七氟烷激活PI_3-K/Akt/P~(70S6K)信号转导通路抑制缺血/再灌注损伤神经元的凋亡

目的研究七氟烷对神经元缺血/再灌注损伤后PI3-K/Akt/P70S6K信号转导通路的影响,探讨七氟烷脑保护机制。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6组:正常培养组(C组)、缺血/再灌注组(I/R组)、缺血/再灌注+2%Sevoflurane组(Sevo组)、缺血/再灌注+2%Sevoflurane+10μmol.L-1LY294002(PI3-K拮抗剂)组(LY组)、缺血/再灌注+2%Sevoflurane+10μmol.L-1Tric irib in(Akt拮抗剂)组(Tri组)、缺血/再灌注+2%Sevoflurane+10 nmol.L-1Rapamyc in(P70S6K拮抗剂)组(Rap组)。C组神经元按正常培养方法培养。Sevo组在神经元缺糖缺氧的同时接受2%Sevoflurane麻醉。LY组、Tri组和Rap组在神经元进行缺糖同时分别加入LY294002、Tric irib in或Rapamyc in使其终浓度分别为10μmol.L-1、10μmol.L-1或10 nmol.L-1后同Sevo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、神经元凋亡和PI3-K、Akt和P70S6K蛋白表达的检测。结果Sevo组PI3K、Akt、P70S6K蛋白表达增加,神经元存活率增加、神经元凋亡率降低(vsI/R组,P<0.01)。LY组PI3K、Akt和P70S6K表达降低,神经元存活率降低、神经元凋亡率增加(vsSevo组,P<0.05或P<0.01);Tri组Akt和P70S6K表达降低,神经元存活率降低、神经元凋亡率增加(vsSevo组,P<0.05或P<0.01);Rap组P70S6K表达降低,神经元存活率降低、神经元凋亡率增加(vsSevo组,P<0.01)。结论Sevoflurane激活了PI3-K/Akt/P70S6K信号通路,在海马神经元缺血/再灌注损伤过程中抑制了神经元凋亡,保护了神经元。

Sevoflurane activates PI3-K/Akt/P70S6K kinase cell-survival signaling pathways and protects neuron against ischemia-reperfusion

Aim To investigate effects of Sevoflurane on PI_3-K/Akt/P~(70S6K) cell-survival signal transduction pathways after neuron ischemia-reperfusion,and explore neuroprotection mechanisms of sevoflurane.Methods Newborn(24～48 h)Wister 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·ml~(-1) 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~(-1) cytosine arabinoside on day 4 in culture to minimize glial growth.One-half of the medium was replaced twice a week with medium containing DMEM(4.5 g·L~(-1) glucose)/F12(1 ∶1),5% fetal bovine serum and 5% horse serum.Cells were used after 7 days. For ischemia-reperfusion(oxygen glucose deprivation,OGD)experiments,cultures were washed three times in a glucose-free balanced salt solution(BSS)and placed in deoxygenated glucose-free medium and sealed under 95% N_2-5% CO_2 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% O_2-5% CO_2.Experimental group cells were respectively carried out OGD,OGD+2% Sevoflurane,OGD+2% Sevoflurane +10 μmol·L~(-1) LY294002,OGD+2% Sevoflurane +10 μmol·L~(-1) Triciribin,and OGD+2% Sevoflurane +10 nmol·L~(-1) Rapamycin.Control cells were cultured normally.Group Sevo was carried out OGD meanwhile anesthesized with 2% sevoflurane.Group LY,Tri and Rap cells was carried out OGD meanwhile culture medium was added 10 μmol·L~(-1) LY294002,10 μmol·L~(-1) Triciribin or 10 nmol·L~(-1) Rapamycin,and anesthesized with 2% sevoflurane.Compound remained present throughout the duration of the experiment until analysis 24 h later.Neuron viability and apoptosis were measured.The protein expression of PI_3-K,Akt and P~(70S6K) were detected.Results Sevoflurane enhanced expression of PI_3-K,Akt and P~(70S6K),meanwhile increased neuron viability and decreased neuron apoptosis(vs group I/R,P<0.01).LY294002 inhibited PI_3-K,Akt and P~(70S6K),as well increased neuron apoptosis and decreased neuron viability(vs group Sevo,P<0.05 orP<0.01).Triciribin inhibited Akt and P~(70S6K),increased neuron apoptosis and decreased neuron viability(vs group Sevo,P<0.05 orP<0.01).Rapamycin inhibited P~(70S6K),and increased neuron apoptosis and decreased neuron viability(vs group Sevo,P<0.01).Conclusion Sevoflurane activites PI_3-K/Akt/P~(70S6K) kinase signal transduction pathways,and inhibits hippocampus neuron apoptosis,and protects neuron against ischemia-reperfusion injury.