缺氧条件下γ-氨基丁酸能系统对视网膜色素上皮的保护作用

doi:10.6040/j.issn.1671-7554.0.2025.1124

摘要/Abstract

摘要： 目的探讨缺氧条件下γ-氨基丁酸(gama-aminobutyric acid, GABA)能系统在人视网膜色素上皮(retinal pigment epithelium, RPE)细胞中的作用及其机制,为防治缺氧相关视网膜疾病提供理论依据。方法分离并培养人RPE细胞,采用RT-PCR法、Western blotting法检测GABA相关分子［谷氨酸脱羧酶65/67(glutamic acid decarboxylase 65/67, GAD65/67)、γ-氨基丁酸转氨酶(GABA transaminase, GABA-T)、囊泡性GABA转运体(vesicular GABA transporter, VGAT)、GABA转运体1/2(GABA transporters 1/2, GAT-1/2)］的表达。利用免疫荧光技术检测人RPE细胞及人视网膜组织中GAD67、GABA-T和GAT-2的表达与定位。将RPE细胞暴露于缺氧环境中(1% O₂,37 ℃,48 h),检测GAD67、GABA-T和GAT-2表达变化、单层细胞通透性和紧密连接蛋白(zonula occludens-1, ZO-1)水平。将细胞分为常氧空载体对照组、常氧基因过表达组、低氧空载体组以及低氧基因过表达组。通过腺病毒载体分别过表达GAD67、GABA-T或GAT-2,评估其对缺氧诱导的RPE细胞屏障功能损伤的影响。结果人RPE细胞表达GAD67、GABA-T和GAT-2,但未检测到GAD65、VGAT和GAT-1的表达。与常氧对照组相比,低氧组各基因蛋白GAD67［(17.54±6.89)%,t=2.595,P<0.05］、GABA-T［(18.64±3.56)%,t=3.563,P<0.01］和GAT-2［(31.79±8.49)%,t=4.147,P<0.01］表达显著下调;同时单层RPE细胞的通透性增加［(78.83±34.79)%,t=-3.222,P<0.05］, ZO-1水平降低［(22.24±8.31)%,t=3.217,P<0.01］。与低氧空载体对照组相比,低氧过表达GAD67或GABA-T组中升高的RPE细胞通透性减轻了(21.97±11.02)%和(21.40±7.65)%,差异有统计学意义(F_GAD67=14.146, F_GABA-T=35.715, P_GAD67<0.05, P_GABA-T<0.01)。与低氧空载体对照组相比,低氧GAT-2 过表达组在无外源 GABA 添加时未表现出明显保护作用(组间P=0.001, P>0.05,),但在加入外源 GABA 后, GAT-2过表达使得缺氧诱导下的RPE细胞通透性下降了(34.49±14.70)%(F=20.084, P<0.01)。此外,GAD67、GABA-T和GAT-2的过表达均未能逆转缺氧导致的ZO-1蛋白降低。结论人RPE细胞表达GABA能系统。在缺氧条件下,GABA能相关蛋白表达降低并伴随屏障功能损伤。增强GAD67、GABA-T表达可显著改善缺氧诱导的RPE细胞屏障功能异常,GAT-2过表达在补充外源GABA时亦有保护作用,提示GABA能系统可能成为改善缺氧相关视网膜疾病RPE屏障功能的潜在靶点。

关键词: 视网膜色素上皮细胞, γ-氨基丁酸, γ-氨基丁酸能系统, 缺氧, 屏障功能, 紧密连接蛋白

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

中图分类号:

R774.1

曲忠花,迟令懿,马志勇,程振英,王旭平. 缺氧条件下γ-氨基丁酸能系统对视网膜色素上皮的保护作用[J]. 山东大学学报 (医学版), 2025, 63(12): 96-104.

QU Zhonghua, CHI Lingyi, MA Zhiyong, CHENG Zhenying, WANG Xuping. Protective role of GABAergic system in retinal pigment epithelium under hypoxia[J]. Journal of Shandong University (Health Sciences), 2025, 63(12): 96-104.

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