α-硫辛酸对小鼠卵巢颗粒细胞氧化应激损伤和凋亡的影响

α-lipoic acid protects mouse ovarian granulosa cells from oxidative stress and apoptosis

目的探讨α-硫辛酸对小鼠颗粒细胞培养过程中抗氧化水平和抗凋亡水平的影响。方法收集经PMSG处理的小鼠卵巢颗粒细胞,经原代培养24h后,添加终浓度为100μmol/L的α-硫辛酸继续培养24h,测定氧化水平SOD、MDA和GSH-Px参数,评价抗氧化能力,采用Trizol法提取各组细胞的RNA,进行RT-PCR法检测α-硫辛酸对caspase-3和caspase-9基因的表达情况的影响,同时利用细胞免疫荧光和ELISA法检测caspase-3和caspase-9蛋白表达,并分析α-硫辛酸降低细胞凋亡的作用。结果分离培养的小鼠卵巢颗粒细胞α-硫辛酸添加后,SOD和GSH-Px活性升高,MDA含量降低(P<0.05);经过RT-PCR扩增技术获得长度为180bp的特异性caspase-3产物,获得长度为152bp的特异性caspase-9产物,α-硫辛酸添加可显著性降低caspase-3和caspase-9mRNA的表达(P<0.05);免疫荧光细胞化学染色显示颗粒细胞caspase-3和caspase-9蛋白阳性染色定位于细胞胞质,分别呈现绿色荧光和红色荧光,细胞核呈蓝色荧光;ELISA结果显示α-硫辛酸添加后caspase-3和caspase-9酶活性显著下调(P<0.05)。结论小鼠颗粒细胞培养过程中添加α-硫辛酸可明显改变细胞抗氧化能力,对卵巢颗粒细胞培养过程中的氧化应激诱导的细胞凋亡起到一定的保护作用。

Objective To study the protective effects of α-lipoic acid on oxidative stress and apoptosis of mouse ovary granulose cells cultured in vitro. Methods Granulosa cells were collected from PMSG-treated mice and cultured in DMEM/F12 medium for 24 hl then the cell medium was supplemented with 100μmol/L α-lipoic acid for 24 h culture. After α-lipoic acid treatment, SOD, MDA and GSH-Px activities in cell supernatant were measured. Additionally, RT-PCR, immunocytochemistry and Western blot were performed to detect the expressions of caspase-3 and caspase-9, Immunofluorescence and ELISA were used to evaluate the expressions of caspase-3 and caspase-9 at the protein level in whole granulosa cell lysate protein in both groups. Results α-lipoic acid supplementation increased SOD and GSH-Px activities and decreased MDA content notably （ P 〈 0. 05）. PCR products of caspase-3 and caspase-9 in granulosa cells were analyzed using RT-PCR and Real-time PCR. The expected 180 bp product was verified for caspase-3 and 152bp for caspase-9 by electrophoresis and gel imager in α-lipoic acid group and control group. Expressions of caspase-3 and caspase-9 at mRNA level showed a decreased tendency in α-lipoic acid group （P（0.05）. For immunofluorescence analysis, caspase-3 and caspase-9 protein of granulosa cells was detected. Cell-specific positive staining was located in the cytoplasm of the granulosa cells which showed green fluorescence for caspase-3 and caspase-9 proteins cell nuclei showed blue fluorescence. In α-lipoic acid group it has a lower bands density values than the control group by Western blot analysis （P〈0.05）. ELISA assay showed a significant decrease of caspase-3 and caspase-9 activities in α-lipoic acid group （P〈0.05）. Oonclusion α-lipoic acid can significantly change the anti-oxidation ability of mouse ovarian granulosa cells, which suggests that it has protective and antioxidant effects on oxidative stress and apoptosis during the culture of mouse ovarian granulosa cells.