Western blot analysis of high-mobility group box-1 (HMGB1), extracellular signal-regulated kinase 1 and 2 (ERK1/2), cleaved caspase-3, synaptophysin, tyrosine hydroxylase, glutamine synthetase, and glyoxalase 1 in rat retinas. β-Actin was used as a housekeeping control. Protein expression of ERK1/2 activation (phosphorylation) was quantified by Western blot analysis using phospho- (P-) ERK1/2 specific antibody and was adjusted to the protein levels of unphosphorylated ERK1/2 antibody in each sample. There is a significant increase in the expression of HMGB1 (a), activated ERK1/2 (b), and cleaved caspase-3 (c) and a significant decrease in the expression of synaptophysin (d), tyrosine hydroxylase (e), glutamine synthetase (f), and glyoxalase 1 (g) in the retinas of diabetic rats compared to nondiabetic controls. Glycyrrhizic acid (GA) significantly attenuated diabetes-induced upregulation of HMGB1 (a), activated ERK1/2 (b), and cleaved caspase-3 (c) and diabetes-induced downregulation of synaptophysin (d), tyrosine hydroxylase (e), GS (f), and glyoxalase 1 (g). Glutamate assay revealed a significant increase in glutamate levels in the retinas of diabetic rats compared to nondiabetic controls. The levels of glutamate in the GA-treated diabetic rats were significantly less than those in the untreated diabetic rats (h). Each experiment was repeated at least 3 times with fresh samples. A representative set of samples is shown. Results are expressed as mean ± SD of at least 6 rats in each group. *P < 0.05 compared with nondiabetic control rats. ^# P < 0.05 compared with diabetic rats.

Western blot analysis of rat retinas. Intravitreal administration of high-mobility group box-1 (HMGB1) induced a significant upregulation of the expression of HMGB1 (a), activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) (b), and cleaved caspase-3 (c) and a significant downregulation of the expression of synaptophysin (d), tyrosine hydroxylase (e), glutamine synthetase (f), and glyoxalase 1 (g) compared to intravitreal administration of phosphate buffer saline (PBS). Glutamate assay revealed that injection of HMGB1 tended to increase retinal glutamate expression, but this was not significant (h). Each experiment was repeated at least 3 times with fresh samples. A representative set of samples is shown. Results are expressed as mean ± SD of at least 6 rats in each group. *P < 0.05 compared with PBS.

Gene expression of high-mobility group box-1 (HMGB1) in the retinas was quantified by RT-PCR using a primer given in the Materials and Methods section and was adjusted to the mRNA level of β-actin in each sample. Each measurement was performed at least 3 times. Results are expressed as mean ± SD of at least 6 rats in each group. GA: glycyrrhizic acid; PBS: phosphate buffer saline *P < 0.05 compared with nondiabetic controlled rats. **P < 0.05 compared with PBS. ^# P < 0.05 compared with diabetic rats.