LI Liechuan,SHEN Ming,NING Caibo,et al.Induction of granulosa cell autophagy and effects in apoptosis by hydrogen peroxide in porcine ovaries[J].Journal of Nanjing Agricultural University,2016,39(5):814-818.[doi:10.7685/jnau.201601001]





Induction of granulosa cell autophagy and effects in apoptosis by hydrogen peroxide in porcine ovaries
李烈川 申明 宁彩波 管志强 蒋毅 吴望军 刘红林
南京农业大学动物科技学院, 江苏 南京 210095
LI Liechuan SHEN Ming NING Caibo GUAN Zhiqiang JIANG Yi WU Wangjun LIU Honglin
College of Animal Sciences and Technology, Nanjing Agricultural University, Nanjing 210095, China
oxidative stressautophagyapoptosisporcine ovarian granulosa cells3-MA
[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.


[1] Matsuda-Minehata F,Inoue N,Goto Y,et al. The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules[J]. Journal of Reproduction and Development,2006,52(6):695-705.
[2] Klionsky D J. The molecular machinery of autophagy:unanswered questions[J]. Journal of Cell Science,2005,118(1):7-18.
[3] Cuervo A M,Wong E. Chaperone-mediated autophagy:roles in disease and aging[J]. Cell Research,2014,24(1):92-104.
[4] Wong A S,Cheung Z H,Ip N Y. Molecular machinery of macroautophagy and its deregulation in diseases[J]. Biochimica et Biophysica Acta(BBA):2011,1812(11):1490-1497.
[5] Castino R,Bellio N,Follo C,et al. Inhibition of PI3k class Ⅲ-dependent autophagy prevents apoptosis and necrosis by oxidative stress in dopaminergic neuroblastoma cells[J]. Toxicological Sciences,2010,117(1):152-162.
[6] Ding Y,Kim J K,Kim S I,et al. TGF-β1 protects against mesangial cell apoptosis via induction of autophagy[J]. Journal of Biological Chemistry,2010,285(48):37909-37919.
[7] Dodson M,Liang Q,Johnson M S,et al. Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells[J]. Autophagy,2013,9(12):1996-2008.
[8] Lock R,Debnath J. Extracellular matrix regulation of autophagy[J]. Current Opinion in Cell Biology,2008,20(5):583-588.
[9] Wu W,Zhang X,Hu X,et al. Lactate down-regulates matrix systhesis and promotes apoptosis and autophagy in rat nucleus pulposus cells[J]. Journal of Orthopaedic Research,2014,32(2):253-261.
[10] Orrenius S. Reactive oxygen species in mitochondria-mediated cell death[J]. Drug Metabolism Reviews,2007,39(2/3):443-455.
[11] Shen M,Lin F,Zhang J,et al. Involvement of the up-regulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress[J]. Journal of Biological Chemistry,2012,287(31):25727-25740.
[12] Wu Y,Tan H,Shui G,et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on classⅠand Ⅲ phosphoinositide 3-kinase[J]. Journal of Biological Chemistry,2010,285(14):10850-10861.
[13] Song C,Song C,Tong F. Autophagy induction is a survival response against oxidative stress in bone marrow-derived mesenchymal stromal cells[J]. Cytotherapy,2014,16(10):1361-1370.
[14] Choi J,Jo M,Lee E,et al. Induction of apoptotic cell death via accumulation of autophagosomes in rat granulosa cells[J]. Fertility and Sterility,2011,95(4):1482-1486.
[15] Choi J Y,Jo M W,Lee E Y,et al. The role of autophagy in follicular development and atresia in rat granulosa cells[J]. Fertility and Sterility,2010,93(8):2532-2537.
[16] Chen J,Ni B,Li B,et al. The responses of autophagy and apoptosis to oxidative stress in nucleus pulposus cells:implications for disc degeneration[J]. Cellular Physiology and Biochemistry,2014,34(4):1175-1189.


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更新日期/Last Update: 1900-01-01