3,4,5,6-四羟基酮对缺氧/复氧所致PC12神经细胞凋亡的保护作用涉及DDAH/ADMA途径

3,4,5,6-tetrahydroxyxanthone protects against hypoxia/reoxygenation-induced apoptosis of PC12 cells: role of the DDAH/ADMA pathway

目的研究3,4,5,6-四羟基酮对缺氧/复氧所致大鼠肾上腺嗜铬细胞瘤(PC12)细胞凋亡的保护作用与二甲基精氨酸-二甲胺水解酶(DDAH)/非对称性二甲基精氨酸(ADMA)通路的关系。方法将体外培养的PC12细胞分为正常对照组、缺氧/复氧组和3个剂量的酮(3、10、30μmol.L-1)处理组。用hoechst 33342染色和膜联蛋白Ⅴ(AnnexinⅤ)+碘化丙啶(PI)流式细胞仪法检测细胞凋亡率,用高效液相色谱(HPLC)法测定DDAH活性及ADMA的浓度;用活性氧及caspase-3试剂盒检测细胞内活性氧(ROS)生成及caspase-3活性。结果缺氧/复氧处理PC12神经细胞能增加ROS生成,降低DDAH活性,升高ADMA水平,激活caspase-3诱导细胞凋亡。外源性ADMA(3、10和30μmol.L-1)能激活caspase-3并诱导PC12神经细胞凋亡。3,4,5,6-四羟基酮(3、10和30μmol.L-1)能抑制缺氧/复氧所致的PC12神经细胞凋亡,抑制ROS生成,增加DDAH活性,降低ADMA水平,抑制caspase-3活性。结论3,4,5,6-四羟基酮对缺氧/复氧所致PC12神经细胞凋亡具有保护作用,其机制与抑制氧化应激调节DDAH/ADMA途径有关。

Objective To observe the inhibitory effect of 3, 4, 5, 6-tetrahydroxyxanthone （Xan） on hypoxia/reoxyge-nation （H/R） -induced apoptosis in PC12 （pheochromocytoma） cells and to investigate the relationship between anti apoptotic action of Xan and the DDAH/ADMA pathway. Methods The apoptosis of PC12 was detected by Hoechst33342 staining or flow cytometry （FCM） with Annecin V ＋ Propidium Iodide （PI）. The activity of DDAH and level of ADMA were measured by high-performance liquid chromatography （HPLC）. The activity of reactive oxygen species （ROS） and caspase-3 were determined by fluorescent ROS detection kit and colorimetric caspase-3 assay kit, re spectively. Results Six-hour hypoxia （1% O2 ） and consequent 24-hour reoxygenation significantly increased the apoptotic death of PC12 cells, concomitantly with increasing intracellular ROS production and caspase-3 activity, which was attenuated by pretreatment with Xan （3, 10 or 30 μmol · L^-1 ）. The decrease in DDAH activity and the increase in ADMA level were also observed after H/R treatment. Furthermore, incubation with exogenous ADMA could increase caspase-3 activity and induce the apoptosis of PC12 cells. Pretreatment with Xan could markedly attenuate the decrease in DDAH activity and the increase in ADMA level. Conclusion Xan protects against H/R induced apoptosis via inhibiting caspase-3-dependent apoptotic signaling pathway, which may be related to improving the DDAH/ADMA pathway by reduction of oxidative stress.