摘要
目的探讨脱氟烷(desflurane)的脑保护作用及其与神经元缺血/再灌注损伤后血红素氧合酶-1(HO-1)表达的信号转导通路的关系。方法将96孔和6孔培养板上培养7d的海马神经元随机分为7组:正常培养组(C组)、缺血/再灌注组(I/R组)、缺血/再灌注+6%desflurane组(D6组)、缺血/再灌注+12%desflurane组(D12组)、缺血/再灌注+12% desflurane+tin-protoporphyrin(HO-1抑制剂)组(T组)、缺血/再灌注+12% desflurane+U-0126(ERK抑制剂)组(U组)、缺血/再灌注+12% desflurane+rapamycin(RS6K抑制剂)组(R组)。C组神经元按正常培养方法培养。I/R组神经元进行缺糖缺氧后复糖复氧处理。D6组和D12组在神经元缺糖缺氧的同时接受6%或12%desflurane麻醉。T组、U组和R组在神经元进行缺糖同时分别加入tin-protoporphyrin、U-0126或Rapamycin使其终浓度均为10μmol·L-1后同D12组处理。96孔培养板的神经元进行细胞存活力的检测。6孔培养板的神经元进行神经元纯度鉴定、神经元凋亡、HO-1-mRNA表达、磷酸化ERK1/2和P90RSK蛋白表达的检测。结果缺血/再灌注后神经元存活率降低,凋亡率增加,HO-1-mRNA的表达增加,PERK1/2和PP90RSK蛋白表达增加(vsC组,P<0.01)。D6组PERK1/2和PP90RSK蛋白表达增加,HO-1-mRNA表达增加,神经元存活率增加、神经元凋亡率降低(vsI/R组,P<0.01)。D12组PERK1/2和PP90RSK蛋白表达增加,HO-1-mRNA表达增加,神经元存活率增加、神经元凋亡率降低(vsI/R组或D6组,P<0.01或P<0.05)。T组PERK1/2和PP90RSK蛋白表达变化不明显(vsD12组,P>0.05),HO-1-mRNA表达降低,神经元存活率降低、神经元凋亡率增加(vsD12组,P<0.01)。U组PERK1/2和PP90RSK表达降低,HO-1-mRNA表达降低,神经元存活率降低、神经元凋亡率增加(vsD12组,P<0.05或P<0.01)。R组PERK1/2蛋白表达变化不明显(vsD12组,P>0.05),PP90RSK表达降低,HO-1-mRNA表达降低,神经元存活率降低,神经元凋亡率增加(vsD12组,P<0.05或P<0.01)。结论Desflurane通过ERK1/2/P90RSK信号转导通路激活HO-1,在海马神经元缺血/再灌注时抑制了神经元凋亡,保护了神经元。
Aim To investigate signal pathway associated with hemeoxygenase-1 （ HO-1 ） expression in desflurane-induced after neuron ischemia-reperfusion, and explore neuroprotection mechanisms of desfiurane. Methods Newborn （24 - 48 h） Wistar rats were decapitated and hippocampus tissue was dissected and cut into 1 mm × 1 mm × 1 mm pieces. Then digestion with 0. 125% trypsin, centrifuged at 800 r · min^-1 for 5 min at 4℃, and suspended in a medium containing DMEM supplemented to 25 mmol · L^-1 glucose, 10% fetal bovine serum, 10% horse serum, and 2 mmol · L^-1 glutamine. Cells were plated at 1.0 × 10^5 · L^-1 on poly-Dlysine-treated 96-well （ 100 μl · well^-1 ） plates as well 6-well （2 ml · well^- 1 ） plates and were treated with 10 μmol · L^-1 cytosine arabinoside on day 4 to minimize glial growth. One-half of the medium was replaced twice a week with medium consisting of DMEM （4.5 g · L^-1 glucose）/F12（1 ： 1）, 5% fetal bovine serum and 5% horse serum. Cells were used after growing for 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 95% 02 -5% CO2. Experimental group cells were respectively carried out OGD （ group I/R）, OGD ＋ 6% Desflurane （ group D6 ）, OGD ＋ 12% Desflurane （group D12）, OGD ＋ 12% Desflurane ＋ tin-pretoporphyfin （groupT） , OGD ＋ 12% Desflurane ＋ U-0126 （ groupU ）, OGD ＋ 12% Desflurane ＋ Rapamycin （group R ）. Control cells were cultured normally. Group D6 , group D12 was carried out OGD meanwhile anesthesized with 6% or 12% Desflurane respectively. Group T, group U and group R cells was carried out OGD meanwhile culture medium was added 10 μmol · L^-1 tin-protoporphyfin, U-0126 or Raparnycin respectivly, and anesthesized with 12% 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-1-mRNA, phosphorylated ERK1/2 （ p^ERK1/2 ） and p^90RSK （pp^90RSK） protein were detected. Results Desflurane enhanced expression of HO-I-mRNA, p^ERK1/2 and pp^90RSK, meanwhile increased neuron viability and decreased neuron apoptosis （vs group C and I/R, P 〈 0.01 ）. Tin-protoporphyrin inhibited HO-I-mRNA, and increased neuron apoptosis and decreased neuron viability（vs group D12,P 〈0. 05 or P 〈0. 01 ）. U-0126 inhibited p^ERK1/2, pp^90RSK and HO-I-mRNA, increased neuron apoptosis and decreased neuron viability （vs group D12,P 〈0. 05 orP 〈0. 01 ）. Rapamycin inhibited pp^90RSK and HO-I-mRNA, increased neuron apoptosis and decreased neuron viability（vs group D12 ,P 〈 0. 05 or P 〈 0. 01 ）. Conclusion Desflurane up-regulates hemeoxygenase-1 through ERK1/2/ p^90RSK kinase cel-survival signaling pathway and protects neuron against ischemia-reperfusion injury.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2007年第3期390-394,共5页
Chinese Pharmacological Bulletin
