Rg between PCA and GLO1. (D) Result of SASA between PCA and GLO1.