Abstract: Objective To investigate the role and mechanism of the γ-aminobutyric acid-ergic(GABAergic)system in human retinal pigment epithelial(RPE)cells under hypoxic conditions, and to provide a theoretical basis for the prevention and treatment of hypoxia-related retinal diseases. Methods Human RPE cells were isolated and cultured. The expression of GABA-related molecules-glutamic acid decarboxylase 65/67(GAD65/67), GABA transaminase(GABA-T), vesicular GABA transporter(VGAT), and GABA transporters 1/2(GAT-1/2)-was detected by RT-PCR and Western blotting. Immunofluorescence was employed to examine the expression and localization of GAD67, GABA-T, and GAT-2 in cultured human RPE cells and human retinal tissue. Cells were exposed to a hypoxic environment(1% O₂, 37 ℃, 48 h), and changes in GAD67, GABA-T, and GAT-2 expression, monolayer permeability, and zonula occludens-1(ZO-1)level were analyzed. RPE cells were divided into four groups: empty vector + normoxia, gene overexpression + normoxia, empty vector + hypoxia, and gene overexpression + hypoxia. Adenoviral vectors were used to overexpress GAD67, GABA-T, and GAT-2, and their protective effects against hypoxia-induced barrier dysfunction in RPE cells were evaluated. Results RT-PCR and Western blotting analyses revealed that human RPE cells expressed GAD67, GABA-T, and GAT-2, whereas GAD65, VGAT, and GAT-1 were undetectable. Compared with the normoxic group, hypoxia significantly downregulated GAD67 ［(17.54±6.89)%, t=2.595, P<0.05］, GABA-T ［(18.64±3.56)%, t=3.563, P<0.01］, and GAT-2 ［(31.79±8.49)%, t=4.147, P<0.01］; increased RPE monolayer permeability ［(78.83±34.79)%, t=-3.222, P<0.05］; and reduced ZO-1 levels ［(22.24±8.31)%, t=3.217, P<0.01］. Compared with the hypoxic empty vector, overexpression of GAD67 or GABA-T alleviated the hypoxia-induced increase in RPE cell permeability by(21.97±11.02)% and(21.40±7.65)%, respectively. The differences were statistically significant(F_GAD67=14.146, F_GABA-T=35.715, P_GAD67<0.05, P_GABA-T<0.01). In contrast to the hypoxic empty vector control group, GAT-2 overexpression did not exhibit a significant protective effect in the absence of exogenous GABA(intergroup P=0.001, P>0.05). However, with the addition of exogenous GABA, GAT-2 overexpression reduced the hypoxia-induced RPE cell permeability by(34.49±14.70)%(F=20.084, P<0.01). None of the overexpressed genes reversed hypoxia-induced reduction of ZO-1 protein. Conclusions Human RPE cells express key components of the GABAergic system. Hypoxia reduces the expression of GABA-related proteins and impairs barrier integrity. Upregulation of GAD67 or GABA-T expression significantly alleviates hypoxia-induced barrier dysfunction in RPE cells. Overexpression of GAT-2 also exhibits a protective effect when supplemented with exogenous GABA. These findings suggest that the GABAergic system may serve as a potential target for improving RPE barrier function in hypoxia-related retinal diseases.

Key words: Retinal pigment epithelial cells, γ-aminobutyric acid, GABAergic system, Hypoxia, Barrier function, Tight junction protein