[The role of the HIF-1 signaling pathway in retinal ganglion cell injury caused by the OPTN (E50K) mutation].

Objective: To explore the role and mechanism of the hypoxia-inducible factor-1 (HIF-1) pathway in rat retinal precursor R28 cell injury caused by the OPTN (E50K) mutation. Methods: This experimental study was conducted from November 2023 to October 2024. The retinas of 18-month-old wild-type (WT) mice and normal tension glaucoma mice with the OPTN (E50K) mutation were extracted for proteomic analysis. In vitro models carrying the genes of green fluorescent protein (GFP), GFP-OPTN (WT) and GFP-OPTN (E50K) were constructed by transfecting R28 cells with adeno-associated viruses. Western blotting and PCR were used to detect the protein and mRNA expressions of apoptotic index B-lymphocytoma-2-associated X protein (BAX), HIF-1α, and energy metabolism enzymes including glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). R28 cells were treated with the HIF-1α inhibitor Lificiguat at concentrations of 0, 25, 50, 75 and 100 μmol/L for 12, 24 and 48 hours, respectively. According to the cell counting kit-8 detection results, 75 μmol/L Lificiguat was selected to treat the cells for 24 hours. The protein and mRNA expression changes of HIF-1α, GLUT1, LDHA and BAX were detected after the addition of Lificiguat. The statistical analysis was performed using the one-way analysis of variance and Tukey test. Results: There were 1 564 differentially expressed proteins in the retinas of mice in the experimental group compared with those in the control group, and these were significantly enriched in the HIF-1 signaling pathway. The protein expression levels of BAX, HIF-1α, GLUT1 and LDHA in OPTN (E50K) mutant R28 cells were 1.28±0.15, 1.54±0.21, 1.28±0.15 and 1.20±0.16, respectively. They were significantly increased compared with those in R28 cells transfected with the GFP-OPTN (WT) gene (0.96±0.04, 0.87±0.07, 0.90±0.10, 0.87±0.02; all P<0.05). The mRNA expression levels of BAX, HIF-1α, GLUT1 and LDHA in OPTN (E50K) mutant R28 cells were 1.20±0.06, 2.89±0.21, 2.37±0.22 and 1.27±0.22, respectively. They were also significantly increased compared with those in R28 cells transfected with the GFP-OPTN (WT) gene (0.87±0.14, 0.88±0.23, 1.24±0.18, 0.94±0.07; all P<0.05). The protein expression levels of HIF-1α, LDHA and BAX in OPTN (E50K) mutant R28 cells treated with Lificiguat were 0.62±0.11, 0.65±0.15 and 0.76±0.03, respectively. They were significantly decreased compared with those in OPTN (E50K) mutant R28 cells untreated with Lificiguat (0.88±0.04, 0.92±0.04, 1.08±0.07; all P<0.05). The mRNA expression levels of HIF-1α, LDHA and BAX in OPTN (E50K) mutant R28 cells treated with Lificiguat were 1.06±0.06, 1.11±0.14 and 1.01±0.01, respectively. They were also significantly decreased compared with those in OPTN (E50K) mutant R28 cells untreated with Lificiguat (1.34±0.09, 1.45±0.14, 1.12±0.00; all P<0.05). The protein expression levels of GLUT1 in OPTN (E50K) mutant R28 cells without and with Lificiguat were 0.99±0.10 and 1.09±0.01, while the mRNA expression levels were 1.23±0.06 and 1.31±0.13, respectively. The expression of the GLUT1 protein and mRNA in OPTN (E50K) mutant R28 cells slightly increased after treatment with Lificiguat, and there was no statistically significant difference (all P>0.05). Conclusions: The OPTN (E50K) mutation affects the expression of energy metabolism enzymes GLUT1 and LDHA in R28 cells by regulating the HIF-1 pathway, inducing apoptosis of R28 cells. Lificiguat can effectively inhibit the overexpression of HIF-1α caused by the OPTN (E50K) mutation and protect R28 cells.