异丙酚预先给药对缺氧复氧鼠脑神经元的保护作用

Protective effects of propofol pretreatment on rat neurons against anoxic-reoxygenated injury

目的观察异丙酚预先给药对缺氧复氧鼠脑神经元神经细胞活力、一氧化氮(NO)产量、热休克蛋白(Hsp70)和Hsp70 mRNA表达的影响,探讨异丙酚有无脑保护作用及其机制。方法 培养12 d的胎鼠大脑神经元,随机分为四组:Ⅰ组(正常对照组);Ⅱ组(缺氧复氧组);Ⅲ组(14 μmol/L异丙酚预处理组);Ⅳ组(56 μmol/L.异丙酚预处理组)。Ⅲ组和Ⅳ组于缺氧前1 h分别换入含有14μmol/L和56 μmol/L异丙酚的培养液,随后置入95％N2+5％CO2培养箱中缺氧30 min。四组于复氧的1、2、4、6、24 h(分别记为,T1、T2、T4、T6、T24)分别用MTT(改良四甲基偶氮唑盐)细胞酶学分析法和硝酸还原酶法测定神经元神经细胞活力(用OD值表示)和NO产量,同时四组于复氧的1、3、8、24、48、72 h分别用原位杂交法和免疫组化法观察Hsp70 mRNA及Hsp70阳性细胞百分率。结果 与Ⅰ组相比,Ⅱ组T1-24各时点OD值降低;Ⅲ组、Ⅳ组T1、T2时点OD值升高;与Ⅱ组相比,Ⅲ组、Ⅳ组OD值均升高;Ⅲ组、Ⅳ组间差异无显著性。在T1、T2及T4时点,与Ⅰ组相比,Ⅱ组NO产量增高;与Ⅱ组相比,Ⅲ组、Ⅳ组NO产量降低;Ⅰ组与Ⅲ组、Ⅰ组与Ⅳ组、Ⅲ组与Ⅳ组间差异无显著性。与I组相比,Ⅱ组Hsp70mRNA及Hsp70阳性细胞百分率增高,且开始增高的时间分别为3 h和8 h,高峰时间分别为24 h和48h;

Objective To investigate the effects of propofol on cultured primary neurons isolated from fetal Wistar rats against anoxic-reoxygenated injury and its neuro-protective mechanisms at cellular level. Methods Neuronal cells were isolated from brains of fetal Wistar rats and cultured for 12 days. The cultured neuronal cells were randomly divided into 4 groups : (Ⅰ) control group; (Ⅱ) anoxic-reoxygenated group in which neurons were exposed to 95% N2 + 5% CO2 at 37·for 3O min; (Ⅲ) propofol pretreatmenl group in which propofol was added to the culture medium (the final concentrations of propofol were 14 μmol·L-1) 1 h before exposure to anoxia. (Ⅳ) propofol pretreatment group (the final concentrations of propofol were 56 μmol·L-1) . Neuronal activity was detected by MTT analysis and NO output was assayed with nitrate reductase method at 1, 2, 4, 6 and 24 h after reoxygenation. The synthesis of heat shock protein (Hsp)70 mRNA was measured by in situ hybridization technique and the synthesis of Hsp70 was measured by immuno-histochemical technique at 1, 3, 8, 24, 48 h and 72 h after reoxygenation.Results (1) 30-min anoxia decreased neuronal activity, increased NO output and significantly increased the synthesis of Hsp70 mRNA and Hsp70. The expression of Hsp70 mRNA reached its peak at 24 h after reoxygenation and that of Hsp70 at 48 h after reoxygenation. (2) Propofol pretreatment significantly increased neuronal activity at 1, 2, 4, 6 and 24 h after reoxygenation and decreased NO output at 1, 2 and 4 h after reoxygenation compared with those in anoxia-reoxygenated group. (3) The synthesis of Hsp70 mRNA was significantly increased and accelerated by pretreatment with both concentrations of propofol but the synthesis of Hsp70 was significantly increased and accelerated by pretreatment with 56μmol·L-1 propofol only. Conclusion Propofol pretreatment can inhibit anoxia-reoxygenation injury to neurons by decreasing NO output and increasing expression of Hsp70 through inducing the synthesis of Hsp70 at both transcription and translation levels.