二氮嗪预处理复合低温对大鼠海马神经元缺氧复氧后损伤的减轻作用

Diazoxide preconditioning combined with subsequent hypothermia alleviates anoxia-re-oxygenation injury: experiment with rat hippocampal neurons

目的探讨二氮嗪预处理后复合低温能否进一步减轻大鼠海马神经元缺氧复氧后损伤及其可能机制。方法将离体培养大鼠海马神经元随机分为37、34、30及22℃4个温度组,每温度组又分为二氮嗪0μmol/L和250μmol/L 2个剂量组,共8组;每组36孔细胞或8皿细胞。比较DZ预处理后不同温度中缺氧4 h复氧后24 h各组海马神经元的生存率、丙二醛的含量、细胞内钙离子浓度。结果22、30、34℃低温组250μmol/L二氮嗪时存活率分别为(94±8)%、(73±9)%及(67±8)%,37℃组为(57±6)%,均P<0.01;22℃存活率最高(P<0.01)。22℃组丙二醛浓度、钙离子荧光值均低于其他各组(均P<0.01)。二氮嗪复合低温各组生存率均高于单纯低温组(P<0.01),丙二醛含量、钙离子荧光值又较单纯低温组进一步降低(均P<0.01)。其中22℃与二氮嗪相结合的保护效果最佳(P<0.01)。结论低温可通过抑制脂质过氧化和钙超载而减轻大鼠海马神经元缺氧复氧后损伤,二氮嗪复合低温能够进一步增强以上保护作用,且二氮嗪复合深低温的保护作用最强。

Objective To observe the alleviative effects of preconditioning with diazoxide （ DZ）, a mitochondrial ATP-sensitive potassium channel opener, combined with subsequent hypothermia on anoxia-re- oxygenation injury. Methods Hippocampal neurons of new-born SD rat were cultured. DZ of the concentration of 250 μmol/L was added into the culture fluid. The group of preconditioned cultured neurons were re-divided into 4 subgroups to be treated by hypothermia of different levels： 37℃, 34℃, 30℃, and 22℃, and then were derived of oxygen for 4 hours. The group of neurons without addition of DZ was redivided into 4 subgroup too to be exposed to different temperatures and then underwent anoxia-re-oxygenation as described above. Thus the experiment was performed on 8 subgroups in total. Twenty-four hours after reoxygenation the neurons were stained with dimethyl sulfoxide and their levels of absorptivity were measured so as to calculate the survival rate. Modified thiobarbital method was used to detect the concentration of malondialdehyde （MDA） in the culture fluid. Fluorescence probe Fluo-3-AM was added to detect the Ca^2± concentration in the neurons. Results The survival rates of the DZ-preconditioned neurons of the 22℃, 30℃ , and 34℃ subgroups were （94 ± 8 ） %, （73 ± 9 ） %, （67 ± 8 ） % respectively, all significantly higher than that of the 37℃ subgroup （57 ± 6%, all P 〈 0.01 ）, with significant differences between the 22℃ subgroup and the 30℃ and 34℃ subgroups （ both P 〈 0. 01 ）. The survival rates of the non-DZ- preconditioned neurons of the 22℃, 30℃, and 34℃subgroups were 66 ± 10%, 52 ± 910%, 48 ± 9% respectively, all significantly higher than that of the 37℃ subgroup （41% ± 7%, all P 〈 0.01 ）, with significant differences between the 22℃ subgroup and the 30℃ and 34℃ subgroups （both P 〈 0.01 ）. The survival rates at different temperatures in the DZ-preconditioned group were all significantly higher than those in the corresponding non-preconditioned subgroups （ all P 〈 0.01 ）. The MDA concentration in the culture fluid of the 22℃ subgroup was significantly lower than those of the 30℃ , 34℃, and 37℃ subgroups in both the DZ-preconditioned and non-preconditioned groups （all P 〈 0.01 ）. The MDA concentrations of the preconditioned subgroups at different temperatures were all significantly lower than those of the corresponding non-preconditioned subgroups （all P 〈 0.01 ）. The fluorescent value of the intracellular Ca^2＋ of the 22℃ subgroup was significantly lower than those of the 30℃ , 34℃, and 37 ℃ subgroups in both the DZ- preconditioned and non-preconditioned groups （ all P 〈 0.01 ）. The fluorescent value of the intracellular Ca^2＋ of the preconditioned subgroups at different temperatures were all significantly lower than those of the corresponding non-preconditioned subgroups （ all P 〈 0.01 ）. Conclusion Hypothermia alleviates the anoxia-re-oxygenation injury： DZ preconditioning enhances the neuroprotection by hypothermia.