依达拉奉在庆大霉素诱导MDCK细胞凋亡中的保护作用
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摘要
为研究依达拉奉(edaravone,EDa)在庆大霉素(gentamicin,GM)诱导MDCK细胞凋亡中的保护作用,本研究用4 mmol/L GM和40?mol/L EDa单独或联合处理MDCK细胞24 h,Annexin V-FITC/PI双染检测细胞凋亡率;Hochest 33258染色观察细胞核形态变化;比色法检测总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)活力和丙二醛(MDA)含量;荧光染色检测活性氧(ROS)含量;Western-blot检测Bax与Bcl-2表达量,caspase-9、caspase-3和PARP蛋白活化情况,细胞色素C(Cyt C)和凋亡诱导因子(AIF)释放情况。结果显示,与对照组相比,EDa组无显著性影响。与GM组相比,GM+EDa组细胞凋亡率显著或极显著性下降(P<0.05或P<0.01);T-SOD和GSH-PX活力显著性下降(P<0.05);CAT活力极显著性上升(P<0.01);MDA含量极显著性下降(P<0.01);ROS含量显著性下降(P<0.05);Bcl-2/Bax比值显著性升高(P<0.05);caspase-9、caspase-3和PARP蛋白的活化水平显著或极显著性降低(P<0.05或P<0.01);Cyt C与AIF含量显著性降低(P<0.05)。结果表明,依达拉奉在庆大霉素通过线粒体凋亡途径诱导MDCK细胞凋亡中起保护作用。
In order to investigate the protective machanism of edaravone(EDa) on apoptosis induced by gentamicin(GM) in MDCK cells,MDCK cells were treated with 4 mmol/L GM and 40 ?mol/L EDa for 24 h respectively or unitedly.The apoptotic rate was detected by Annexin V-FITC/PI double staining.The changes of nuclear morphology were observed by Hochest 33258 staining.T-SOD,GSH-PX,CAT activity and MDA content were detected by colorimetric assay.ROS content was detected by fluorescent staining.The expression of Bax and Bcl-2,the activation of caspase-9,caspase-3 and PARP protein,the release of Cytochrome(Cyt C)and apoptosis-induced factor(AIF) were detected by Western-blotting.In result,the Eda group was no significant effect compared with the control group.Compared with the GM group,the apoptotic rate of the GM+EDa group was significantly or extremely ignificantly decreased(P<0.05 or P<0.01).The T-SOD and GSH-PX activities were significantly decreased(P<0.05).CAT activity was extremely significantly increased(P<0.01).MDA content decreased extremely significantly(P<0.01).ROS content decreased significantly(P<0.05).Bcl-2/Bax ratio increased significantly(P<0.05).The activation level of caspase-9,caspase-3 and PARP protein was significantly or extremely significantly decreased(P<0.05 or P<0.01).The contents of Cyt C and AIF were significantly decreased(P<0.05).These findings suggested that EDa can relieve oxidative damage to supperess the gentamicin-induced apoptosis through mitochondrial apoptotic pathway.
In order to investigate the protective machanism of edaravone(EDa) on apoptosis induced by gentamicin(GM) in MDCK cells,MDCK cells were treated with 4 mmol/L GM and 40 ?mol/L EDa for 24 h respectively or unitedly.The apoptotic rate was detected by Annexin V-FITC/PI double staining.The changes of nuclear morphology were observed by Hochest 33258 staining.T-SOD,GSH-PX,CAT activity and MDA content were detected by colorimetric assay.ROS content was detected by fluorescent staining.The expression of Bax and Bcl-2,the activation of caspase-9,caspase-3 and PARP protein,the release of Cytochrome(Cyt C)and apoptosis-induced factor(AIF) were detected by Western-blotting.In result,the Eda group was no significant effect compared with the control group.Compared with the GM group,the apoptotic rate of the GM+EDa group was significantly or extremely ignificantly decreased(P<0.05 or P<0.01).The T-SOD and GSH-PX activities were significantly decreased(P<0.05).CAT activity was extremely significantly increased(P<0.01).MDA content decreased extremely significantly(P<0.01).ROS content decreased significantly(P<0.05).Bcl-2/Bax ratio increased significantly(P<0.05).The activation level of caspase-9,caspase-3 and PARP protein was significantly or extremely significantly decreased(P<0.05 or P<0.01).The contents of Cyt C and AIF were significantly decreased(P<0.05).These findings suggested that EDa can relieve oxidative damage to supperess the gentamicin-induced apoptosis through mitochondrial apoptotic pathway.
引文
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[16]王小洪,丁冬怡,孙志颖,等.迷迭香酸甲酯对6-OHDA所致脑内氧化损伤的影响[J].扬州大学学报:农业与生命科学版,2017,38(4):30-34.WANG Xiao-hong,DING Dong-yi,SUN Zhi-ying,et al.Effect of methylrosmarinate on the oxidative stress of mice induced by 6-OHDA[J].Journal of Yangzhou University:Agricultural and Life Science Edition,2017,38(4):30-34.(in Chinese)
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[18]袁波.α-硫辛酸对庆大霉素所致大鼠耳毒性损害的保护作用[D].沈阳:中国医科大学,2005.YUAN Bo.α-lipoic acid protection for entamicin-induced wistar rat cochleotoxicity[D].Shenyang:China Medical University,2005.(in Chinese)
[19]HENGARTNER M O.The biochemistry of apoptosis[J].Nature,2000,407(6805):770-776.
[20]顾晰.庆大霉素对小鼠耳蜗螺旋神经节细胞损伤机制研究[D].福州:福建医科大学,2009.GU Xi.Study of the injury mechanism of cultured spiral ganglion cells from mouse cochlea induced by gentamicin[D].Fuzhou:Fujian Medical University,2019.(in Chinese)
[2]WATANABE T,TAHARA M,TODO S.The novel antioxidant edaravone:from bench to bedside[J].Cardiovascular Therapeutics,2008,26(2):101-114.
[3]YOSHIDA H,YANAI H,NAMIKI Y,et al.Neuroprotective effects of edaravone:a novel free radical scavenger in cerebrovascular injury[J].Cns Drug Reviews,2010,12(1):9-20.
[4]QIN H,BUCKLEY J A,LI X,et al.Inhibition of the JAK/STAT pathway protects againstα-synuclein-induced neuroinflammation and dopaminergic neurodegeneration[J].Journal of Neuroscience the Official Journal of the Society for Neuroscience,2016,36(18):5144.
[5]QI F,LI A,INAGAKI Y,et al.Induction of apoptosis by cinobufacini preparation through mitochondria-and Fas-mediated caspase-dependent pathways in human hepatocellular carcinoma cells[J].Food&Chemical Toxicology,2012,50(2):295-302.
[6]MORIO Y,TSUJI M,INAGAKI M,et al.Ethanol-induced apoptosis in human liver adenocarcinoma cells(SK-Hep1):Fas-and mitochondria-mediated pathways and interaction with MAPK signaling system[J].Toxicology in Vitro,2013,27(6):1820-1829.
[7]赵彦超,顾耘.细胞凋亡通路研究进展[J].现代医学,2013(4):285-288.ZHAO Yan-chao,GU Yun.Progress in research on apoptosis pathway[J].Modern Medical Journal,2013(4):285-288.(in Chinese)
[8]ABDEL MONEIM A E,OTHMAN M S,AREF A M.Azadirachta indica attenuates cisplatin-induced nephrotoxicity and oxidative stress[J].Biomed Research International,2014:647131.
[9]张敏,沈建明.依达拉奉减轻大鼠肾小管上皮细胞氧化应激与凋亡作用[J].医药导报,2015(7):875-878.ZHANG Min,SHEN Jian-ming.Protection of edararone on oxidative stress and apoptosis 40 renal tubular epithelial cells in rats[J].Herald of Medicine,2015(7):875-878.(in Chinese)
[10]ZOU P,SONG J,JIANG B,et al.Epigallocatechin-3-gallate protects against cisplatin nephrotoxicity by inhibiting the apoptosis in mouse[J].International Journal of Clinical&Experimental Pathology,2014,7(8):4607-4616.
[11]颜玉文,魏中琦,鞠婧婧,等.细胞松弛素H对MPP+诱导PC12细胞凋亡的保护作用[J].扬州大学学报:农业与生命科学版,2014,35(1):16-21.YAN Yu-wen,WEI Zhong-qi,JU Jing-jing,et al.Protective effects of cytochalasin H on MPP+-induced apoptosis in PC12 cells[J].Journal of Yangzhou Universi ty:Agricultural and Life Science Edition,2014,35(1):16-21.(in Chinese)
[12]卫玮,韩兵社,关丽英,等.活性氧激活线粒体凋亡通路是亚硒酸钠诱导NB4细胞凋亡的重要机制[J].中国医学科学院学报,2007,29(3):324-328.WEI Wei,HAN Bing-she,GUAN Li-ying,et al.Mitochondrial transmembrane potential loss caused by reactive oxygen species plays a major role in sodium selenite-induced apoptosis in NB4 cells[J].Acta Academiae Medicinae Sinicae,2007,29(3):324-328.(in Chinese)
[13]汪延辉,刘毅,郭利明,等.番茄红素及维生素E和C合用对庆大霉素大鼠肾毒性预防作用的比较[J].中国药理学与毒理学杂志,2008,22(6):436-439.WANG Yan-hui,LIU Yi,GUO Li-ming,et al.Effect comparison of lycopene and combination of vitamins E and C on gentamicin-induced nephrotoxicity in rats[J].Chinese Journal of Pharmacology and Toxicology,2008,22(6):436-439.(in Chinese)
[14]王洪娇.还原型谷胱甘肽对庆大霉素致大鼠耳蜗毛细胞凋亡及磷酸化JNK的影响[D].沈阳:中国医科大学,2007.WANG Hong-jiao.Effects of glutathione on gentamicin-induced cochlear hair cell apoptosis and phosphorylated JNK in rat[D].Shenyang:China Medical University Graduate School,2007.(in Chinese)
[15]ORTIZ-ESPEJO M,GIL-CAMPOS M,MESA M D,et al.Alterations in the antioxidant defense system in prepubertal children with a history of extrauterine growth restriction[J].European Journal of Nutrition,2014,53(2):607-615.
[16]王小洪,丁冬怡,孙志颖,等.迷迭香酸甲酯对6-OHDA所致脑内氧化损伤的影响[J].扬州大学学报:农业与生命科学版,2017,38(4):30-34.WANG Xiao-hong,DING Dong-yi,SUN Zhi-ying,et al.Effect of methylrosmarinate on the oxidative stress of mice induced by 6-OHDA[J].Journal of Yangzhou University:Agricultural and Life Science Edition,2017,38(4):30-34.(in Chinese)
[17]KARAHAN I,ATESSAHIN A,YILMAZ S,et al.Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats[J].Toxicology,2005,215(3):198-204.
[18]袁波.α-硫辛酸对庆大霉素所致大鼠耳毒性损害的保护作用[D].沈阳:中国医科大学,2005.YUAN Bo.α-lipoic acid protection for entamicin-induced wistar rat cochleotoxicity[D].Shenyang:China Medical University,2005.(in Chinese)
[19]HENGARTNER M O.The biochemistry of apoptosis[J].Nature,2000,407(6805):770-776.
[20]顾晰.庆大霉素对小鼠耳蜗螺旋神经节细胞损伤机制研究[D].福州:福建医科大学,2009.GU Xi.Study of the injury mechanism of cultured spiral ganglion cells from mouse cochlea induced by gentamicin[D].Fuzhou:Fujian Medical University,2019.(in Chinese)