过氧化氢对成肌细胞的氧化损伤作用研究
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摘要
旨在以体外培养的小鼠成肌细胞(C2C12)为研究对象,探讨氧化损伤可能的路径。本试验分为3个处理组,每个处理组6个重复。处理组分别为:对照组(Control),以DMEM培养基培养的细胞作为空白对照,培养24 h;吡咯烷二硫代氨基甲酸酯(PDTC)组(PDTC),以每个孔1 mL的PDTC(20μmol·L~(-1))孵育细胞24 h;H_2O_2组(H_2O_2),加入0.5 mmol·L~(-1)的H_2O_2处理细胞24 h。检测各组细胞的存活率、ROS水平与凋亡率,采用荧光定量PCR和蛋白免疫印记法检测细胞凋亡和自噬相关基因的表达以及NF-κB信号通路相关因子的表达。结果表明,与对照组相比,PDTC可显著提高细胞的总凋亡率(P<0.05),可显著上调细胞半胱天冬蛋白酶-3(caspase-3)、caspase-6、caspase-9的mRNA表达量以及caspase-3蛋白表达水平(P<0.05),显著上调Beclin 1的mRNA表达量和微管相关蛋白1轻链3(LC3)-II/I的值以及LC3-II蛋白表达水平(P<0.05),显著下调核因子kappa B副族1(p50)、B型白细胞/2型淋巴细胞样蛋白(Bcl-2)、v-rel禽网状内皮增生病病毒癌基因同源物A(RelA)和环氧合酶(Cox)-2的mRNA表达量以及NF-κB蛋白表达水平(P<0.05);H_2O_2可显著提高细胞的ROS水平和细胞总凋亡率(P<0.05),可显著上调caspase-3、caspase-6、caspase-8、caspase-9的mRNA表达量以及caspase-3蛋白表达水平(P<0.05),显著上调Beclin 1和LC3-II/LC3-I的mRNA表达量以及LC3-II蛋白表达水平(P<0.05),显著提高Bcl-2相关X蛋白(Bax)的mRNA表达量(P<0.05),显著下调p50、Bcl-2、RelA和Cox-1、Cox-2的mRNA表达量以及核因子kappa B(NF-κB)蛋白表达水平(P<0.05)。结果提示,H_2O_2会引起C2C12细胞的ROS升高,并且能够通过抑制NF-κB信号通路介导细胞凋亡和自噬的发生,这与NF-κB因子的特异性抑制剂PDTC的作用相类似。
The change of C2 C12 cells exposed to H_2O_2 was investigated and the possible path of oxidative damage was explored in this study. There was 3 treatments, and 6 repeats in each treatment. C2 C12 cells attached to the substrate and cultured in DMEM for 24 h were treated as control(n = 6); the medium was added with PDTC(20 μmol·L~(-1), PDTC treatment, n = 6) for 24 h or H_2O_2(0.5 mmol·L~(-1), H_2O_2 treatment, n = 6) for 24 h. The viability, ROS level and apoptosis rate of cells in different treatments were detected. The expression of genes related to cell apoptosis and autophagy and factors related to NF-κB signaling pathway were determined by real-time PCR and Western blot. The results showed that cells in PDTC treatment had significantly higher total apoptosis rate, mRNA expression of caspase-3, caspase-6, caspase-9 and the protein expression of total caspase-3, mRNA expression of Beclin1, LC3-II/LC3-I and the protein expression level of total LC3-II(P<0.05), and had significantly lower mRNA expression of p50, Bcl-2, RelA, Cox-2 and protein expression of total NF-κB compared to that in the control treatment(P<0.05). Moreover, cells in H_2O_2 treatment exhibited significantly higher ROS level and total apoptosis rate(P<0.05), mRNA expression of caspase-3, caspase-6, caspase-8, caspase-9 and protein expression of total caspase-3(P<0.05), mRNA expression of Beclin 1, LC3-II/LC3-I and protein expression of total LC3-II(P<0.05), mRNA expression of Bax(P<0.05), and had significantly lower mRNA expressions of p50, Bcl-2, RelA, Cox-1, Cox-2 and protein expression of total NF-κB(P<0.05) compared to that in the control treatment. These results indicated that H_2O_2 could promote the ROS formation, mediated the apoptosis and autophagy processes by suppressing the NF-κB signaling pathway in C2 C12 cells, which was similar with the role of PDTC, the inhibitor of NF-κB factor.
The change of C2 C12 cells exposed to H_2O_2 was investigated and the possible path of oxidative damage was explored in this study. There was 3 treatments, and 6 repeats in each treatment. C2 C12 cells attached to the substrate and cultured in DMEM for 24 h were treated as control(n = 6); the medium was added with PDTC(20 μmol·L~(-1), PDTC treatment, n = 6) for 24 h or H_2O_2(0.5 mmol·L~(-1), H_2O_2 treatment, n = 6) for 24 h. The viability, ROS level and apoptosis rate of cells in different treatments were detected. The expression of genes related to cell apoptosis and autophagy and factors related to NF-κB signaling pathway were determined by real-time PCR and Western blot. The results showed that cells in PDTC treatment had significantly higher total apoptosis rate, mRNA expression of caspase-3, caspase-6, caspase-9 and the protein expression of total caspase-3, mRNA expression of Beclin1, LC3-II/LC3-I and the protein expression level of total LC3-II(P<0.05), and had significantly lower mRNA expression of p50, Bcl-2, RelA, Cox-2 and protein expression of total NF-κB compared to that in the control treatment(P<0.05). Moreover, cells in H_2O_2 treatment exhibited significantly higher ROS level and total apoptosis rate(P<0.05), mRNA expression of caspase-3, caspase-6, caspase-8, caspase-9 and protein expression of total caspase-3(P<0.05), mRNA expression of Beclin 1, LC3-II/LC3-I and protein expression of total LC3-II(P<0.05), mRNA expression of Bax(P<0.05), and had significantly lower mRNA expressions of p50, Bcl-2, RelA, Cox-1, Cox-2 and protein expression of total NF-κB(P<0.05) compared to that in the control treatment. These results indicated that H_2O_2 could promote the ROS formation, mediated the apoptosis and autophagy processes by suppressing the NF-κB signaling pathway in C2 C12 cells, which was similar with the role of PDTC, the inhibitor of NF-κB factor.
引文
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[11] 陈伟,林映才,马现永,等.一些抗氧化剂的抗/促氧化作用及其机制[J].动物营养学报,2012,24(4):595-605.CHEN W,LIN Y C,MA X Y,et al.Anti/pro-oxidative functions of antioxidants and their mechanisms[J].Chinese Journal of Animal Nutrition,2012,24(4):595-605.(in Chinese)
[12] YIN J,REN W K,WU X S,et al.Oxidative stress-mediated signaling pathways:a review[J].J Food Agric Environ,2013,11(2):132-139.
[13] LEE J C,SON Y O,CHOI K C,et al.Hydrogen peroxide induces apoptosis of BJAB cells due to formation of hydroxyl radicals via intracellular iron-mediated Fenton chemistry in glucose oxidase-mediated oxidative stress[J].Mol Cells,2006,22(1):21-29.
[14] ESSICK E E,WILSON R M,PIMENTEL D R,et al.Adiponectin modulates oxidative stress-induced autophagy in cardiomyocytes[J].PLoS One,2013,8(7):e68697.
[15] 韩飞,周孟良.过氧化氢诱导HepG2细胞产生氧化应激细胞模型的建立[J].食品科学,2011,32(5):55-57.HAN F,ZHOU M L.An experimental HepG2 cell model of hydrogen peroxide induced DNA oxidative injury[J].Food Science,2011,32(5):55-57.(in Chinese)
[16] YAZAKI T,HIRADATE Y,HOSHINO Y,et al.L-arnitine improves hydrogen peroxide-induced impairment of nuclear maturation in porcine oocytes[J].Anim Sci J,2013,84(5):395-402.
[17] 杨金莲.酒精刺激激活NF-κB信号通路促进肝癌的生长和转移[D].合肥:安徽医科大学,2012.YANG J L.NF-κB activation mediates ethanol-promoted liver cancer progression[D].Hefei:Anhui Medical University,2012.(in Chinese)
[18] 程何祥,张荣庆,马颖艳,等.过氧化氢诱导乳鼠心肌细胞凋亡及半胱天冬酶-3抑制剂的作用[J].第四军医大学学报,2001,22(21):1953-1956.CHENG H X,ZHANG R Q,MA Y Y,et al.Apoptosis of neonatal rat cardiomyocytes induced by exogenous hydroxyl free radical and the effect of caspase-3 antagonist[J].Journal of the Fourth Military Medical University,2001,22(21):1953-1956.(in Chinese)
[19] 肖卫民,蒋碧梅,石永忠,等.过氧化氢诱导C2C12细胞凋亡的机制初探[J].医学临床研究,2004,21(11):1288-1291.XIAO W M,JIANG B M,SHI Y Z,et al.Preliminary investigation on the mechanism of C2C12 cell apoptosis induced by hydrogen peroxide[J].Journal of Clinical Research,2004,21(11):1288-1291.(in Chinese)
[20] JIANG B M,XIAO W M,SHI Y Z,et al.Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells[J].Free Radical Bio Med,2005,39(5):658-667.
[21] WYLLIE A H."Where,O death,is thy sting?" A brief review of apoptosis biology[J].Mol Neurobiol,2010,42(1):4-9.
[22] KIFFIN R,BANDYOPADHYAY U,CUERVO A M.Oxidative stress and autophagy[J].Antioxid Redox Sign,2006,8(1-2):152-162.
[23] 易蓓,袁海汀,许永会,等.NF-κB抑制剂PDTC对人骨髓瘤U266细胞增殖和凋亡的影响[J].中国病理生理杂志,2017,33(7):1177-1183.YI B,YUAN H T,XU Y H,et al.Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells[J].Chinese Journal of Pathophysiology,2017,33(7):1177-1183.(in Chinese)
[24] BYUN Y J,KIM S K,KIM Y M,et al.Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway[J].Neurosci Lett,2009,461(2):131-135.
[25] ZHANG H Y,KONG X X,KANG J S,et al.Oxidative stress induces parallel autophagy and mitochondria dysfunction in human glioma U251 cells[J].Toxicol Sci,2009,110(2):376-388.
[26] YU L,WAN F Y,DUTTA S,et al.Autophagic programmed cell death by selective catalase degradation[J].Proc Natl Acad Sci USA,2006,103(13):4952-4957.
[27] SCHERZ-SHOUVAL R,SHVETS E,FASS E,et al.Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4[J].EMBO J,2007,26(7):1749-1760.
[28] GAO H,LIN L,HAQ I U,et al.Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells[J].Biochem Biophys Res Commun,2016,473(1):311-316.
[29] YUAN L,WEI S P,WANG J,et al.Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53,PI3K/Akt,JNK,and p38 signaling pathways in HepG2 cancer cells[J].J Agric Food Chem,2014,62(23):5390-5400.
[30] YIN J,DUAN J L,CUI Z J,et al.Hydrogen peroxide-induced oxidative stress activates NF-κB and Nrf2/Keap1 signals and triggers autophagy in piglets[J].RSC Adv,2015,5(20):15479-15486.
[31] 刘庆羊,孙自强,赵鸿雁,等.自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制[J].畜牧兽医学报,2018,49(8):1761-1769.LIU Q Y,SUN Z Q,ZHAO H Y,et al.Effects of autophagy on the apoptosis of osteoclasts and osteoclast precursors mediated by osteoprotegerin[J].Acta Veterinaria et Zootechnica Sinica,2018,49(8):1761-1769.(in Chinese)
[32] 胡林,南良康,张敏,等.猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析[J].畜牧兽医学报,2018,49(6):1274-1281.HU L,NAN L K,ZHANG M,et al.The mechanism of porcine Beclin1 gene on autophagy and apoptosis[J].Acta Veterinaria et Zootechnica Sinica,2018,49(6):1274-1281.(in Chinese)
[33] 张勇,崔岩.NF-κB在细胞凋亡中的调节作用和应用前景[J].动物营养学报,2011,23(5):715-719.ZHANG Y,CUI Y.NF-κB:modulation in apoptosis and application prospects[J].Chinese Journal of Animal Nutrition,2011,23(5):715-719.(in Chinese)
[34] CHEN X X,ZHANG L,LI J L,et al.Hydrogen peroxide-induced change in meat quality of the breast muscle of broilers is mediated by ROS generation,apoptosis,and autophagy in the NF-κB signal pathway[J].J Agric Food Chem,2017,65(19):3986-3994.
[35] CHEN X,GU R,ZHANG L,et al.Induction of nuclear factor-κB signal-mediated apoptosis and autophagy by reactive oxygen species is associated with hydrogen peroxide-impaired growth performance of broilers[J].Animal,2018,12(12):2561-2570.
[2] 邱小忠,余磊,廖华,等.活性氧促使L6成肌细胞的分化[J].第一军医大学学报,2005,25(11):1384-1386.QIU X Z,YU L,LIAO H,et al.Oxidative stress-induced differentiation of L6 myoblasts[J].Journal of First Military Medical University,2005,25(11):1384-1386.(in Chinese)
[3] FLANDIN P,DONATI Y,BARAZZONE-ARGIROFFO C,et al.Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle[J].FEBS Lett,2005,579(16):3411-3415.
[4] SIMON H U,HAJ-YEHIA A,LEVI-SCHAFFER F.Role of reactive oxygen species (ROS) in apoptosis induction[J].Apoptosis,2000,5(5):415-418.
[5] SCHERZ-SHOUVAL R,ELAZAR Z.Regulation of autophagy by ROS:physiology and pathology[J].Trends Biochem Sci,2011,36(1):30-38.
[6] JIANG Y,ZHANG W H,GAO F,et al.Micro-encapsulated sodium butyrate attenuates oxidative stress induced by corticosterone exposure and modulates apoptosis in intestinal mucosa of broiler chickens[J].Anim Prod Sci,2014,55(5):587-594.
[7] YIN Y,LU L,WANG D T,et al.Astragalus polysaccharide inhibits autophagy and apoptosis from peroxide-induced injury in C2C12 myoblasts[J].Cell Biochem Biophys,2015,73(2):433-439.
[8] LI H,MALHOTRA S,KUMAR A.Nuclear factor-kappa B signaling in skeletal muscle atrophy[J].J Mol Med,2008,86(10):1113-1126.
[9] 赵敏孟.胚蛋注射丙酮酸肌酸调控肉鸡能量代谢和肌肉发育的作用机理研究[D].南京:南京农业大学,2017.ZHAO M M.Regulatory mechanism of in ovo feeding of creatine pyruvate on energy metabolism and muscle development of broiler chickens[D].Nanjing:Nanjing Agricultural University,2017.(in Chinese)
[10] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[11] 陈伟,林映才,马现永,等.一些抗氧化剂的抗/促氧化作用及其机制[J].动物营养学报,2012,24(4):595-605.CHEN W,LIN Y C,MA X Y,et al.Anti/pro-oxidative functions of antioxidants and their mechanisms[J].Chinese Journal of Animal Nutrition,2012,24(4):595-605.(in Chinese)
[12] YIN J,REN W K,WU X S,et al.Oxidative stress-mediated signaling pathways:a review[J].J Food Agric Environ,2013,11(2):132-139.
[13] LEE J C,SON Y O,CHOI K C,et al.Hydrogen peroxide induces apoptosis of BJAB cells due to formation of hydroxyl radicals via intracellular iron-mediated Fenton chemistry in glucose oxidase-mediated oxidative stress[J].Mol Cells,2006,22(1):21-29.
[14] ESSICK E E,WILSON R M,PIMENTEL D R,et al.Adiponectin modulates oxidative stress-induced autophagy in cardiomyocytes[J].PLoS One,2013,8(7):e68697.
[15] 韩飞,周孟良.过氧化氢诱导HepG2细胞产生氧化应激细胞模型的建立[J].食品科学,2011,32(5):55-57.HAN F,ZHOU M L.An experimental HepG2 cell model of hydrogen peroxide induced DNA oxidative injury[J].Food Science,2011,32(5):55-57.(in Chinese)
[16] YAZAKI T,HIRADATE Y,HOSHINO Y,et al.L-arnitine improves hydrogen peroxide-induced impairment of nuclear maturation in porcine oocytes[J].Anim Sci J,2013,84(5):395-402.
[17] 杨金莲.酒精刺激激活NF-κB信号通路促进肝癌的生长和转移[D].合肥:安徽医科大学,2012.YANG J L.NF-κB activation mediates ethanol-promoted liver cancer progression[D].Hefei:Anhui Medical University,2012.(in Chinese)
[18] 程何祥,张荣庆,马颖艳,等.过氧化氢诱导乳鼠心肌细胞凋亡及半胱天冬酶-3抑制剂的作用[J].第四军医大学学报,2001,22(21):1953-1956.CHENG H X,ZHANG R Q,MA Y Y,et al.Apoptosis of neonatal rat cardiomyocytes induced by exogenous hydroxyl free radical and the effect of caspase-3 antagonist[J].Journal of the Fourth Military Medical University,2001,22(21):1953-1956.(in Chinese)
[19] 肖卫民,蒋碧梅,石永忠,等.过氧化氢诱导C2C12细胞凋亡的机制初探[J].医学临床研究,2004,21(11):1288-1291.XIAO W M,JIANG B M,SHI Y Z,et al.Preliminary investigation on the mechanism of C2C12 cell apoptosis induced by hydrogen peroxide[J].Journal of Clinical Research,2004,21(11):1288-1291.(in Chinese)
[20] JIANG B M,XIAO W M,SHI Y Z,et al.Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells[J].Free Radical Bio Med,2005,39(5):658-667.
[21] WYLLIE A H."Where,O death,is thy sting?" A brief review of apoptosis biology[J].Mol Neurobiol,2010,42(1):4-9.
[22] KIFFIN R,BANDYOPADHYAY U,CUERVO A M.Oxidative stress and autophagy[J].Antioxid Redox Sign,2006,8(1-2):152-162.
[23] 易蓓,袁海汀,许永会,等.NF-κB抑制剂PDTC对人骨髓瘤U266细胞增殖和凋亡的影响[J].中国病理生理杂志,2017,33(7):1177-1183.YI B,YUAN H T,XU Y H,et al.Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells[J].Chinese Journal of Pathophysiology,2017,33(7):1177-1183.(in Chinese)
[24] BYUN Y J,KIM S K,KIM Y M,et al.Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway[J].Neurosci Lett,2009,461(2):131-135.
[25] ZHANG H Y,KONG X X,KANG J S,et al.Oxidative stress induces parallel autophagy and mitochondria dysfunction in human glioma U251 cells[J].Toxicol Sci,2009,110(2):376-388.
[26] YU L,WAN F Y,DUTTA S,et al.Autophagic programmed cell death by selective catalase degradation[J].Proc Natl Acad Sci USA,2006,103(13):4952-4957.
[27] SCHERZ-SHOUVAL R,SHVETS E,FASS E,et al.Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4[J].EMBO J,2007,26(7):1749-1760.
[28] GAO H,LIN L,HAQ I U,et al.Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells[J].Biochem Biophys Res Commun,2016,473(1):311-316.
[29] YUAN L,WEI S P,WANG J,et al.Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53,PI3K/Akt,JNK,and p38 signaling pathways in HepG2 cancer cells[J].J Agric Food Chem,2014,62(23):5390-5400.
[30] YIN J,DUAN J L,CUI Z J,et al.Hydrogen peroxide-induced oxidative stress activates NF-κB and Nrf2/Keap1 signals and triggers autophagy in piglets[J].RSC Adv,2015,5(20):15479-15486.
[31] 刘庆羊,孙自强,赵鸿雁,等.自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制[J].畜牧兽医学报,2018,49(8):1761-1769.LIU Q Y,SUN Z Q,ZHAO H Y,et al.Effects of autophagy on the apoptosis of osteoclasts and osteoclast precursors mediated by osteoprotegerin[J].Acta Veterinaria et Zootechnica Sinica,2018,49(8):1761-1769.(in Chinese)
[32] 胡林,南良康,张敏,等.猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析[J].畜牧兽医学报,2018,49(6):1274-1281.HU L,NAN L K,ZHANG M,et al.The mechanism of porcine Beclin1 gene on autophagy and apoptosis[J].Acta Veterinaria et Zootechnica Sinica,2018,49(6):1274-1281.(in Chinese)
[33] 张勇,崔岩.NF-κB在细胞凋亡中的调节作用和应用前景[J].动物营养学报,2011,23(5):715-719.ZHANG Y,CUI Y.NF-κB:modulation in apoptosis and application prospects[J].Chinese Journal of Animal Nutrition,2011,23(5):715-719.(in Chinese)
[34] CHEN X X,ZHANG L,LI J L,et al.Hydrogen peroxide-induced change in meat quality of the breast muscle of broilers is mediated by ROS generation,apoptosis,and autophagy in the NF-κB signal pathway[J].J Agric Food Chem,2017,65(19):3986-3994.
[35] CHEN X,GU R,ZHANG L,et al.Induction of nuclear factor-κB signal-mediated apoptosis and autophagy by reactive oxygen species is associated with hydrogen peroxide-impaired growth performance of broilers[J].Animal,2018,12(12):2561-2570.