动物营养学报 >

2018 , Vol. 30 >Issue 7: 2632 - 2640

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.07.023

分子营养 Molecular Nutrition

白藜芦醇通过沉默调节蛋白1-解偶联蛋白2信号通路降低小鼠睾丸间质细胞TM3的氧化损伤

展开
  • 1. 湖南农业大学动物医学院, 长沙 410128;
    2. 湖南农业大学动物科学技术学院, 长沙 410128
张晓春(1992-),女,辽宁丹东人,硕士研究生,从事营养对动物繁殖调控研究。E-mail:987052094@qq.com

收稿日期: 2018-01-03

  网络出版日期: 2018-07-20

基金资助

国家自然科学基金项目（31772819，31572591）；中国博士后科学基金第七批特别资助（2014T70770）

收起

Resveratrol Attenuates Acute Oxidative Injury in Mouse Leydig Cell TM3 via Silent Information Regulator 1/Uncoupling Protein 2 Signaling Pathway

Expand
  • 1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
    2. College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China

Received date: 2018-01-03

  Online published: 2018-07-20

Fold

摘要

本试验旨在研究白藜芦醇（RES）对氧化损伤小鼠睾丸间质细胞TM3的保护作用，并探索其可能的作用机制。首先，用不同浓度（0、150、200、250、300 μmol/L）的过氧化氢（H2O2）处理小鼠睾丸间质细胞TM3 8 h，确定建立氧化损伤细胞模型的适宜H2O2浓度；然后，用不同浓度（0、2.5、5.0和10.0 μmol/L）的RES分别处理正常细胞24 h，确定RES安全浓度；最后，用安全浓度的RES处理氧化损伤细胞24 h。在整个培养过程中采用iCELLigence实时无标记细胞功能分析仪监测细胞的增殖情况；待安全浓度的RES处理氧化损伤细胞结束后采用2'，7'-二氯荧光素二乙酸酯（DCFH-DA）探针法检测细胞中活性氧（ROS）的含量，实时荧光定量PCR（RT-qPCR）和蛋白质印迹（Western blotting）法分别检测沉默调节蛋白1（SIRT1）/解偶联蛋白2（UCP2）信号通路中关键因子SIRT1和UCP2 mRNA和蛋白质的相对表达量。结果表明：1）用浓度为150 μmol/L及以上的H2O2处理细胞8 h后，极显著降低细胞存活率（P<0.01），因此确定150 μmol/L为建立氧化损伤细胞模型的适宜H2O2浓度。2）用10.0 μmol/L及以下浓度的RES处理正常细胞24 h后，细胞存活率均无显著变化（P>0.05）；用2.5、5.0和10.0 μmol/L的RES处理氧化损伤细胞24 h后，细胞存活率均显著提高（P<0.05），且各浓度RES组间无显著差异（P>0.05）。因此，选用5.0 μmol/L为RES的安全浓度。3）用5.0 μmol/L的RES处理氧化损伤细胞24 h后，细胞中ROS的含量极显著降低（P<0.01）；细胞中SIRT1 mRNA和蛋白质的相对表达量极显著增加（P<0.01），而UCP2 mRNA和蛋白质的相对表达量则显著或极显著降低（P<0.01或P<0.05）。由此可见，适宜浓度的RES可激活SIRT1同时抑制UCP2的表达，UCP2通过负反馈调节方式减少细胞内ROS的生成，从而在一定程度上抑制TM3细胞的氧化损伤。

关键词： 白藜芦醇; 氧化损伤; 沉默调节蛋白1; 解偶联蛋白2; 小鼠睾丸间质细胞TM3

本文引用格式

张晓春, 陈指龙, 方娟, 黎陈, 伍小松, 杨青 . 白藜芦醇通过沉默调节蛋白1-解偶联蛋白2信号通路降低小鼠睾丸间质细胞TM3的氧化损伤[J]. 动物营养学报, 2018 , 30(7) : 2632 -2640 . DOI: 10.3969/j.issn.1006-267x.2018.07.023

Abstract

This experiment was conducted to investigate the protective effect of resveratrol (RES) on oxidative-damaged mouse Leydig cell TM3, and to explore its possible mechanism. Firstly, TM3 cells were treated with different concentrations (0, 150, 200, 250 and 300 μmol/L) of hydrogen peroxide (H2O2), and the optimal concentration of H2O2 was selected to establish the oxidative-damaged cell model. Secondly, the normal cells were treated with different concentrations (0, 2.5, 5.0 and 10.0 μmol/L) of RES for 24 h to select the safe concentration of RES. Finally, the oxidative-damaged cells were treated with safe concentration of RES for 24 h. Cell proliferation was real-time monitored by iCELLigence cell function analyzer during the whole culture process. After the oxidative-damaged cells treated by safe concentration of RES, the reactive oxygen species (ROS) content in cell was detected by 2',7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe method, and the real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting method were used to detect the mRNA and protein relative expression levels of silent information regulator 1 (SIRT1) and uncoupling protein 2 (UCP2) which were the key factors in SIRT1/UCP2 signal pathway, respectively. The results showed as follows:1) the cell viability was extremely significantly reduced after treatment with a concentration of 150 μmol/L or more of H2O2 for 8 h (P<0.01). So, the concentration of 150 μmol/L was selected as the optimal concentration of H2O2 to establish the oxidative-damaged cell model. 2) The cell viability had no significant change when the normal cells treated by 10.0 μmol/L or less RES for 24 h (P>0.05); the cell viability was significantly increased when the oxidative-damaged cells treated by 2.5, 5.0 and 10.0 μmol/L RES for 24 h (P<0.05), and no significant difference among different concentration RES groups (P>0.05). So, the concentration of 5.0 μmol/L was selected as the safe concentration. 3) When the oxidative-damaged cells were treated by 5 μmol/L of RES for 24 h, the content of ROS in cells was extremely significantly increased (P<0.01); the relative expression levels of SIRT1 mRNA and protein were extremely significantly up-regulated (P<0.01), while the relative expression levels of UCP2 mRNA and protein were extremely significantly or significantly down-regulated (P<0.01 or P<0.05). It is concluded that RES can activate the expression of SIRT1 and inhibit the expression of UCP2; meanwhile, UCP2 can attenuate the production of ROS in cells through a negative feedback regulation, thereby inhibiting the oxidative injury of TM3 cells to some extent.

Key words： resveratrol; oxidative injury; SIRT1; UCP2; mouse Leydig cell TM3

参考文献

[1] ASADI N,BAHMANI M,KHERADMAND A,et al.The impact of oxidative stress on testicular function and the role of antioxidants in improving it:a review[J].Journal of Clinical and Diagnostic Research,2017,11(5):IE01-IE05.

[2] 陈指龙,张晓春,薛立群,等.环磷酰胺致小鼠生精障碍作用研究[J].动物医学进展,2017,38(5):34-38.

[3] PANGENI R,SAHNI J K,ALI J,et al.Resveratrol:review on therapeutic potential and recent advances in drug delivery[J].Expert Opinion on Drug Delivery,2014,11(8):1285-1298.  

[4] MALHOTRA A,BATH S,ELBARBRY F.An organ system approach to explore the antioxidative,anti-inflammatory,and cytoprotective actions of resveratrol[J].Oxidative Medicine and Cellular Longevity,2015,2015:803971.

[5] 张成,耿照玉,赵晓惠.白藜芦醇的生物学功能及其在畜禽生产中的应用[J].动物营养学报,2017,29(11):3837-3843.

[6] 王慧君.白藜芦醇对高糖高胆固醇饮食小鼠睾丸Leydig细胞睾酮合成障碍的保护作用研究[D].硕士学位论文.合肥:安徽医科大学,2014.

[7] 成海建,游伟,靳青,等.白藜芦醇通过激活去乙酰化酶1/腺苷一磷酸激活的蛋白激酶信号通路影响牛脂肪细胞凋亡[J].动物营养学报,2017,29(12):4398-4407.

[8] 周曦,易龙,金鑫,等.SIRT1/UCP2通路在白藜芦醇抑制血管内皮细胞氧化应激损伤中的作用[J].第三军医大学学报,2013,35(16):1671-1675.

[9] HSU C P,ZHAI P Y,YAMAMOTO T,et al.Silent information regulator 1 protects the heart from ischemia/reperfusion[J].Circulation,2010,122(21):2170-2182.  

[10] MATTIASON G,SULLIVAN P G.The emerging functions of UCP2 in health,disease,and therapeutics[J].Antioxidants & Redox Signaling,2006,8(1/2):1-38.

[11] 张润蔚,王玲,张春晓,等.过氧化氢诱导斜带石斑鱼原代肝细胞氧化损伤模型的构建[J].动物营养学报,2017,29(4):1227-1232.

[12] 丁逍,刘琳,陈迪,等.过氧化氢诱导原代大鼠睾丸间质细胞氧化损伤模型的建立[J].南京农业大学学报,2014,37(4):99-104.

[13] 乔卫龙,李炜弘,史年刚,等.线粒体能量代谢与疾病关联机制的研究进展[J].云南中医中药杂志,2016,37(4):61-62.

[14] 韩孝先,刘泽宇,周庆彪,等.线粒体在炎症调控中的作用研究进展[J].癌变·畸变·突变,2017,29(6):467-470,475.

[15] 李海.线粒体解耦联蛋白2与能量代谢[J].内江师范学院学报,2010,25(8):52-55.

[16] 郑贵浪.UCP2在脓毒症大鼠心肌细胞线粒体中的作用及初步机制探讨[D].博士学位论文.广州:南方医科大学,2016.

[17] JI F,SHEN T J,ZOU W Z,et al.UCP2 Regulates embryonic neurogenesis via ROS-mediated yap alternation in the developing neocortex[J].Stem Cells,2017,35(6):1479-1492.  

[18] BRAND M D.Uncoupling to survive?The role of mitochondrial inefficiency in ageing[J].Experimental Gerontology,2000,35(6/7):811-820.

[19] ZHANG K J,SHANG Y L,LIAO S Y,et al.Uncoupling protein 2 protects testicular germ cells from hyperthermia-induced apoptosis[J].Biochemical and Biophysical Research Communications,2007,360(2):327-332.  

[20] 王晓娜,朱春芳,张家燕,等.精子的氧化损伤与线粒体解偶联蛋白2(UCP2)表达的关联性[J].医学研究杂志,2017,46(7):62-66.

[21] BORDONE L,MOTTA M C,PICARD F,et al.Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic β cells[J].PLoS Biology,2006,4(9):e295.

[22] 徐静,李楠,王俊红,等.二甲双胍对2型糖尿病合并非酒精性脂肪肝大鼠肝脏SIRT1、UCP2表达的影响[J].中南大学学报(医学版),2013,38(9):882-887.

[23] 张玉佩,孔怡琳,杨钦河,等.SIRT1/UCP2通路在脂肪变性HepG2细胞线粒体能量代谢中的调控机制[J].中国老年学杂志,2016,36(3):513-516.

[24] FISLER J S,WARDEN C H.Uncoupling proteins,dietary fat and the metabolic syndrome[J].Nutrition & Metabolism,2006,3:38.
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2