硫化氢对氧糖剥夺/复氧诱导小鼠脑皮层神经元损伤的体外观察

doi:10.3969/j.issn.0529-1356.2014.03.004

摘要/Abstract

摘要：

目的探讨硫化氢（H₂S）对氧糖剥夺/复氧（OGD/R）诱导神经元损伤的影响。方法小鼠皮层神经元用无糖厄尔氏平衡盐溶液（EBSS）于2% O₂、5% CO₂、93% N₂ 37℃培养4h，换用neurobasal + B27培养基于37℃ 5%CO₂培养箱中继续培养12h，建立OGD/R模型。以硫氢化钠(NaHS)作为H₂S的供体，采用细胞计数试剂盒(CCK-8)检测细胞存活率；用fura-2/AM和显微荧光成像系统检测神经元胞质钙离子浓度（［Ca ²⁺ ］i）；利用比色法检测乳酸脱氢酶（LDH）释放率；碘化丙啶（PI）染色检测细胞损伤。结果 200、300与600 μmol/L NaHS预处理30min再OGD/R，神经元存活率显著提高（n=4）；300 μmol/L NaHS预处理后再OGD/R，［Ca ²⁺］i（n=5）、LDH释放率（n=4）和细胞损伤率（n=6）均低于OGD/R组，且使用10 μmol/L钙螯合剂BAPTA也降低OGD/R诱导的LDH释放率和细胞损伤率。结论 H₂S减轻OGD/R所致皮层神经元损伤，其机制与H₂S抑制OGD/R诱导神经元钙超载有关。

关键词: 氧糖剥夺/复氧, 硫化氢, 细胞损伤, 钙超载, 显微荧光成像, 小鼠

Abstract:

Objective To explore the effects of H₂S on neuronal injuries induced by oxygen glucose deprivation/reoxygenation (OGD/R) in cortical neurons. Methods For OGD, the primary cultured cortical neurons were incubated with glucose-free EBSS media for 4h in N₂/CO₂/O₂ (93%/5%/2%) atmosphere. Thereafter, the media were replaced by Neurobasal/B27 culture media and the neurons were incubated for 12 h in a 5% CO₂ incubator at 37℃. NaHS was used as a H₂S donor and cell survival rate was determined by cell counting kit 8(CCk-8). ［Ca ²⁺ ]i was determined using fura-2/AM and fluorescence microscopic imaging systems. The release rate of lactate dehydrogenase (LDH) was determined by lactate dehydrogenase assay kit, and cell damage was analyzed by staining of propidium iodide (PI). Results After pretreated with 200, 300 and 600 μmol/L sodium hydrosulfide (NaHS) for 30min before OGD/R, the cell survival rate of neurons significantly increased (n=4). ［Ca ²⁺］I(n=5), LDH release rate (n=4) and cell damage percentage (n=6) in the neuron pretreated with 300 μM NaHS were significantly lower than those in ODG/R cells. Treatment with 10μmol/L calcium chelator BAPTA also reduced the LDH release rate and cell damage percentage induced by ODG/R in neurons. Conclusion The results indicate that H₂S may inhibit the OGD/R induced damage in cortical neurons via reducing calcium overload of neurons.

Key words: Oxygen-glucose deprivation/reoxygenation, Hydrogen sulfide, Cell injury, Calcium overload, Fluorescence microscopic imaging, Mouse

伍吉云魏慈照徐悦青刘路宽张洋萍魏楚蓉刘星罗友根*. 硫化氢对氧糖剥夺/复氧诱导小鼠脑皮层神经元损伤的体外观察[J]. 解剖学报, 2014, 45(3): 316-320.

WU Ji-yun WEI Ci-zhao XU Yue-qing LIU Lu-kuan ZHANG Yang-ping WEI Chu-rong MAO Mu-hua LUO You-gen*. Effects of hydrogen sulfide on mouse cortical neuronal injuries induced by oxygen gloucose deprivation/reoxygenation in vitro[J]. AAS, 2014, 45(3): 316-320.

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