阿魏酸钠防护晶状体上皮细胞氧化损伤的信号转导机制

目的: 探讨阿魏酸钠对氧化损伤的晶状体上皮细胞内 Ca²⁺ 、环磷酸腺苷(cAMP) 、环磷酸鸟苷(cGMP) 的影响, 从细胞信号转导角度揭示天然药物防治白内障的细胞和分子学机制。方法: 采用牛晶状体上皮细胞进行晶状体上皮细胞(LEC) 原代和传代培养, 以含有过氧化氢(H₂O₂) 的培养液孵育 LEC复制氧化损伤模型, 并加入阿魏酸钠单体共同孵育。分别采用四甲基偶氮唑兰(MTT) 比色测定法观察在不同时间和浓度条件下 LEC 活性变化, 采用荧光分光光 度 计 测 定 不 同 时 间 细 胞 内 钙 离 子 浓 度(Ca²⁺]_i) 以及放射免疫分析法测定不同时间 LEC内cAMP 、cGMP 的含量变化。结果: H₂O₂ 组可引起LEC 吸光度值(A)明显下降(P<0.01), 阿魏酸钠能明显增强氧化损伤的LEC活性, 并呈剂量-效应关系和时间-效应关系。氧化损伤的 LECCa²⁺]_i 升高(P<0.01);阿魏酸钠可以降低由 H₂O₂ 引起的细胞Ca²⁺]_i 的升 高, 并呈时间-效应关系。H₂O₂ 组cAMP 浓度升高;cGMP 浓度下降(P<0.01);阿魏酸钠使 cAMP 水平下调, cGMP 水平上升(P<0.01), 并呈时间-效应关系。结论: 阿魏酸钠可明显抑制 LEC氧化损伤及凋亡, 其作用机制可能是通过钙信号系统、cAMP 信号系统、cGMP 信号系统及其相互作用来调节生物学效应。

Effects of anti-apoptosis drugs on signal transduction of lens epithelial cell damaged by oxidative stress

AIM: To investigate the prevention ef-fects of sodium ferulate (SF) on lens epithelial cells (LEC) damaged by oxidative stress, and to study the changes of intracellular-free calcium, cyclic adenosine monophosphate (cAMP) and cyclic guanine monophos-phate (cGMP). METHODS: Bovine LEC were primarily cultured and sub-cultured, and used for experiment in generation 3.LEC were incubatedwith hydrogen peroxide (H₂O₂). SF 25, 50, and 100 mg°L^-1 were added into the LEC damaged by oxidative stress and incubated for 12, 24 and 36 hours, respectively. The LEC activities were observed by 3-(4, 5-dimethy-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. Intracellular-free calcium content of LEC was detected by spectrofluo-rophotometer using Fura-2/AM as an indicator.Intracel-lular cAMP and cGMP concentrations of LEC were deter-mined by radioimmunoassay (RIA). RESULTS: The ab-sorbance values (A values) in LEC of H₂O₂ groupsobvi-ously reduced compare with that in the control group(P< 0.01). The activities of LEC treated with SF obvious dose-dependent and time-dependent increased.The intra-cellular-free calcium contents of LEC in H₂O₂ group by spectrofluorophotometer increased compared with that in the control group (P<0.01). However, it obvious time-dependent decreased in SF groups (P<0.01). The cAMP concentration of LEC increased and the cGMP con-centration of LEC decreased in H₂O₂ group by radioim-munoassay compared with that in the control group (P< 0.01). The cAMP concentration of LEC decreased and the cGMP concentration of LEC obvious time-dependent increased in SF groups (P<0.01). CONCLUSION: SF can protect LEC from oxidative damages and inhibit apoptosis of LEC.The mechanisms of preventing and de-laying cataract formation by four anti-apoptosis drugs may be related to the cross-talk among calcium signal trans-duction system, cAMP signal transduction system and cGMP signal transduction system.