丙烯腈诱导的氧化应激对大鼠睾丸NF-κB信号通路的影响

赵乾龙，罗波艳，潘丽，魏倩，张瑞萍，党瑜慧，李芝兰

兰州大学公共卫生学院，兰州 730000



丙烯腈诱导的氧化应激对大鼠睾丸NF-κB信号通路的影响

赵乾龙，罗波艳，潘丽，魏倩，张瑞萍，党瑜慧，李芝兰*

兰州大学公共卫生学院，兰州 730000

为探索丙烯腈(acrylonitrile, ACN)诱导的氧化应激对大鼠睾丸NF-κB信号通路的影响，将50只SPF级健康SD雄性大鼠按体重随机分为12.5、25、50 mg·kg-1ACN染毒组，50 mg·kg-1ACN+300 mg·kg-1N-乙酰半胱氨酸(N-acetylcysteine, NAC)干预组(NAC干预组)，对照组(给予等体积玉米油)，每组10只，灌胃，1次/天，6天/周，共90 d。可见光分光光度法检测睾丸组织中超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、还原型谷胱甘肽/氧化型谷胱甘肽比值(glutathione/oxidized glutathione, GSH/GSSG)、丙二醛(malondialdehyde, MDA)。免疫荧光染色法检测睾丸核因子-κB(nuclear factor κB, NF-κB)激活及核转移。Western Blot检测睾丸p65、IκB蛋白表达。结果显示，低、高剂量染毒组大鼠睾丸GSH/GSSG比值、GSH-Px酶活性与对照组比较降低(P<0.05)。中、高剂量染毒组大鼠睾丸MDA含量与对照组比较升高(P<0.05)。NAC干预组大鼠睾丸MDA含量与高ACN组比较降低(P<0.05)；NAC干预组大鼠睾丸GSH/GSSG比值与高ACN组比较升高(P<0.05)；免疫荧光结果显示，高ACN组大鼠睾丸NF-κB被激活，并转移入细胞核。NAC干预组与高ACN组比较p65蛋白表达及核转移显著减少。Western Blot结果显示，高剂量染毒组大鼠睾丸p65蛋白表达与对照组比较升高(P<0.05)，IκB蛋白表达与对照组比较降低(P<0.05)；NAC干预组大鼠睾丸p65蛋白表达与高ACN组比较降低，IκB蛋白表达与高ACN组比较升高，差异有统计学意义(P<0.05)。结果表明丙烯腈引起的氧化应激激活了大鼠睾丸生殖细胞NF-κB信号通路。

丙烯腈；大鼠；睾丸；氧化应激；NF-κB；p65；IκB

Received 22 December 2015 accepted 4 June 2016

丙烯腈(acrylonitrile, ACN)，又称乙烯基氰，无色易燃易挥发，具有特殊的杏仁气味，微溶于水，是一种广泛用于合成人造纤维、人造橡胶、塑料制造的化学单体[1]。

研究发现，长期接触ACN的男工精子数量和精子密度显著降低，精子性染色体畸变率升高，染色体二倍体百分率增高，精子DNA损伤严重[2]。耿江[3]研究发现，ACN接触组男工精子总密度、运动精子密度、前向运动精子密度、精子运动速度、直线性及摆动降低。长期接触ACN还可引起男工血清睾酮水平显著下降[4]。

核转录因子κB(nuclear factor κB，NF-κB)是细胞核内重要的转录调节因子，不仅参与机体免疫、炎症、组织损伤修复和胚胎发育等过程还可以调节凋亡相关基因的表达[5]。NF-κB在自由基生物学领域研究中受到相当大的关注，ROS可以激活NF-κB，激活程度也可以被抗氧化剂所调节[6]。NF-κB通常与其抑制剂IκB结合不发生作用，ROS过量产生时会激活IκB激酶复合物IKK，进而降解IκB蛋白[7]，然后NF-κB被游离出来而激活，激活后的NF-κB再转移入细胞核发挥其生物活性作用[8]。研究发现，由ROS激活的NF-κB信号通路能被N-乙酰半胱氨酸(N-acetylcysteine, NAC)特异性地抑制[9]。

本研究通过ACN对大鼠亚慢性染毒，观察大鼠睾丸组织氧化还原酶活性及p65、IκB蛋白表达水平变化，探讨氧化应激对NF-κB信号通路的影响。

1 材料与方法(Materials and methods)

1.1 实验动物及分组

SPF级健康成年SD雄性大鼠50只，体重(250～300) g，购买于甘肃中医学院SPF级实验动物中心(实验动物质量合格证编号SCXK(甘)2011-0001)。实验动物随机分为5组，每组10只，饲养于甘肃中医学院SPF级实验动物中心(实验设施合格证编号SYXK(甘)2011-0001)，自由进食及饮水，适应性饲养1周后，以12.5、25、50 mg·kg-1ACN[10]、50 mg·kg-1ACN+300 mg·kg-1NAC(NAC干预组)灌胃，NAC干预组NAC灌胃30 min后再灌ACN[7]，对照组大鼠给予玉米油，1次/天，6天/周，共90 d。

1.2 药物及试剂

ACN购买于天津四通化工厂(纯度>99%)，NAC(美国Amresco公司)，ECL超敏发光液(美国Thermo公司)，p65兔抗大鼠一抗(美国CST公司)，IκB兔抗大鼠一抗(美国CST公司)，β-actin一抗(武汉伊莱瑞特生物有限公司)，HRP标记山羊抗兔二抗(美国CST公司)。超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽(GSH)测试盒均购买于南京建成生物工程研究所、BCA蛋白、NF-κB核转运试剂盒均购买于碧云天生物技术有限公司。

1.3 氧化还原相关酶的测定

每组随机取6只动物处死后摘取右侧睾丸，置于平皿中用4 ℃生理盐水漂洗去除血液，滤纸擦干，称重后分装成4份。睾丸匀浆的制备：(1)取1份睾丸组织按重量(g):匀浆介质(mL)=1:9的体积比加入0.86%预冷的生理盐水，在冰水浴下制成10%匀浆，2 500 r·min-1离心10 min，取上清，用于SOD、MDA、GSH-Px、BCA检测；(2)取1份睾丸组织按1:4的体积比加入GSH试剂盒自带的匀浆介质，在冰水浴下制成20%匀浆，3 500 r·min-1离心10 min，取上清，用于GSH检测，检测方法均严格按试剂盒说明书进行。另外2份睾丸组织用液氮冷冻后，转移至-80 ℃冰箱保存。

1.4 NF-κB激活-核转录免疫荧光检测

大鼠处死后，①取左侧睾丸，在4%多聚甲醛中固定48 h。②梯度乙醇脱水后用二甲苯透明，然后放入熔融的石蜡中浸透，每次30 min，共3次，然后包埋。③包埋好的石蜡块即进行切片，切片的厚度为5 μm。将制备的石蜡组织切片按照常规的方法进行脱蜡、水化、水合，然后严格按NF-κB激活-核转运检测试剂盒说明书进行操作。

1.5 p65、IκB蛋白Western Blot测定

从-80 ℃冰箱中取出睾丸组织，按照每20 mg组织加入200 μL RIPA(每1 mL RIPA加入10 μL PMSF)裂解液的比例加入裂解液，冰浴中用玻璃匀浆器匀浆，直至组织充分裂解。裂解后的样品在12 000 g·min-1，4 ℃下，离心15 min，取上清，分装30 μL上清用于BCA法测总蛋白，剩余上清按100 μL分装到EP管中，加入稀释液RIPA和5×上样缓冲液后在沸水中变性5 min，然后按每条泳道蛋白含量40 μg蛋白进行SDS-PAGE电泳，然后根据彩虹marker标识的目标蛋白的位置将目标条带转移到PVDF膜上，用5%脱脂奶粉的TBST室温封闭1～2 h，然后加入p65一抗(1:1 000)、IκB一抗(1:1 000)，4 ℃孵育过夜，然后用辣根过氧化酶标记的二抗(1:2 000)室温下孵育2 h，选用β-actin为内参蛋白。加适量ECL超敏发光液然后用凝胶成像仪曝光，保存蛋白条带，然后用Quantity One 4.6.2对蛋白条带灰度值进行分析。

1.6 统计分析

2 结果(Results)

2.1 丙烯腈染毒对大鼠睾丸抗氧化能力及脂质过氧化的影响

单因素方差分析结果示，低、高剂量染毒组大鼠睾丸GSH/GSSG比值、GSH-Px酶活性与对照组比较降低，差异均有统计学意义(P<0.05)。中、高剂量染毒组大鼠睾丸MDA含量与对照组比较升高，差异均有统计学意义(P<0.05)。具体结果见表1。

2.2 NAC对ACN引起睾丸氧化损伤的干预作用

单因素方差分析结果示，NAC预处理后，NAC干预组大鼠睾丸MDA含量与高ACN组比较降低，差异均有统计学意义(P<0.05)；NAC干预组大鼠睾丸GSH/GSSG比值与高ACN组比较升高，差异有统计学意义(P<0.05)；NAC干预组大鼠睾丸GSH-Px活性与高ACN组比较升高，但差异无统计学意义(P>0.05)。见表2。

表1 丙烯腈(ACN)对大鼠睾丸脂质过氧化的影响±s，n=6)

注：*与对照组比较，P<0.05。

Note:*Compared with control, P<0.05.

表2 N-乙酰半胱氨酸(NAC)对ACN染毒引起大鼠睾丸氧化损伤的干预作用±s，n=6)

注：*与对照组比较，P<0.05；#与ACN组比较，P<0.05。

Note:*Compared with control, P<0.05;#Compared with ACN, P<0.05.

2.3 氧化应激对NF-κB信号通路的影响

免疫荧光结果显示，高ACN组大鼠睾丸NF-κB被激活，并转移入细胞核。用NAC提前处理后p65蛋白表达及核转移显著减少。结果见图1。

Western Blot结果示，高剂量染毒组大鼠睾丸p65蛋白表达与对照组比较升高，差异有统计学意义(P<0.05)，NAC干预组大鼠睾丸p65蛋白表达与高ACN组比较降低，差异有统计学意义(P<0.05)。高剂量染毒组大鼠睾丸IκB蛋白表达与对照组比较降低，差异有统计学意义(P<0.05)，NAC干预组大鼠睾丸IκB蛋白表达与高ACN组比较升高，差异有统计学意义(P<0.05)。结果见图2。

图1 ACN染毒后NF-κB激活及核转移的免疫荧光分析注：左边的图表示Cy3标记的p65，中间的图为细胞核DAPI染色，右边的图是合成图，白色箭头表示NF-κB激活并转移入核。倍数为400×。Fig. 1 Immunofluorescence analysis on activation of NF-κB induced by ACNNote: The left graph was p65 labeled with Cy3, the middle graph was nucleus stained with DAPI, and the right graph was merged by the left and middle graphs. The arrows were used to point out the activation and translocation of NF-κB into nucleus. Magnification=400×.

图2 ACN染毒后NF-κB激活的免疫印迹分析注：A，Western Blot p65、IκB-α蛋白条带。B～C，Western Blot p65、IκB-α蛋白条带半定量分析。ACN组为50 mg·kg-1 ACN处理组，ACN+NAC组为50 mg·kg-1 ACN+300 mg·kg-1 NAC共同处理组。*与对照组比较，P<0.05；#与ACN组比较，P<0.05。Fig. 2 Western Blot analysis on activation of NF-κB induced by ACNNote: A, Western Blot bands of p65 and IκB-α proteins; B, semi-quantitative analysis on Western Blot of p65 and IκB-α proteins. ACN stands for 50 mg·kg-1 ACN treatment group; ACN+NAC stands for 50 mg·kg-1 ACN+300 mg·kg-1 NAC treatment group.*Compared with control, P<0.05; # Compared with ACN, P<0.05.

3 讨论(Discussion)

NF-κB是细胞核内重要的转录调节因子，通常与抑制因子IκB相结合，以非活性形式存在于细胞质中，当细胞受到上游刺激因子，如ROS、肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)、细菌脂多糖(LPS)等的作用时而激活[19-21]。本研究免疫荧光检测结果示，高剂量染毒组大鼠睾丸NF-κB被激活，并转移入核；Western Blot结果示，高剂量染毒组大鼠睾丸p65蛋白表达升高、IκB蛋白表达降低。这是因为ACN染毒机体产生大量的ROS，过多的ROS能够刺激IKK蛋白表达升高[6]。IKK激活后，IκB蛋白在激酶复合物IKK的作用下氨基端丝氨酸残基的第32和第36位点被磷酸化，进而被泛素连接酶复合物E3RSIκB-TrCP识别并泛素化，然后被26S蛋白酶体识别并迅速降解[22]。IκB蛋白降解后P65蛋白被游离出来而激活，激活后转移入细胞核，与目的基因结合并促进其转录[8,23]。此外，本研究结果发现加入NAC后ACN染毒组大鼠睾丸NF-κB信号通路激活显著受到抑制，这与李燕等[9]报道的结果一致，可能是因为NAC通过清除过量产生的ROS抑制了氧化应激发生，从而拮抗了NF-κB的活化和其在核内的表达。提示ACN引起的氧化应激激活了大鼠睾丸生殖细胞NF-κB信号通路。

致谢：本课题研究由中央高校基本科研业务费专项资金项目(lzujbky-2014-221)支持。

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Effects of Oxidative Stress Induced via Acrylonitrile on NF-κB Signaling Pathway in Rats Testis

Zhao Qianlong, Luo Boyan, Pan Li, Wei Qian, Zhang Ruiping, Dang Yuhui, Li Zhilan*

School of Public Health, Lanzhou University, Lanzhou 730000, China

In order to study the effects of acrylonitrile (ACN)-induced oxidative stress via NF-κB signaling pathway in rat testis, fifty Sprague-Dawley male rats in SPF degree were randomly divided into 5 groups according to the body weight, and were treated with 0, 12.5, 25, 50 mg·kg-1ACN or 50 mg·kg-1ACN + 300 mg·kg-1N-acetylcysteine (NAC) (NAC intervention group) respectively, via gavage, 6 days per week for 90 days. The activity of testicular superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and contents of maleic dialdehyde (MDA) were determined by visible spectrophotometry. The activation and nuclear translocation of testicular nuclear factor κB (p65) was detected by immunofluorescence. The testicular protein level of p65 and IκB were detected by Western Blot. The results showed that in comparison with control grpup, the ratio of GSH/GSSG and the activity of GSH-Px were significant decreased in low and high concentration of ACN group. The contents of MDA were significant increased in medium and high concentration of ACN groups. In NAC intervention group, the contents of MDA were significant decreased, while the ratio of GSH/GSSG was significantly increased as compared with high concentration of ACN group. Results of immunofluorescence showed that NF-κB pathway was activated, and p65 was transferred into the nucleus in high concentration of ACN group. In contrast, the activation of NF-κB was inhibited by NAC. Results of Western Blot showed that the expression of p65 protein was significantly increased and the expression of IκB protein was significantly decreased in high concentration of ACN group, while these changes were effectively abolished by NAC. Our study indicated that ACN-induced oxidative stress was able to activate NF-κB signaling pathway in rats testis.

acrylonitrile; rats; testis; oxidative stress; NF-κB; p65; IκB

中央高校基本科研业务费专项资金项目(lzujbky-2014-221)

赵乾龙(1989-)，男，硕士研究生，研究方向为妇女劳动卫生与生殖健康，E-mail: zhaoql13@lzu.edu.cn；

*通讯作者(Corresponding author)， E-mail: lizhl@lzu.edu.cn

10.7524/AJE.1673-5897.20151222001

2015-12-22 录用日期：2016-06-04

1673-5897(2016)4-155-06

X171.5

A

简介：李芝兰(1962-)，女，硕士研究生导师，教授，兰州大学公共卫生学院儿少卫生与妇幼保健学研究所所长，研究方向为妇女劳动卫生与生殖健康。

赵乾龙, 罗波艳, 潘丽, 等. 丙烯腈诱导的氧化应激对大鼠睾丸NF-κB信号通路的影响[J]. 生态毒理学报，2016, 11(4): 155-160

Zhao Q L, Luo B Y, Pan L, et al. Effects of oxidative stress induced via acrylonitrile on NF-κB signaling pathway in rats testis [J]. Asian Journal of Ecotoxicology, 2016, 11(4): 155-160 (in Chinese)