摘要
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 explore the role of glutamate transporter-1（GLT-1） in the protective effect of hydrogen sulfide（H2S） against chemical hypoxia-induced injury in PC12 cells.Methods PC12 cells were exposed to cobalt chloride（CoCl2） ,so as to establish a chemical hypoxia-induced cellular insult model.PC12 cells were divided into 6 groups,control group,CoCl2-treated group,H2S group,H2S-protected group,DHK（dihydrokainic acid,a GLT-1 inhibitor） group and DHK-prevented group.The expression of GLT-1 was measured by Western bolt assay.Cell viability was detected by cell counter kit（CCK-8）;The changes in morphology and amount of apoptotic cells were observed by Hoechst 33258 staining;Mitochondria membrane potential（MMP） was tested by rhodamine 123（Rh123） staining and photofluorograph.Results Treatment of PC12 cells with 600 μmol/L CoCl2 for 24h significantly reduced expression of GLT-1.Pretreatment of cells with 400 μmol/L NaHS（a donor of H2S） for 30min prior to exposure to CoCl2 markedly blocked the inhibitory effect of CoCl2 on expression of GLT-1.DHK（400 μmol/L） inhibited the protective effect of H2S against CoCl2-induced PC12 cells injuries,resulting in a decrease in cell viability,an increase in apoptotic cells,and a loss of MMP.Conclusions Upregulating the expression of GLT-1 may be one of the mechanisms responsible for protection of H2S against CoCl2-induced injury in PC12 cells.
出处
《中南医学科学杂志》
CAS
2011年第4期380-384,共5页
Medical Science Journal of Central South China
基金
广东省科技计划资助项目资助(2010B080701035
2008B080703053)
关键词
谷氨酸转运体-1
硫化氢
PC12细胞
活性氧
低氧
glutamate transporter-1
hydrogen sulfide
PC12 cells
reactive oxygen species
hypoxia
