氧化应激在镉抑制小鼠睾丸间质细胞睾酮合成中的作用
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摘要
目的探讨氧化应激在镉抑制小鼠睾丸间质细胞睾酮合成中的作用。方法采用20μmol/L CdCl_2处理小鼠睾丸间质细胞系TM3细胞,在加入CdCl_2 4 h和8 h后分别收集细胞和上清液,等容积磷酸盐缓冲液处理8 h为对照组,采用ELISA法检测上清液中睾酮含量,采用RT-PCR法检测血红素加氧酶-1(HO-1)、超氧化物歧化酶(SOD1)、SOD2和SOD3、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)mRNA的表达水平,采用Western blot法检测HO-1和血红素加氧酶-2(HO-2)蛋白的表达水平。结果 CdCl_2处理4 h和8 h后的细胞上清液中睾酮的含量明显低于对照组(P<0.01);CdCl_2处理组的SOD1、SOD2、SOD3、GSH-Px、CAT mRNA与对照组相比表达水平均降低。此外,CdCl_2处理组中HO-1的mRNA表达量与对照相比明显升高;与此相一致,CdCl_2明显诱导TM3细胞HO-1的蛋白表达,并具有明显的时间-效应关系(P<0.01);结论镉抑制TM3细胞睾酮的合成,氧化应激可能在其中发挥重要作用。
Objective To investigate the effects of oxidative stress on testosterone biosynthesis inhibited by cadmium in mouse Leydig cells. Methods TM3 cells were treated with 20 μmol/L CdCl_2, and collected the cell and the culture supernatant fluid in 4 h and 8 h after CdCl_2 exposure. Testosterone was detected by ELISA assay. The expression of heme oxygenase-1(HO-1), superoxide dismutase 1(SOD1),superoxide dismutase 2(SOD2),superoxide dismutase 3(SOD3),glutathione peroxidase(GSH-Px) and catalase(CAT) mRNA were detected by RT-PCR. The expression levels of HO-1 and heme oxygenase-2(HO-2) proteins were examined by Western blot. Results TM3 cells were incubated with 20μmol/L CdCl_2 for 4 h or 8 h. The testosterone levels in the culture supernatant fluid were significantly lower in Cd-exposure groups(P<0.01) than that in the control group. The mRNA expression of SOD1, SOD2, SOD3, GSH-Px and CAT were all decreased compared with the control group. Cadmium up-regulated significantly the expression of HO-1 mRNA in TM3 cells(P<0.01). Correspondingly, the protein expression of HO-1 was increased markedly in time-effect course in Cd-exposure group(P<0.01). Conclusion Cadmium inhibits the synthesis of testosterone in TM3 cells, and oxidative stress may play an important role of testosterone biosynthesis in mouse Leydig cells.
Objective To investigate the effects of oxidative stress on testosterone biosynthesis inhibited by cadmium in mouse Leydig cells. Methods TM3 cells were treated with 20 μmol/L CdCl_2, and collected the cell and the culture supernatant fluid in 4 h and 8 h after CdCl_2 exposure. Testosterone was detected by ELISA assay. The expression of heme oxygenase-1(HO-1), superoxide dismutase 1(SOD1),superoxide dismutase 2(SOD2),superoxide dismutase 3(SOD3),glutathione peroxidase(GSH-Px) and catalase(CAT) mRNA were detected by RT-PCR. The expression levels of HO-1 and heme oxygenase-2(HO-2) proteins were examined by Western blot. Results TM3 cells were incubated with 20μmol/L CdCl_2 for 4 h or 8 h. The testosterone levels in the culture supernatant fluid were significantly lower in Cd-exposure groups(P<0.01) than that in the control group. The mRNA expression of SOD1, SOD2, SOD3, GSH-Px and CAT were all decreased compared with the control group. Cadmium up-regulated significantly the expression of HO-1 mRNA in TM3 cells(P<0.01). Correspondingly, the protein expression of HO-1 was increased markedly in time-effect course in Cd-exposure group(P<0.01). Conclusion Cadmium inhibits the synthesis of testosterone in TM3 cells, and oxidative stress may play an important role of testosterone biosynthesis in mouse Leydig cells.
引文
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[14] Kang J, Jeong M G, Oh S, et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy[J]. Febs Letters, 2016,588(1):79-85.
[15] Piotrkowski B, Monz N C M, Pagotto R M, et al. Effects of heme oxygenase isozymes on Leydig cells steroidogenesis[J].J Endocrinol,2009, 203(1):155-65.
[2] El-Demerdash F M, Yousef M I, Kedwany F S, et al. Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and beta-carotene[J]. Food Chem Toxicol,2004,42:1563-71.
[3] Monsefi M, Alaee S, Moradshahi A, et al. Cadmium‐induced infertility in male mice[J]. Environ Toxicol, 2010, 25(1):94-102.
[4] Traish A M, Goldstein I, Kim N N. Testosterone and erectile function: from basic research to a new clinical paradigm for managing men with androgen insufficiency and erectile dysfunction[J].Eur Urol, 2007, 52(1):54-70.
[5] 朱文祥,刘洪茂,穆柯瀚,等.镉对小鼠睾丸间质细胞睾酮合成的影响[J].安徽医科大学学报, 2018,53(1):100-4.
[6] Ji Y L, Wang H, Liu P, et al. Pubertal cadmium exposure impairs testicular development and spermatogenesis via disrupting testicular testosterone synthesis in adult mice[J].Reprod Toxicol, 2010, 29(2):176-83.
[7] Khanna S, Mitra S, Lakhera P C, et al. N-acetylcysteine effectively mitigates cadmium-induced oxidative damage and cell death in Leydig cells in vitro[J]. Drug Chem Toxicol, 2016, 39(1):74-80.
[8] Chelikani P, Fita I, Loewen P C. Diversity of structures and properties among catalases[J].Cell Mol Life Sci,2004,61:192-208.
[9] Miao W, Jin Y, Lin X, et al. Differential expression of the main polycyclic aromatic hydrocarbon responsive genes in the extrahepatic tissues of mice[J]. Environ Toxicol Pharmaco,2014,37:885-94.
[10] Domazetovic V, Marcucci G, Iantomasi T,et al.Oxidative stress in bone remodeling: role of antioxidants[J].Clin Cases Miner Bone Metab, 2017,14(2):209-16.
[11] Podkalicka P, Mucha O, Józkowicz A, et al. Heme oxygenase inhibition in cancers: possible tools and targets[J].WspolczesnaOnkol, 2018,22(1):23-32.
[12] Loboda A, Damulewicz M, Pyza E, et al. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism[J].Cell Mol Life Sci,2016,73(17):3221-47.
[13] Soares M P, Bach F H.Heme oxygenase-1: from biology to therapeutic potential[J].Trends Mol Med,2009,15(2):50-8.
[14] Kang J, Jeong M G, Oh S, et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy[J]. Febs Letters, 2016,588(1):79-85.
[15] Piotrkowski B, Monz N C M, Pagotto R M, et al. Effects of heme oxygenase isozymes on Leydig cells steroidogenesis[J].J Endocrinol,2009, 203(1):155-65.