猪黄体不同发育时期活性氧水平及多种抗氧化酶mRNA表达量变化规律的研究
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摘要
试验旨在研究活性氧(ROS)及抗氧化酶基因表达在猪黄体组织发育及退化过程中的变化规律,为诠释猪黄体抗氧化机制补充理论基础并提供新的思路。试验所用卵巢采自延吉屠宰场,将黄体从卵巢剥离后,通过其形态大小将黄体初步分为初期、中期、后期以及白体,然后通过检测孕酮水平准确区分中期及后期黄体;通过冰冻切片及DHE荧光染色技术检测各时期黄体ROS水平,通过实时荧光定量PCR对各时期黄体内锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)以及谷胱甘肽过氧化氢酶(GPx)的mRNA表达量进行检测。结果表明:猪黄体内ROS水平随猪黄体周期性发育进程出现规律性升高,即后期ROS水平显著高于其他时期(P<0.05);中期显著高于初期与白体(P<0.05);而初期与白体间差异不显著(P>0.05)。Mn-SOD、GPx1及GPx4基因的表达水平随黄体的发育进程则出现规律性的下降:初期及白体中这几个基因的表达水平显著高于中期与后期(P<0.05),中期显著高于后期(P<0.05)。对于GPx3基因,其在白体的表达量显著高于其他时期(P<0.05),初期表达水平显著高于中期与后期(P<0.05),中期显著高于后期(P<0.05)。CAT基因表达量不随黄体的发育而变化(P>0.05)。综合上述试验结果,ROS、Mn-SOD及GPx与猪黄体发育及退化有关;而ROS水平与Mn-SOD及GPx的表达水平呈现出相反的规律。
The purpose of the experiment was to study the change law of reactive oxygen species(ROS) and the expression of antioxidant enzymes genes in the development and degradation of porcine corpus luteum,and to supplement the theoretical basis and provide new ideas for interpreting the antioxidant mechanism of porcine corpus luteum.The ovaries used in the experiment were collected from Yanji slaughterhouse.After removed from the ovary,the corpus luteum was initially divided into early corpus luteum,middle corpus luteum,late corpus luteum and white bodies by its size.The middle and late corpus luteum were then accurately separated by detecting progesterone levels.The levels of ROS in the corpus luteum were detected by frozen section and DHE fluorescence staining technique.The amount of expression of manganese superoxide dismutase(Mn-SOD),catalase(CAT) and Glutathione peroxidase(GPx) of antioxidant enzymes were examined by Real-time PCR in each period.The results showed that the ROS level of porcine corpus luteum increased regularly with the periodic development of porcine corpus luteum,that was,ROS level in the late period were significantly higher than that in other periods(P<0.05).And the ROS level in the middle period was significantly higher than that in the initial period and white body(P<0.05),and there was no significant difference between white body and the initial period(P>0.05).The expression of Mn-SOD, GPx1 and GPx4 decreased regularly,they were significantly higher in the initial period and white body than that in the middle and late stage(P<0.05),and their expression in the middle stage was significantly higher than that in the late stage(P<0.05).and the white body was significantly higher than that in other stages(P<0.05).The expression of GPx3 gene in white body was significantly higher than that in other stages(P<0.05),the expression in initial period was significantly higher than that in the middle and later stage(P<0.05),and the expression in the middle stage was significantly higher than that in the late stage(P<0.05).The CAT expression had no significant change with the development of the corpus luteum(P>0.05).In conclusion,ROS,Mn-SOD and GPx were the influencing factors for the development and degradation of corpus luteum,and the level of ROS was opposite to that of Mn-SOD and GPx.
The purpose of the experiment was to study the change law of reactive oxygen species(ROS) and the expression of antioxidant enzymes genes in the development and degradation of porcine corpus luteum,and to supplement the theoretical basis and provide new ideas for interpreting the antioxidant mechanism of porcine corpus luteum.The ovaries used in the experiment were collected from Yanji slaughterhouse.After removed from the ovary,the corpus luteum was initially divided into early corpus luteum,middle corpus luteum,late corpus luteum and white bodies by its size.The middle and late corpus luteum were then accurately separated by detecting progesterone levels.The levels of ROS in the corpus luteum were detected by frozen section and DHE fluorescence staining technique.The amount of expression of manganese superoxide dismutase(Mn-SOD),catalase(CAT) and Glutathione peroxidase(GPx) of antioxidant enzymes were examined by Real-time PCR in each period.The results showed that the ROS level of porcine corpus luteum increased regularly with the periodic development of porcine corpus luteum,that was,ROS level in the late period were significantly higher than that in other periods(P<0.05).And the ROS level in the middle period was significantly higher than that in the initial period and white body(P<0.05),and there was no significant difference between white body and the initial period(P>0.05).The expression of Mn-SOD, GPx1 and GPx4 decreased regularly,they were significantly higher in the initial period and white body than that in the middle and late stage(P<0.05),and their expression in the middle stage was significantly higher than that in the late stage(P<0.05).and the white body was significantly higher than that in other stages(P<0.05).The expression of GPx3 gene in white body was significantly higher than that in other stages(P<0.05),the expression in initial period was significantly higher than that in the middle and later stage(P<0.05),and the expression in the middle stage was significantly higher than that in the late stage(P<0.05).The CAT expression had no significant change with the development of the corpus luteum(P>0.05).In conclusion,ROS,Mn-SOD and GPx were the influencing factors for the development and degradation of corpus luteum,and the level of ROS was opposite to that of Mn-SOD and GPx.
引文
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[17] AL-GUBORY K H,CEBALLOS-PICOT I,NICOLE A,et al.Changes in activities of superoxide dismutase,nitric oxide synthase,glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle[J].BBA-General Subjects,2005,1725(3):348-357.
[18] SUGINO N,TAKIGUCHI S,KASHIDA S,et al.Suppression of intracellular superoxide dismutase activity by antisense oligonucleotides causes inhibition of progesterone production by rat luteal cells[J].Biology of Reproduction,1999,61(4):1133-1138.
[19] RUEDA B R,TILLY K I,HANSEN T R,et al.Expression of superoxide dismutase,catalase and glutathione peroxidase in the bovine corpusluteum:Evidence supporting a role for oxidative stress in luteolysis[J]. Endocrine,1995,3(3):227-232.
[20] NAKAMURA T,ISHIGAMI T,MAKINO N,et al.The down-regulation of glutathione feroxidase causes bovine luteal cell apoptosis during structural luteolysis[J]. Journal of Biochemistry,2001,129(6):937.
[2] JAHAUN A,GLORIA B.A review of the catalytic mechanism of human manganese superoxide dismutase[J].Antioxidants,2018,7(2):25-41.
[3] RHEE S G,LEE S K.Differential function of catalase,glutathione peroxidase and peroxiredoxin in mouse red blood cells[J]. Free Radical Biology and Medicine,2017,112:6.
[4] KINOWAKI Y,KURATA M,ISHIBASHI S,et al.Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma[J].Laboratory Investigation,2018,98(5):609-619.
[5] MIYAMOTO A,SHIRASUNA K,SASAHARA K.Local regulation of corpus luteum development and regression in the cow:Impact of angiogenic and vasoactive factors[J].Domestic Animal Endocrinology,2009,37(3):159-169.
[6] GHAVAMI S,SHOJAEI S,YEGANEH B,et al.Autophagy and apoptosis dysfunction in neurodegenerative disorders[J].Progress in Neurobiology,2014,112:24-49.
[7] PANIERI E,GOGVADZE V,NORBERG E,et al.Reactive oxygen species generated in different compartments induce cell death,survival,or senescence[J].Free Radical Biology & Medicine,2013,57(2):176-187.
[8] GHOSH J,MONDAL S.Nucleic acids and protein content in relation to growth and regression of buffalo (Bubalus bubalis) corpora lutea[J].Animal Reproduction Science,2006,93(3-4):316-327.
[9] SAKUMOTO R,KOMATSU T,KASUYA E.Expression of mRNAs for interleukin-4,interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle[J]. Domestic Animal Endocrinology,2006,31(3):246-257.
[10] GARREL C,CEBALLOS-PICOT I,GERMAIN G,et al.Oxidative stress-inducible antioxidant adaptive response during prostagland in F-2 alpha-induced luteal cell death in vivo[J].Free Radical Research,2007,41(3):251-259.
[11] SHIRASUNA K,MIYAMOTO A.The Life Cycle of the Corpus Luteum[M].Springer International Publishing,2017.
[12] NAKAMURA T,SAKAMOTO K.Reactive oxygen species up-regulates cyclooxygenase-2,p53,and Bax mRNA expression in bovine luteal cells[J].Biochemical & Biophysical Research Communications,2001,284(1):203-210.
[13] DHARMARAJAN A M,HISHEH S,SINGH B,et al.Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro:A novel role for superoxide dismutase in regulating Bax expression[J].Endocrinology,1999,140(6):2555-2561.
[14] ROBERGE S,ROUSSEL J,ANDERSSON D C,et al.TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes[J].Cardiovascular Research,2014,103(1):90-99.
[15] QU B G,WANG H,JIA Y G,et al.Changes in tumor necrosis factor-α,heat shock protein 70,malondialdehyde and superoxide dismutase in patients with different severities of alcoholic fatty liver disease:A prospective observational study[J].Medicine,2015,94(11):e643.
[16] MAEHARA K,HASEGAWA T,ISOBE,KEN-ICHI I.A NF-κB p65 subunit is indispensable for activating manganese superoxide:Dismutase gene transcription mediated by tumor necrosis factor-α[J].Journal of Cellular Biochemistry,2015,77(3):474-486.
[17] AL-GUBORY K H,CEBALLOS-PICOT I,NICOLE A,et al.Changes in activities of superoxide dismutase,nitric oxide synthase,glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle[J].BBA-General Subjects,2005,1725(3):348-357.
[18] SUGINO N,TAKIGUCHI S,KASHIDA S,et al.Suppression of intracellular superoxide dismutase activity by antisense oligonucleotides causes inhibition of progesterone production by rat luteal cells[J].Biology of Reproduction,1999,61(4):1133-1138.
[19] RUEDA B R,TILLY K I,HANSEN T R,et al.Expression of superoxide dismutase,catalase and glutathione peroxidase in the bovine corpusluteum:Evidence supporting a role for oxidative stress in luteolysis[J]. Endocrine,1995,3(3):227-232.
[20] NAKAMURA T,ISHIGAMI T,MAKINO N,et al.The down-regulation of glutathione feroxidase causes bovine luteal cell apoptosis during structural luteolysis[J]. Journal of Biochemistry,2001,129(6):937.