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目的探讨谷氨酸转运体-1(GLT-1)在硫化氢(H2S)保护PC12细胞对抗化学性低氧损伤中的作用。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞建立化学性低氧损伤模型作为研究对象。实验分为6组,正常对照组:未经任何药物处理的PC12细胞;CoCl2处理组:PC12细胞用600μmol/L的CoCl2处理24h或48h;H2S单独处理组;H2S预处理组:400μmol/L NaHS(H2S的供体)提前30min作用PC12细胞,然后与CoCl2一起再作用24h或48h;DHK(一种GLT-1抑制剂)单独处理组;DHK阻断组:在NaHS预处理前30min,给以400μmol/L DHK,然后按H2S预处理组继续处理。应用蛋白免疫印迹法(Western bolt)检测GLT-1蛋白表达;CCK-8比色法测定细胞存活率;Hoechst33258核染色法检测细胞凋亡的形态学改变及数量改变;罗丹明123(Rh123)染色及荧光显微镜照相测定线粒体膜电位(MMP)。结果 600μmol/L CoCl2处理PC12细胞24h可使GLT-1表达明显减少;在CoCl2处理PC12细胞前应用400μmol/L NaHS预处理30min能明显地阻断CoCl2对GLT-1表达的抑制作用;400μmol/L的GLT-1抑制剂DHK能阻断H2S保护PC12细胞对抗CoCl2诱导的损伤作用,使细胞存活率降低,凋亡细胞数量及MMP丢失增多。结论上调GLT-1表达可能是H2S保护PC12细胞对抗CoCl2损伤的作用机制之一。
Objective To investigate the role of glutamate transporter-1 (GLT-1) in the protection of PC12 cells against chemical hypoxia by hydrogen sulfide (H2S). Methods The chemical hypoxia model was established by treating the PC12 cells with cobalt chloride (CoCl 2), a chemical hypoxia mimic agent. The experiment was divided into 6 groups, normal control group: PC12 cells without any drug treatment; CoCl2 treatment group: PC12 cells treated with 600μmol / L CoCl2 for 24h or 48h; H2S alone treatment group; H2S pretreatment group: 400μmol / L NaHS (Donor of H2S) pretreated with PC12 cells 30min before acting with CoCl2 for 24h or 48h; DHK alone (a GLT-1 inhibitor); DHK blocking group: 30min before NaHS pretreatment 400μmol / L DHK, then treated by H2S pretreatment group. The protein expression of GLT-1 was detected by Western blot, the cell viability was determined by CCK-8 colorimetric assay, the morphological changes and number changes of apoptotic cells were detected by Hoechst33258 nuclear staining, the staining of Rh123 And mitochondrial membrane potential (MMP) were measured by fluorescence microscopy. Results The treatment of PC12 cells with 600 μmol / L CoCl2 for 24 h resulted in a significant decrease of GLT-1 expression. Pretreatment with 400 μmol / L NaHS for 30 min before CoCl2 treatment of PC12 cells significantly blocked the inhibition of GLT-1 expression by 400 μmol / L DHT, a GLT-1 inhibitor, can block the protective effect of H2S against CoCl2-induced injury, which leads to the decrease of cell viability and the increase of apoptotic cells and MMPs. Conclusion Upregulation of GLT-1 expression may be one of the mechanisms by which H2S protects PC12 cells against CoCl2 injury.