H_2S通过cAMP介导PI_3-K/Akt/P^(70S6K)通路抑制缺氧/复氧后神经元的凋亡

H_2S inhibits neuron apoptosis induced by anoxia-reoxygenation through cAMP-mediated PI_3-K/Akt/P^(70S6K) kinase cell-survival signaling pathways

目的:研究H2S对缺氧/复氧后神经元存活信号转导通路PI3-K/Akt/P70S6K的影响。方法:取96孔和6孔培养板上培养7d的海马神经元,正常培养组(C组)神经元按正常培养方法培养。NaHS组在进行缺氧/复氧时加入NaHS使其终浓度为150μmol/L。Tri组、Rap组和Tri+Rap组在加入150μmol/LNaHS的同时分别加入triciribin10μmol/L、rapamycin10nmol/L或triciribin10μmol/L+rapamycin10nmol/L。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、cAMP和PI3-K、Akt和P70S6K蛋白表达的检测。结果:NaHS显著增加了cAMP的浓度和PI3K、Akt、P70S6K蛋白的表达,同时增加了神经元存活率、降低了神经元凋亡率(P<0.01vsC组和A/R组)。Triciribin抑制了Akt和P70S6K表达同时降低了神经元存活率、升高了神经元凋亡率(P<0.05,P<0.01vsNaHS组)。Rapamycin抑制了P70S6K表达同时降低了神经元存活率、升高了神经元凋亡率(P<0.05,P<0.01vsNaHS组)。结论:H2S通过cAMP激活了PI3-K/Akt/P70S6K信号通路,抑制缺氧/复氧后海马神经元的凋亡。

AIM： To investigate the effect of hydrogen sulfide on neuron apoptosis through PI3 -K/Akt/P^70S6K cell - survival signal transduction pathways after neuron anoxia - reoxygenation. METHODS： Newborn （ 24 - 48 h） Wistar rats were decapitated. The hippocampus tissue was dissected and cells were suspended. Cells were plated at 1.0 × 10^8 cells/L on poly -dlysine -treated 96 -well （100 μL/well） plates and 6 -well （2 mL/well） plates. Cells were used after 7 days. For anoxia - reoxygenation （ oxygen glucose deprivation, OGD） experiments, cells were washed three times in a glucose- free balanced salt solution （BSS）. They were then placed in deoxygenated glucose -free medium and cultured 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% air, 5% CO2. In experimental group, cells were respectively carried out OGD, OGD ＋ 150 μmol/L NaHS, OGD ＋ 150 μmol/ L NaHS ＋ 10 μmol/L triciribin, OGD ＋ 150 μmol/L NaHS ＋ 10 nmol/L rapamycin and OGD ＋ 150 μmol/L NaHS ＋ 10 μmol/L triciribin ＋ 10 mnol/L rapamycin. Control cells were cultured normally. 24 h later, neuron viability and apoptosis were measured. The level of cAMP and protein expression of PI3 - K, Akt and P^70S6K were detected. RESULTS： NaHS enhanced concentration of cAMP and expression of PI3 - K, Akt and P^70S6K. Meanwhile, increased neuron viability and decreased neuron apoptosis （ P 〈0.01 vs group C or group I/R） were observed. Triciribin inhibited Akt and P^70S6K, as well as increased neuron apoptosis and decreased neuron viability （P 〈 0. 05, P 〈 0. 01 vs group NaHS）. Rapamycin inhibited P^70S6K, as well as increased neuron apoptosis and decreased neuron viability （ P 〈0.05, P 〈0. 01 vs group NaHS）. CONCLUSION： H2S inhibits hippocampus neuron apoptosis and protects neuron from anoxia- reoxygenation injury through cAMP - mediated PI3 - K/Akt/P^70S6K kinase cell - survival signaling pathways.