过氧化氢诱导猪卵巢颗粒细胞自噬及其对凋亡的影响

[目的] 本试验旨在研究抑制氧化应激诱导的自噬对猪卵巢颗粒细胞凋亡的作用。[方法] 采用流式细胞术和细胞计数试剂盒（cell counting kit-8，CCK8）检测不同浓度（0、50、100和150 μmol？L-1）过氧化氢处理12 h对猪颗粒细胞的凋亡诱导作用和细胞活力的影响；Western-blot检测微管相关蛋白1轻链3（LC3，包括LC3-Ⅰ和LC3-Ⅱ）和自噬相关蛋白P62/SQSTM1的表达量变化，转染GFP-LC3质粒检测自噬体的数量；利用3-甲基腺嘌呤（3-MA）提前4 h抑制自噬后再氧化应激12 h，用流式细胞术检测细胞的凋亡率，CCK8检测细胞活力。[结果] 氧化应激12 h后，细胞活力随过氧化氢浓度提高显著降低，凋亡率显著上升。在氧化应激前6 h LC3-Ⅱ相对表达量先增加后减少，P62的相对表达量先减少再增加；转染GFP-LC3质粒后，自噬体数量极显著上调，6 h后显著下降，上述结果表明在氧化应激早期自噬增加，持续氧化应激自噬受到抑制。若提前4 h用3-MA抑制自噬，再用过氧化氢处理12 h，抑制自噬后细胞凋亡率较单独氧化应激组显著下降，细胞活力也显著上升。[结论] 氧化应激能诱导猪卵巢颗粒细胞的凋亡和早期自噬，若抑制早期自噬，凋亡率会下降。
[Objectives] The paper aims to investigate the interplay between autophagy and apoptosis of porcine ovarian granulose cells in the response to oxidative stress. [Methods] We detected apoptosis by flow cytometry,and the cell counting kit-8(CCK8) assay was used to measure cell viability after 12 hours of exposure to H2O2 at different concentrations. The expression of microtubule-associated protein 1 light chain 3(LC3,including LC3-Ⅰ and LC3-Ⅱ) and autophagy adaptor protein p62/SQSTM1 was detected by Western-blot. The number of autophagosomes was demonstrated by transfecting cells with the GFP-LC3 plasmid. Moreover,after 4 hours of 3-methyladenine(3-MA) treatment and 12 hours of oxidative stimuli,granulosa cell viability was detected by CCK8,and apoptosis rate was calculated by flow cytometry. [Results] The results showed that a 12 hours H2O2 exposure decreased cell viability evidently and significantly increased the percentage of apoptotic cells. The relative expression of LC-Ⅱ increased first and then decreased within 6 hours of oxidative stress stimuli while the expression tendency of P62 was on the contrary. The relative expression of LC-Ⅱ increased first and then decreased within 6 hours of oxidative stress stimuli while the expression tendency of P62 was on the contrary. Similarly,GFP-LC3 transfection significantly promoted the formation of autophagic puncta,which reduced dramatically after 6 hours of H2O2 treatment. These data may indicate that autophagy in porcine ovarian granulosa cells was activated at the early stage of oxidative stress,but would be restrained under prolonged oxidative stress. Furthermore,apoptotic rates in cells treated with 3-MA for 4 hours before 12 hours of H2O2 exposure decreased markedly conpared to H2O2-only-treated cells,and cell viability increased significantly in the 3-MA treated group. [Conclusions] Our findings demonstrate that early stage of oxidative stress promotes autophay,and the inhibition of autophay may reduce apoptosis in the later stage