细胞凋亡抑制剂防护晶状体上皮细胞氧化损伤及信号转导机制

Apoptosis Inhibitors Protect Lens Epithelial Cell Damaged by Oxidative Stress and its Mechanism of Signal Transduction

目的:探讨细胞凋亡抑制剂阿魏酸钠(SF)、五味子乙素(Sch B)、菊花(FC)、车前子(SP)对过氧化氢(H2O2)所致氧化损伤的晶状体上皮细胞(LEC)活性以及细胞内钙(Ca2+)、环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)的影响,研究四种细胞凋亡抑制剂防治白内障的细胞信号转导机制。方法:将传代培养的牛LEC与H2O2共同孵育复制氧化损伤的模型,同时加入SF和Sch B单体及FC和SP水提取液孵育后,采用四甲基偶氮唑蓝法(MTT)检测LEC活性,采用荧光分光光度法检测LEC内钙离子的浓度([Ca2+]i)、放射免疫分析法检测LEC内cAMP和cGMP含量,并探讨不同浓度和不同孵育时间(12h、24h、36h)的影响。结果:①H2O2组LEC活性下降,SF、Sch B、FC、SP均可明显增强氧化损伤的LEC活性(P<0.01),呈浓度依赖关系和时间依赖关系。②H2O2组LEC的[Ca2+]i升高,SF、Sch B、FC、SP均可显著降低氧化损伤LEC的[Ca2+]i(P<0.01),呈时间-效应关系。③H2O2组LEC的cAMP浓度升高、cGMP浓度下降,SF、Sch B、FC、SP均可使氧化损伤的LEC的cAMP水平显著降低(P<0.01)、cGMP水平显著升高(P<0.01),也呈时间-效应关系。结论:四种细胞凋亡抑制剂SF、Sch B、FC、SP可明显抑制LEC氧化损伤及凋亡,通过[Ca2+]i、cAMP、cGMP信号转导及相互作用调节生物学效应可能是其主要的细胞和分子生物学机制。

Objective： To investigate the prevention of four apoptosis inhibitors on lens epithelial cell （LEC） damaged by oxidative stress and mechanism of signal transduction. Methods Sub-cultured bovine LEC were incubated with hydrogen peroxide（H2O2） and deferent concentrations of sodium ferulate （ SF, 25μg/ml, 50μg/ml, 100μg/ml）, schisandrin B （ Sch B, 12.5μg/ml, 25μg/ml, 50μg/ml）, flos ehrysanthemi （ FC, 0.2mg/ml. 0.4mg/ml.0.8mg/ml）, semen plantaginis（SP, 2mg/ml, 4mg/ml, 8mg/ml） for 12h, 24h and 36 h respectively. The LEC activities were observed by methyl thiazolyl tetrazolium assay （MTT）. LEC [Ca^2＋]i were detected by spectrofluorophotometer. Intracellular cyclic adenosine monophosphate （cAMP） and cyclic guanine monophosphate （cGMP） of LEC were determined by radioimmunoassay. Results： （1）The activities of LEC increased in deferent concentrations of SF, Sch B, FC and SP groups compared with H2O2 group（P〈0.01）, shown dose-dependent and time-dependent. （2） The [Ca^2＋]i of LEC decreased in SF, Sch B, FC and SP groups compared with H2O2 group （P〈0.01）, shown time-dependent. （3）The LEC cAMP decreased and The LEC cGMP increased in SF, Sch B, FC and SP groups compared with H2O2 group（P〈0.01）, shown time-dependent. Conclusion： （1） SF, Sch B, FC and SP can protect LEC against oxidative damages and increase activities of LEC. （2）The calcium, cAMP and cGMP signal transduction system and the cross-talk may be the mechanisms of SF, Sch B, FC and SP preventing and delaying cataract formation.