硫化氢通过抑制iNOS-NO通路对抗化学性缺氧引起的PC12细胞损伤

【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24h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30min，应用400µmol/LNaHS（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60min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60min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。

Hydrogen Sulfide Protects PC12 Cells against Chemical Hypoxia-induced Injury by Inhibiting iNOS-NO Pathway

【Objective】 To investigate whether hydrogen sulfide (H2S) protected PC12 cells against chemical hypoxia-induced injury by inhibiting iNOS-NO pathway.【Method】 PC12 cells were treated with cobalt chloride (CoCl2) 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; Apoptotic rate was evaluated by propidium iodide staining and flow cytometry (FCM); Nitrite accumulation, an indicator of nitrogen monoxidum (NO) production, was measured in cell culture 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 µmol/L CoCl2 for 24h significantly enhanced iNOS expression. Pretreatment with 400 µmol/LNaHS(a donor of H2S) for 30 min prior to exposure of PC12 cells to 600 µmol/L CoCl2 not only inhibited CoCl2-induced increase in expression of iNOS and NO production, but also protected PC12 cells against injuries induced by 600 µmol/L CoCl2, enhancing cell viability and decreasing amount of apoptotic cells. Similarly, pretreatment with L-Canavanine (10 µmol/L), an inhibitor of iNOS for 60 min prior to exposure of PC12 cells to CoCl2 also conferred the same cytoprotective effect of H2S. In addition, pretreatment with SB203580, an inhibitor of p38MAPK, for 60 min prior exposure of PC12 cells to 600 µmol/L CoCl2 could also down-regulate the expression of iNOS induced by CoCl2. 【Conclussions】The iNOS-NO pathway mediates CoCl2-induced injury and H2S can protect PC12 cells against chemical hypoxia-induced injury by inhibiting iNOS-NO pathway.