环境高温诱导巴马香猪睾丸细胞凋亡并激活Nrf2/HO-1抗氧化信号通路

doi:10.11843/j.issn.0366-6964.2018.06.007

摘要/Abstract

摘要：

旨在通过研究高温环境对巴马香猪阴囊温度分布和睾丸组织中抗氧化和细胞凋亡相关蛋白表达的影响，筛选巴马香猪热敏感性指标。选取7月龄性成熟雄性巴马香猪12头，随机分成对照组和高温组。高温组饲养环境温度为24 h内从25℃→40℃→25℃循环变温，连续8 d，第8天试验结束后，取睾丸器官，采用免疫组织化学方法检测抗氧化通路标志蛋白（nuclear factor erythroid 2-related factor 2，Nrf2）、抗氧化蛋白（heme oxygenase 1，HO-1）和细胞凋亡标志蛋白（cleaved caspase 3，c-caspase 3）的组织表达情况。结果表明，高温组猪阴囊表面平均温度显著升高（P<0.05），升高幅度达到3.7℃。高温组睾丸间质细胞、生精上皮细胞Nrf2和HO-1蛋白表达显著升高（P<0.05），并且间质细胞核Nrf2蛋白表达明显升高。高温组猪睾丸间质和睾丸生精上皮细胞核阳性表达c-caspase 3蛋白细胞百分比显著升高（P<0.05），其中睾丸生精上皮细胞核阳性表达c-caspase 3蛋白细胞百分比升高更为明显。综上表明，40℃环境温度已明显超过了巴马香猪阴囊的最大热调节能力范围，导致睾丸温度升高，诱使睾丸组织细胞凋亡增加。同时，环境高温也激活了猪睾丸间质细胞Nrf2抗氧化信号通路和HO-1抗氧化蛋白的高效表达，这为寻找应对热应激导致公猪精液品质下降问题提供潜在的有效分子靶点。

关键词: 环境高温, Nrf2, HO-1, 睾丸, 巴马香猪

Abstract:

The objective of this study was to determine the heat sensitivity index for Bama miniature pigs by investigating the effect of ambient high temperature on scrotal temperature distributions and testicular protein expressions related to the antioxidative capacity and cellular apoptosis in the pigs. Twelve 7-month-old mature Bama miniature pigs were selected and randomly allotted to a thermal neutral(TN)group and a heat-treated(HT)group. The HT group was exposed to high ambient temperature, which was changed from 25 to 40℃ and back to 25℃ during each 24 h period for 8 d. At the end of the experiment, testes were collected and measured for the protein expression of nuclear factor erythroid 2-related factor 2(Nrf2), heme oxygenase 1(HO-1)and cleaved caspase 3 (c-caspase 3)using immunohistochemical staining. Results showed that, the HT group pigs presented a significant increase in scrotal surface temperature(P<0.05), which was 3.7℃ higher than the TN group pigs. In the HT group, the testicular protein expression of Nrf2 and HO-1 increased significantly in both the testicular interstitial cells and the germ line cells of seminiferous epithelium(P<0.05). Moreover, prominent nuclear accumulation of Nrf2 protein was observed in the testicular interstitial cells. In the HT group, there was an increased percentage of the c-caspase 3-positive nucleuses in the testicular interstitial cells and germ line cells of seminiferous epithelium(P<0.05), respectively. The percentage of the c-caspase 3-positive nucleuses in the testicular interstitial cells increased more sharply than that in the germ line cells of seminiferous epithelium. In conclusion, the capacity of scrotal thermal regulation was overwhelmed in the 40℃ ambient temperature, which increased the testicular temperature and caused the testicular cell apoptosis. High ambient temperature also activated Nrf2 signal pathway and increased HO-1 expression, indicating that the testicular Nrf2/HO-1 signal pathway might be a potential target for new strategies to protect semen quality from high ambient temperature-induced damage in boars.

Key words: ambient high temperature, Nrf2, HO-1, testis, Bama miniature pig

中图分类号:

S828.8

李延森, 李照见, 贾潇潇, 李春梅. 环境高温诱导巴马香猪睾丸细胞凋亡并激活Nrf2/HO-1抗氧化信号通路[J]. 畜牧兽医学报, 2018, 49(6): 1154-1162.

LI Yan-sen, LI Zhao-jian, JIA Xiao-xiao, LI Chun-mei. Ambient High Temperature Induced Cellular Apoptosis and Nrf2/HO-1 Signal Pathway in Testes of Bama Miniature Pigs[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1154-1162.

参考文献

[1] 李延森, 曹云, 王爱华, 等. 南通地区不同品种公猪夏季精液品质变化[J]. 畜牧与兽医, 2014, 46(10):40-42.
LI Y S, CAO Y, WANG A H, et al. The changes in boar semen quality of different type strains in the area of Nantong during hot summer[J]. Animal Husbandry & Veterinary Medicine, 2014, 46(10):40-42. (in Chinese)
[2] 李延森, 沈祥星, 李春梅. 母猪发情和产仔性能与环境温度变化相关性分析[J]. 畜牧兽医学报, 2016, 47(6):1133-1139.
LI Y S, SHEN X X, LI C M. Correlation analysis between the ambient temperatures and reproductive performance of sows[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(6):1133-1139. (in Chinese)
[3] PAUL C, TENG S, SAUNDERS P T K. A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death[J]. Biol Reprod, 2009, 80(5):913-919.
[4] ZHANG M Q, JIANG M, BI Y, et al. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice[J]. PLoS One, 2012, 7(7):e41412.
[5] SPORNM B, LIBY K T. Nrf2 and cancer:The good, the bad and the importance of context[J]. Nat Rev Cancer, 2012, 12(8):564-571.
[6] BELLK F, AL-MUBARAK B, FOWLER J H, et al. Mild oxidative stress activates Nrf2 in astrocytes, which contributes to neuroprotective ischemic preconditioning[J]. Proc Natl Acad Sci U S A, 2011, 108(1):E1-E2.
[7] SOARESM P, BACH F H. Heme oxygenase-1:From biology to therapeutic potential[J]. Trends Mol Med, 2009, 15(2):50-58.
[8] SHEN B, WANG W, DING L, et al. Nuclear factor erythroid 2-related factor 2 rescues the oxidative stress induced by di-N-butylphthalate in testicular Leydig cells[J]. Hum Exp Toxicol, 2015, 32(2):145-152.
[9] SHIRAISHI K, NAITO K. Increased expression of Leydig cell haem oxygenase-1 preserves spermatogenesis in varicocele[J].Hum Reprod, 2005, 20(9):2608-2613.
[10] BAE W J, HA U S, CHOI J B, et al. Protective effect of decursin extracted from Angelica gigas in male infertility via Nrf2/HO-1 signaling pathway[J]. Oxid Med Cell Longev, 2016, 2016:5901098.
[11] 颜培实, 李如治. 家畜环境卫生学[M]. 北京:高等教育出版社, 2011.
YAN P S, LI R Z. Domestic animals and environmental hygiene[M]. Beijing:China Higher Education Press, 2011. (in Chinese)
[12] YU J, YIN P, LIU F H, et al. Effect of heat stress on the porcine small intestine:A morphological and gene expression study[J]. Comp Biochem Physiol A, 2010, 156(1):119-128.
[13] LI Y S, HUANG Y, PIAO Y G, et al. Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis[J]. Reprod Biol Endocrin, 2013, 11:23.
[14] LI Y S, WANG A H, TAYA K, et al. Declining semen quality and steadying seminal plasma ions in heat-stressed boar model[J]. Reprod Med Biol, 2015, 14(4):171-177.
[15] MAHER J, YAMAMOTO M. The rise of antioxidant signaling-the evolution and hormetic actions of Nrf2[J]. Toxicol Appl Pharmacol, 2010, 244(1):4-15.
[16] LI Y S, CAO Y, WANG F, et al. Scrotal heat induced the Nrf2-driven antioxidant response during oxidative stress and apoptosis in the mouse testis[J].Acta Histochem, 2014, 116(5):883-890.
[17] LI Y S, CAO Y, WANG F, et al. Tert-butylhydroquinone attenuates scrotal heat-induced damage by regulating Nrf2-antioxidant system in the mouse testis[J]. Gen Comp Endocrinol, 2014, 208:12-20.
[18] CHEN H L, JIN S Y, GUO J J, et al. Knockout of the transcription factor Nrf2:Effects on testosterone production by aging mouse Leydig cells[J]. Mol Cell Endocrinol, 2015, 409:113-120.
[19] STOCKER R. Induction of haem oxygenase as a defence against oxidative stress[J].Free Radic Res Commun, 1990, 9(2):101-112.
[20] KOMATSU T, TABATA M, KUBO-IRIE M, et al. The effects of nanoparticles on mouse testis Leydig cells in vitro[J]. Toxicol in vitro, 2008, 22(8):1825-1831.
[21] OZAWA N, GODA N, MAKINO N, et al. Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress[J]. J Clin Invest, 2002, 109(4):457-467.
[22] ALAM J, COOK J L. How many transcription factors does it take to turn on the heme oxygenase-1 gene?[J]. Am J Respir Cell Mol Biol, 2007, 36(2):166-174.
[23] AGARWAL A, JOHN A R, ALVAREZ J G. Studies on men's health and fertility[M]. New Jersey:Humana Press, 2012.
[24] HARDY M P, GANJAM V K. Stress, 11β-HSD, and Leydig cell function[J]. J Androl, 1997, 18(5):475-479.
[25] 范小瑞, 张禛, 席华明, 等. 热应激对猪睾丸Cyt-C和Caspase-3表达的影响[J]. 中国农业科学, 2017, 50(5):924-931.
FAN X R, ZHANG Z, XI H M, et al. Effect of heat stress on the expression of Cyt-C and Caspase-3 in boar testis[J]. Scientia Agricultura Sinica, 2017, 50(5):924-931. (in Chinese)