异丙酚预处理对大鼠海马神经元凋亡的影响

Effects of propofol preconditioning on anoxia-reoxygenation induced apoptosis in rat hippocampal neurons

目的探讨异丙酚预处理对缺氧/复氧后大鼠海马神经元凋亡的影响。方法取离体培养的大鼠海马神经元,随机分成5组:正常组(A)组。CoCl2组(B)组:加入300μmol·L-1(下同)CoCl2处理4h,然后更换正常的培养基培养24h,之后更换无血清的培养基培养。脂肪乳组(C)组:加入10%脂肪乳剂90μl预处理1h后同B组。异丙酚组(D)组:加入100μmol·L-1异丙酚1h后同B组。7-NI组(E)组:如D组,但加入CoCl2的同时加入3·3μl7-NI(25g·L-1)处理4h。MTT法测定细胞增殖,流式细胞技术测定细胞的凋亡。RT-PCR技术检测神经型一氧化氮合酶和凋亡相关基因的表达情况。结果脂肪乳组与CoCl2组比较,各项指标相似(P>0·05);异丙酚可增加神经元的增殖能力,减少凋亡(P<0·05或P<0·01),上调神经型一氧化氮合酶和凋亡抑制基因bcl-2的表达,下调促凋亡基因cyclinD1的表达。而这些调节作用可被神经型一氧化氮合酶抑制剂7-NI抑制(P<0·05或P<0·01)。结论100μmol·L-1异丙酚预处理对缺氧/复氧后大鼠海马神经元有保护作用,神经型一氧化氮合酶和凋亡相关基因在其中起重要作用。

Aim To determine what effects propofol exerts on apoptosis in hippocampal neurons caused by anoxia-reoxygenation.Methods Newborn SD rats(<24 h after birth) weighting 5 to 6 grams were decapitated,and hippocampal tissue was isolated and hippocampal neurons were prepared by digestion with tryp-sin.After being cultured for 9 to 10 d,the hippocampal neurons were randomly assigned to one of 5 groups:Control group(group A);CoCl2 group(group B),treated with 300 μmol·L-1(the other groups were same)CoCl2 for 4 h,cultured with normal medium for 24 h,then stimulated with no serum media;intralipid group(group C),pretreated the neurons with 10% intralipid 90 μl for 1 h and then treated as group B;propofol group(group D),pretreated the neurons with 100 μmol·L-1 propofol for 1 hour,then treated as group B;7-NI group(group E),treated as group D,but CoCl2 and 3.3 μl 7-NI(25 g·L-1) were added at the same time.MTT method and FACS were used to detect the proliferation and apoptosis of neurons.RT-PCR method was used to show the expression of neuronal nitric oxide synthesis and apoptosis related gene in propofol preconditioning.Results The results were compared between intralipid group and CoCl2 group,and the difference was not significant(P>0.05).When pretreated with propofol,neurons proliferation increased and apoptosis decreased(P<0.05 or P<0.01).Neuronal nitric oxide synthesis was up-regulated after preconditioning;meanwhile,Bcl-2 gene was up-regulated and cyclinD1 gene was down-regulated.But these regulation can be suppressed by inhibitor of neuronal nitric oxide synthesis(P<0.05 or P<0.01).Conclusion Preconditioning with 100 μmol·L-1 propofol can protect hippocampal neurons against anoxia-reoxygenation injury,neuronal nitric oxide synthesis and apoptosis related gene play an important part in these protective effect.