五味子乙素对晶状体上皮细胞内Ca^(2+) cAMP cGMP的影响

Effect of Schezandrin B to intracellular-free calcium cAMP cGMP in Lens Epithelial Cell

目的:研究旨在探讨五味子乙素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,并加入五味子乙素单体共同孵育。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:①H2O2组可引起LEC吸光度值(A)明显下降(P<0.01),五味子乙素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。②氧化损伤的LEC[Ca2+]i升高(P<0.01);五味子乙素可以降低由H2O2引起的细胞[Ca2+]i的升高,并呈时间-效应关系。③H2O2组cAMP浓度升高;cGMP浓度下降(P<0.01);五味子乙素使cAMP水平下调,cGMP水平上升(P<0.01),并呈时间-效应关系。结论:五味子乙素可明显抑制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。

Objective ：To investigate the prevention of schezandrin B（ Sch B） on lens epithelial cell （LEC） damaged by oxidative stress, and to study the changes of intracellular - free calcium, cyclic adenosine monophosphate （cAMP） and cyclic guanine monophosphate （cGMP）. Methods ： Bovine LEC were primarily cultured and sub -cultured, and used for experiment in generation 3. LEC were incubated with hydrogen peroxide （H202 ）. Sch B 25μg/mL, 50μg/mL, 100μg/mL 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 - diphenyhetrazolium bromide（MTT） assay. Intracellular - free calcium content of LEC were detected by spectrofluorophotometer using Fura - 2/AM as an indicator. Intracellular cAMP and cGMP concentrations of LEC were determined by radioimmunoassay （RIA）. Results ; ①The results of MTT test showed that the absorbance values（ A values ） in LEC of H2O2 groups reduced obviously to compare with control group（ P 〈 0.01）. The activities of LEC treated with Sch B increased, and showing obvious dose - dependent and time - dependent. ②The intracellular - free calcium contents of LEC in H2O2 group by spectrefluorephotometer increased compared with control group （ P 〈 0.01 ） ; however, it decreased in Sch B groups showing obvious time - dependent （ P 〈 0.01 ）. ③The cAMP concentration of LEC increased and the cGMP concentration of LEC decreased in H2O2 group by radioimmunoassay significantly compared with control group （ P 〈0.01 ） ; however,the cAMP concentration of LEC decreased and The cGMP concentration of LEC increased in Sch B groups , showing obvious time - dependent（ P 〈0.01 ）. Conclusion ： Sch B protected LEC from oxidative damages and inhibit apoptosis of LEC. It is possible that the mechanisms of preventing and delaying cataract formation by four anti - apoptosis drugs may be the cress - talk among calcium signal transduction system, cAMP signal transduction system and eGMP signal transduction system.