摘要
【目的】探讨硫化氢(H2S)是否通过抑制iNOS-NO通路对抗化学性缺氧诱导的PC12细胞损伤。【方法】应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞建立化学性缺氧损伤模型。应用CCK-8比色法检测细胞存活率;Hoechst33258染色法观察细胞凋亡的形态学改变;PI染色流式细胞仪检测细胞凋亡率;Griess试剂盒检测细胞培养液中的亚硝酸盐(NO的代谢物)的浓度;Western blot法检测iNOS蛋白的表达水平。【结果】应用600μmol/L CoCl2处理PC12细胞24 h可使诱导型一氧化氮合酶(iNOS)表达明显增多;在应用600μmol/L CoCl2处理PC12细胞前30 min,应用400μmol/L NaHS(H2S的供体)预处理细胞不仅可明显地抑制CoCl2诱导的iNOS表达及NO生成的增多,还能保护PC12细胞对抗600μmol/L CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目减少;在CoCl2损伤PC12细胞前60 min应用iNOS抑制剂L-Canavanine(10μmol/L)预处理也能产生类似NaHS的作用。SB203580(p38MAPK特异性抑制剂)预处理60 min也可以下调CoCl2引起的iNOS高表达。【结论】iNOS-NO通路介导CoCl2引起PC12细胞的损伤作用;H2S通过抑制iNOS-NO通路对抗化学性缺氧诱导的PC12细胞损伤。
[Objective] To investigate whether hydrogen sulfide （H2S） protected PC I 2 cells against chemical hypoxia-induced injury by inhibiting iNOS-NO pathway. [Method] PC12 cells were treated with cobalt chloride （COC12） to set up a chemical hypoxia- induced cellular injury model. Cell viability was tested by Cell Counter Kit （CCK-8）; morphological changes of apoptotic cells were detected by Hoechst33258 staining; Apoptotie rate was evaluated by propidium iodide staining and flow cytometry （FCM） ; Nitrite accumulation, an indicator of nitrogen monoxidum （NO） production, was measured in cell cuhure supernatants using the Griess reagent; the expression of the inducible enzyme of NO （iNOS） was determined by Western blot assay. [Results]Exposure of PC12 cells to 600 p^mol/L COC12 for 24 h significantly enhanced iNOS expression. Pretreatment with 400 p.mol/LNaHS （a donor of H2S） for 30 min prior to exposure of PC12 cells to 600 p.m01/L COC12 not only inhibited CoC12-induced increase in expression of iNOS and NO production, but also protected PC12 cells against injuries induced by 600 ~mol/L COC12, enhancing cell viability and decreasing amount of apoptotic cells. Similarly, pretreatment with L-Canavanine （ 10 p, mol/L）, an inhibitor of iNOS for 60 rain prior to exposureof PC12 cells to CoC1= also conferred the same cytoprotective effect of H2S. In addition, pretreatment with SB203580, an inhibitor of p38MAPK, for 60 rain prior exposure of PC12 cells to 600μmol/L COC12 could also down-regulate the expression of iNOS induced by COC12. [Conclussions] The iNOS-NO pathway mediates CoC12-induced injury and H2S can protect PC12 cells against chemical hypoxia- induced injury by inhibiting iNOS-NO pathway.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2011年第6期741-746,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省科技计划项(2010B080701035
2008B080703053)
