硫辛酸对体外培养的多环芳烃诱导的 Müller 细胞毒性的保护作用。
Protective effects of lipoic acid on chrysene-induced toxicity on Müller cells in vitro.
作者信息
Mansoor Saffar, Gupta Navin, Luczy-Bachman Georgia, Limb G Astrid, Kuppermann Baruch D, Kenney M Cristina
机构信息
Gavin Herbert Eye Institute, School of Medicine, University of California, Irvine, CA 92697, USA.
出版信息
Mol Vis. 2013;19:25-38. Epub 2013 Jan 7.
PURPOSE
This study evaluates the toxic effects of chrysene (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro and investigates whether the inhibitor lipoic acid can reverse the chrysene-induced toxic effects.
METHODS
MIO-M1 cells were exposed to varying concentrations of chrysene with or without lipoic acid. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3/7 activity was measured by a fluorochrome assay. Lactate dehydrogenase (LDH) release was quantified by an LDH assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with a 2',7'-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay. Intracellular ATP content was determined by the ATPLite kit.
RESULTS
MIO-M1 cells showed significantly decreased cell viability, increased caspase-3/7 activity, LDH release at the highest chrysene concentration, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to 300, 500, and 1,000 µM chrysene compared with the control. Pretreatment with 80 µM lipoic acid reversed loss of cell viability in 500-µM-chrysene-treated cultures (24.7%, p<0.001). Similarly, pretreatment with 80 µM lipoic acid before chrysene resulted in decreased caspase-3/7 activities (75.7%, p<0.001), decreased ROS/RNS levels (80.02%, p<0.001), increased ΔΨm values (86%, p<0.001), and increased ATP levels (40.5%, p<0.001) compared to 500-µM-chrysene-treated cultures.
CONCLUSIONS
Chrysene, a component of cigarette smoke, can diminish cell viability in MIO-M1 cells in vitro by apoptosis at the lower concentrations of Chrysene (300 and 500 µM) and necrosis at the highest concentration. Moreover, mitochondrial function was particularly altered. However, lipoic acid can partially reverse the cytotoxic effect of chrysene. Lipoic acid administration may reduce or prevent Müller cell degeneration in retinal degenerative disorders.
目的
本研究评估了苯并[a]芘（香烟烟雾中的一种成分）对体外 Müller 细胞（MIO-M1）的毒性作用，并研究抑制剂硫辛酸是否能逆转苯并[a]芘诱导的毒性作用。
方法
MIO-M1 细胞暴露于不同浓度的苯并[a]芘，同时或不同时添加硫辛酸。通过台盼蓝染料排斥试验测定细胞活力。通过荧光染料试验测定半胱天冬酶-3/7 活性。通过乳酸脱氢酶（LDH）试验定量 LDH 释放。用 2',7'-二氯二氢荧光素二乙酸酯染料试验测定活性氧/氮物种（ROS/RNS）的产生。使用 JC-1 试验测量线粒体膜电位（ΔΨm）。通过 ATPLite 试剂盒测定细胞内 ATP 含量。
结果
与对照组相比，MIO-M1 细胞在暴露于 300、500 和 1000 μM 苯并[a]芘后，细胞活力显著降低，半胱天冬酶-3/7 活性增加，在最高苯并[a]芘浓度下 LDH 释放增加，ROS/RNS 水平升高，ΔΨm 值降低，细胞内 ATP 含量降低。用 80 μM 硫辛酸预处理可逆转 500 μM 苯并[a]芘处理培养物中的细胞活力丧失（24.7%，p<0.001）。同样，在苯并[a]芘处理前用 80 μM 硫辛酸预处理导致与 500 μM 苯并[a]芘处理培养物相比，半胱天冬酶-3/7 活性降低（75.7%，p<0.001），ROS/RNS 水平降低（80.02%，p<0.001），ΔΨm 值增加（86%，p<0.001），ATP 水平增加（40.5%，p<0.001）。
结论
苯并[a]芘作为香烟烟雾的一种成分，在较低浓度（300 和 500 μM）的苯并[a]芘作用下可通过凋亡降低体外 MIO-M1 细胞的活力，在最高浓度下可导致坏死。此外，线粒体功能发生了特别改变。然而，硫辛酸可部分逆转苯并[a]芘的细胞毒性作用。给予硫辛酸可能会减少或预防视网膜退行性疾病中的 Müller 细胞变性。
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