摘要
目的研究脱氟烷(desflurane)诱导氧糖剥夺损伤神经元血红素氧合酶-1表达的信号转导通路,探讨脱氟烷脑保护机制.方法将96孔和6孔培养板上培养7 d的大鼠海马神经元随机分为6组(n=12):正常培养组(C组),神经元按正常培养方法培养;氧糖剥夺组(I/R组),神经元缺糖缺氧后复糖复氧处理;氧糖剥夺+6%desflurane组(Des组),神经元缺糖缺氧的同时接受6%desflurane麻醉;氧糖剥夺+6%desflurane+10μmol/L tin-protoporphyrin(HO-1抑制剂)组(Tin组)、氧糖剥夺+6%desflurane+10μmol/L LY 294002(PI3-K抑制剂)组(LY组)、氧糖剥夺+6%desflurane+10μmol/L Triciribin(Akt抑制剂)组(Tri组)神经元进行缺糖同时分别加入tin-protoporphyrin、LY 294002或Triciribin使其终浓度均为10μmol/L后同D组处理.96孔培养板的神经元进行细胞存活率的检测.6孔培养板的神经元进行神经元纯度鉴定、神经元凋亡、HO-1mRNA表达、PI3-K和Akt蛋白表达的检测.结果Des组PI3K和Akt蛋白表达增加,HO-1mRNA表达增加,神经元存活率增加、凋亡率降低(vs I/R组,P<0.01).Tin组PI3K和Akt蛋白表达变化不明显(vs Des组,P>0.05),HO-1mRNA表达降低,神经元存活率降低、凋亡率增加(vs Des组,P<0.01).LY组PI3K和Akt表达降低,HO-1mRNA表达降低,神经元存活率降低、凋亡率增加(vs Des组,P<0.05或P<0.01).Tri组PI3K表达变化不明显(vs Des组,P>0.05),Akt表达降低,HO-1mRNA表达降低,神经元存活率降低、凋亡率增加(vs Des组,P<0.05或P<0.01).结论Desflurane通过PI3-K/Akt信号转导通路诱导大鼠海马神经元HO-1表达,保护了神经元.
Objective To investigate the signal pathway of HO-1 expression in desflurane-induced after neuronischemia-reperfusion, and explore neuroprotection mechanisms of desflurane. Methods Newborn （24 -48 h） Wistar rats were decapitated and hippoeampus tissue was dissected. Then digestion with 0.125% trypsin, and suspended in a medium containing DMEM supplemented to 25 mmol/L glucose, 10% fetal bovine serum, 10% horse serum, and 2 mmol/L glutamine. Cells were cultured at 1.0 × 10^5/mL on poly -dlysine -treated 96 - well （100 μL/well） plates as well 6 - well （2 mL/well） plates. Cultures were treated with 10 μmoL/L cytosine arabinoside on day 4 in culture to minimize glial growth. One - half of the medium was replaced twice a week. Cells were used after culture 7 days. For ischemia- reperfusion （oxygen glucose deprivation, OGD） experiments, cultures were washed three times in a glucose -free balanced salt solution （BSS）. They were then placed in deoxygenated glucose - free medium and sealed under 95% N2 - 5% CO2 in an anaerobic chamber equilibrated to 37 ℃ and 100% humidity for 45 min. OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 21% O2-5% CO2. Experimental group cells were respectively carried out OGD （group I/R）, OGD＋6% Desflurane （group Des）, OGD＋6% Desflurane ＋10 ixmoL/L tin-protoporphyrin（group Tin）, OGD＋6% Desflurane ＋10 μmol/L LY 294002 （group LY）, OGD＋6% Desflurane ＋10 μmol/L Triciribin （group Tri）. Control cells were cultured normally. Group Des was carried out OGD meanwhile anesthesized with 6% Desflurane. Group Tin, LY and Tri cells was carried out OGD meanwhile culture medium was added 10 μmoL/L tin-protoporphyrin, 10 μmoL/L LY294002 or 10 μmoL/L Triciribin respectivly, and anesthesized with 6% Desflurane. Compound remained present throughout the duration of the experiment until analysis 24 h later.Neuron viability and apoptosis were measured. The expression of HO-1mRNA, PI3-K and Akt protein were detected. Results Desflurane enhanced expression of HO-1mRNA, PI3-K and Akt, meanwhile increased neuron viability and decreased neuron apoptosis （vs group I/R, P 〈 0.01 ）. Tin-protoporphyrin inhibited HO-1-mRNA, as well increased neuron apoptosis and decreased neuron viability （vs group Des, P 〈 0.01 ）. LY 294002 inhibited PI3-K, Akt and HO-1mRNA, increased neuron apoptosis and decreased neuron viability （vs group Des, P〈 0.05 or P〈 0.01 ）. Triciribin inhibited Akt and HO-1-mRNA, increased neuron apoptosis and decreased neuron viability （vs group Des, P 〈 0.01 ）. Conclusion Desflurane via PI3-K/Akt cell-survival signaling pathways regulates hemeoxygenase-1 and protects neuron against ischemi- a-reperfusion injury.
出处
《昆明医学院学报》
2008年第2期51-55,共5页
Journal of Kunming Medical College
