外源性硫化氢对大鼠海马神经元氧糖缺失/恢复损伤的作用  

The effects of exogenous hydrogen sulfide on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration

作　　者：刘蓓蓓[1] 潘昊[1] 陈娣[1] 谢学猛[1] 张锦程[1] 杨光田[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院急诊科,武汉430030

出　　处：《中华急诊医学杂志》2014年第7期770-775,共6页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金（81071526）

摘　　要：目的 研究外源性硫化氢（hydrogen sulfide,H2S）对大鼠海马神经元氧糖缺失/恢复（oxygen-glucose deprivation and recovery,OGD/R）损伤的作用,并探讨其作用机制.方法 孕16～18d胎鼠海马神经元原代培养,培养7d后NSE免疫组化法作神经元鉴定,培养第8天时氧糖缺失1h恢复24 h造成缺氧/复氧损伤,缺氧同时给予硫氢化钠（sodium hydrosulphide,NaHS）干预.将大鼠海马神经元随机（随机数字法）分为3组：正常培养组（Ⅰ组）、OGD/R组（Ⅱ组）、OGD/R＋ NaHS组（Ⅲ组）,Ⅲ组又根据NaHS浓度分为Ⅲ1-5亚组;NaHS浓度分别为25、50、100、200、400 μmol/L.MTT法测定各组细胞活力,比色法检测各组培养液中乳酸脱氢酶（lactatedehydrogenase,LDH）活性,流式法检测各组细胞凋亡,RT-PCR法检测各组细胞HIF-1 αmRNA表达.结果 与正常培养组比较,模型组细胞活力明显降低,培养液中LDH漏出增多,凋亡加重,HIF-1α mRNA表达增高（P＜0.01）;与模型组比较,Ⅲ1组细胞活力、培养液中LDH漏出、凋亡、HIF-1α mRNA表达的差异无统计学意义（P＞0.05）;与模型组比较,Ⅲ2-4组细胞活力明显增高,培养液中LDH漏出减轻,凋亡减轻,HIF-1α mRNA表达增高（P＜0.01）;与模型组比较,Ⅲ5组细胞活力明显降低,培养液中LDH漏出增多,凋亡加重,HIF-1α mRNA表达降低（P＜0.01）.结论 低浓度H2S（25 μmol/L）对海马神经元OGD/R损伤无明显影响;中浓度H2S （50～200μmol/L）可以减轻海马神经元OGD/R损伤,增加细胞活力,减轻细胞凋亡;但高浓度H2S （400μmol/L）则可加重其损伤;海马神经元OGD/R损伤后HIF-1 αmRNA表达增强,适当浓度H2S（50～200 μmol/L）可能通过上调HIF-1α的表达发挥保护作用.Objective To investigate the effects of exogenous hydrogen sulfide （H2S） on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration （OGD/R） and explore its mechanism.Methods Hippocampus neurons were isolated from embryonic day 16-18 （E16-18） rat embryos.Hippocampus was immediately removed and digested with 0.25％ trypsin.The neurons were isolated and cultured at 37 ℃ for 7 days and neuron-specific enolase （NSE） was detected by immunohistochemical staining method to identify neurons.At 8th day,the neurons were placed in deoxygenated glucose-free medium and exposed to 95％ N2-5％ CO2 in an air tight chamber for 1 hour,and then replaced the glucose-free medium with original medium,and returned the cultures to a standard incubator in 5％ CO2 at 37 ℃ and incubated for another 24 h.The neurons were divided into 3 groups：group Ⅰ control; group Ⅱ OGD/R,and group ⅢOGD ＋ NaHS,the latter was further divided into 5 subgroups：groups Ⅲ1-5 with 25,50,100,200,400 μmol/L NaHS added,respectively.Then cell viability was quantified by MTT method,the level of lactate dehydrogenase （LDH） were detected,apoptosis was measured by Annexin V FITC/PI Apoptosis Kits,and RT-PCR was used to assay HIF-1 α mRNA in neurons in all groups.Results Compared with control group,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅱ were significantly increased,the cell viability was significantly decreased （P ＜ 0.01）.There were no significant differences in the LDH level,apoptosis and expression of HIF-1 α mRNA and the cell viability between group Ⅱ and group Ⅲ1 （P ＞ 0.05）.Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅲ2-4 were significantly increased,the cell viability was significantly increased （P ＜ 0.01）.Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1 α mRNA in the group Ⅲ 5 were significantly decreased,the cell viability was significantly decreased （P ＜ 0.01）.Co

关 键 词：硫化氢 氧糖缺失 恢复 海马神经元 凋亡 缺氧诱导因子-1Α 

分 类 号：R741[医药卫生—神经病学与精神病学]