南京农业大学学报  2016, Vol. 39 Issue (2): 262-268   PDF    
http://dx.doi.org/10.7685/jnau.201508001
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文章信息

徐伟风, 朴元国, 李延森, 李春梅. 2016.
XU Weifeng, PIAO Yuanguo, LI Yansen, LI Chunmei. 2016.
日粮中大豆异黄酮对环境内分泌干扰物3-甲基-4-硝基酚所致公鸡肝损伤的影响
Effects of dietary soybean isoflavones on environmental endocrine disruptor 3-methyl-4-nitrophenol induced liver injury in roosters
南京农业大学学报, 39(2): 262-268
Journal of Nanjing Agricultural University, 39(2): 262-268.
http://dx.doi.org/10.7685/jnau.201508001

文章历史

收稿日期:2015-08-01
日粮中大豆异黄酮对环境内分泌干扰物3-甲基-4-硝基酚所致公鸡肝损伤的影响
徐伟风, 朴元国, 李延森, 李春梅     
南京农业大学动物科技学院, 江苏 南京 210095
摘要[目的] 本文旨在研究大豆异黄酮(soybean isoflavone,SI)对环境内分泌干扰物3-甲基-4-硝基酚(3-methyl-4-nitrophenol,PNMC)所致公鸡肝损伤的影响及其作用机制。[方法] 将1日龄健康白羽公鸡48只随机分为3组(每组4个重复,每个重复4只):基础饲粮组(对照组)、基础饲粮+饮水添加10 mg·L-1 PNMC组(PNMC组)、基础饲粮+饮水添加10 mg·L-1 PNMC+饲料添加100 mg·kg-1SI组(PNMC+SI组),试验期共42 d,分别于21和42 d采样。记录公鸡的生长性能和肝脏质量,并计算肝脏指数;检测肝脏组织中总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)、谷胱甘肽(GSH)及过氧化氢(H2O2)含量;HE染色观察肝脏组织病理变化。[结果] 与对照组相比,PNMC组在21和42 d公鸡体质量均显著降低(P<0.05),22~42 d和1~42 d日增体质量极显著降低(P<0.01),1~21 d、22~42 d、1~42 d料重比及采食量升高(P<0.01),42 d肝脏质量及肝组织匀浆中GSH含量降低(P<0.05);与PNMC组比较,PNMC+SI组在21和42 d公鸡体质量显著增加(P<0.05),1~21 d、22~42 d日增体质量极显著上升(P<0.01),22~42 d、1~42 d采食量极显著降低(P<0.01),1~21 d、22~42 d、1~42 d料重比极显著下降(P<0.01);42 d肝组织匀浆中GSH含量显著升高(P<0.05);而肝组织中T-SOD、CAT活性及MDA、H2O2含量均无显著差异(P>0.05);组织切片表明PNMC组和对照组相比,肝组织多处出现坏死及不同程度的核固缩,而PNMC+SI组肝组织损伤明显减轻,趋向于对照组,缓解了肝脏组织的病理变化。[结论] 大豆异黄酮能够缓解3-甲基-4-硝基酚引起的公鸡肝损伤,增强公鸡肝脏抗氧化能力。
关键词大豆异黄酮     3-甲基-4-硝基酚     肝脏     公鸡     抗氧化能力    
Effects of dietary soybean isoflavones on environmental endocrine disruptor 3-methyl-4-nitrophenol induced liver injury in roosters
XU Weifeng, PIAO Yuanguo, LI Yansen, LI Chunmei     
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
Abstract: [Objectives] The aim of the paper is to investigate the effects of soybean isoflavones(SI)on liver injury induced by environmental endocrine disruptor 3-methyl-4-nitrophenol(PNMC)in roosters and its mechanism. [Methods] Forty-eight healthy white roosters(1 day-old)were randomly divided into 3 groups(4 replicates per group and 4 birds per replicate):basal diet group(control group),basal diet+10 mg·L-1 PNMC in water group(PNMC group),basal diet+10 mg·L-1 PNMC in water+100 mg·kg-1 SI in diet group(PNMC+SI group). The trial lasted 42 days and samples were collected on 21 and 42 days,respectively,and the growth performance was recorded. On the final day,livers were sampled and weighed to calculate the liver index. Activities of T-SOD,CAT and contents of MDA,GSH and H2O2 in liver tissues were assessed. In addition,morphological changes of livers were observed using HE staining. [Results] Compared with the control group,body weights in PNMC group decreased on 21 and 42 days(P<0.05);ADG reduced during 22-42 d and 1-42 d;while ADFI and F/G significantly increased during 1-21 d,22-42 d and 1-42 d(P<0.01);liver weight and the content of GSH were significantly lower on 42 days(P<0.05). Compared with PNMC group,final body weight in PNMC+SI group significantly increased(P<0.01);ADG was higher during 1-21 d and 22-42 d;and F/G markedly decreased during 1-21 d,22-42 d and 1-42 d(P<0.01);meanwhile,the GSH content in PNMC+SI group was significantly enhanced on 42 days(P<0.05),while contents of T-SOD,CAT,MDA,H2O2 of livers in PNMC group and PNMC+SI group showed no significant difference compared with those in control group(P>0.05). Multiple hepatocyte necrosis and different degrees of nuclear condensation were observed in PNMC group,while in PNMC+SI group less serious damage was shown similar to the situation of control,alleviating the pathological changes in liver tissues. [Conclusions] These results suggested that SI could attenuate liver injury caused by PNMC,improve growth performance,and increase antioxidant ability in the liver of PNMC exposed roosters.
Keywords: soybean isoflavone     3-methyl-4-nitrophenol     liver     rooster     antioxidant capacity    

环境内分泌干扰物是指环境中能对机体健康产生不利影响或使其后代内分泌功能发生改变的外源性化学物质[1]。3-甲基-4-硝基酚(3-methyl-4-nirophenol,PNMC),是一种重要的有机合成原料,也是大气污染物柴油车尾气排放的颗粒物质的主要活性成分之一[2, 3],具有内分泌干扰作用。另外,农作物种植过程中大量使用杀虫剂,使得杀虫剂在环境中广泛存在,而PNMC也是杀虫剂杀螟硫磷的降解产物,环境中50%的杀螟硫磷都能降解为PNMC[4]。广泛存在于环境中的PNMC,严重威胁着人类及动植物的健康。

大豆异黄酮(soybean isoflavone,SI)为豆科植物大豆的成熟种子,是一类从大豆中分离的生物活性成分。近年来研究发现,SI具有广泛的药理学功效,能预防某些慢性疾病如乳腺癌[5]、前列腺癌[6]等;治疗雌激素缺乏引起的疾病如改善妇女更年期综合症[7]、骨质疏松[8]等;此外,还有降血脂[9]、抗动脉粥样硬化[10]、抗炎[11]等多种功效。大豆异黄酮的基本骨架为3-苯基苯并二氢呋喃,主要有黄豆苷元、染料木素[12]。目前已有研究表明,多种黄酮类化合物如葛根总黄酮[13]、银杏黄酮[14]、毛橘红总黄酮[15]等对肝脏脂质过氧化损伤具有抑制作用,对多种损伤因素引起的肝损伤和肝纤维化具有保护作用[16, 17, 18]。本试验通过研究日粮添加SI对PNMC引起公鸡肝损伤的缓解作用,为SI在家禽抗应激中的应用提供试验依据。

1 材料与方法 1.1 主要材料

主要试剂:大豆异黄酮(纯度80%,华北制药股份有限公司);3-甲基-4-硝基酚(3-methyl-4-nitrophenol,PNMC,纯度99.9%,成都科隆化学品有限公司);总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性和谷胱甘肽(GSH)、丙二醛(MDA)、过氧化氢(H2O2)、蛋白质含量测试盒(南京建成生物工程研究所);苏木精-伊红(HE)染液(南京建成生物科技有限公司)。

主要仪器:组织包埋机YD-6D、展片机KD-P(浙江省金华市医疗设备厂);石蜡切片机Leica RM2235和烘片机Leica HI1220(上海徕卡仪器有限公司);光学显微镜Nikon YS100和倒置显微镜(日本Nikon株式会社);酶标仪(美国Thermo公司);5417R型台式冷冻高速离心机(德国Eppendorf公司);低温冷冻离心机J2-MI型(德国Beckman公司);虚拟显微镜(日本Olympus公司)。

1.2 试验方法 1.2.1 动物分组与处理

试验为单因素试验设计,将48只1日龄健康白羽公鸡随机分为3组,每组设置4个重复,每个重复4只鸡,试验期为42 d。试验基础日粮采用玉米-豆粕型日粮,分1~21 d和22~42 d两阶段进行饲养。日粮组成和营养水平见表 1。对照组饲喂基础饲粮,PNMC组向饮水中添加10 mg · L-1 PNMC,PNMC+SI组向饮水中添加10 mg · L-1 PNMC且在饲粮中添加100 mg · kg-1SI。试验阶段采用笼养,24 h光照,自由饮水和采食,对照组与试验组采用相同的免疫程序进行免疫以及相同的饲养管理方式进行饲养。

表 1 基础饲粮组成以及营养水平 Table 1 Composition and nutrient levels of basal diets
组成
Composition
生长时段Growth stage组成
Composition
生长时段Growth stage
1~21 d22~42 d1~21 d22~42 d
原料组成/% Ingredients composition营养组成2/% Nutrient composition
玉米Maize55.1060.80 代谢能3Metabolisable energy12.5613.34
豆粕Soybean meal32.2023.70 粗蛋白Crude protein21.8819.41
玉米蛋白粉Corn gluten meal4.505.50 钙Calcium0.970.81
豆油Soybean oil3.205.00 有效磷Available phosphorus0.330.39
预混料1 Premix5.005.00 赖氨酸Lysine1.181.06
蛋氨酸Methionine0.500.43
蛋氨酸+胱氨酸Methionine+cysteine0.870.76
注: 1)预混料为每kg饲粮提供:维生素A 24 mg;维生素D3 6 mg,维生素E 20 mg,维生素K 1.3 mg,维生素B1 2.2 mg,维生素B2 8 mg,维生素B3 40 mg,维生素B6 4 mg,维生素B12 0.013 mg,生物素0.04 mg,叶酸1 mg, 杆菌肽锌30 mg,胆碱400 mg,泛酸钙10 mg,铁80 mg, 铜8.0 mg,锌65 mg,锰110 mg,碘1.1 mg,硒0.3 mg;2)营养水平为计算值;3)代谢能单位为MJ · kg-1
Note: 1)Premix provided per kg of diet:vitamin A 24 mg,vitamin D3 6 mg,vitamin E 20 mg,vitamin K 1.3 mg,vitamin B1 2.2 mg,vitamin B2 8 mg,vitamin B3 40 mg,vitamin B6 4 mg,vitamin B12 0.013 mg,vitamin H 0.04 mg,folic acid 1 mg,bacitracin zinc 30 mg,choline 400 mg,calcium pantothenate 10 mg,Fe 80 mg,Cu 8.0 mg,Zn 65 mg,Mn 110 mg,I 1.1 mg,Se 0.3 mg;2)Nutrient levels are calculated values;3)The unit of metabolisable energy is MJ · kg-1.
1.2.2 样品采集

试验分别于21和42 d采样,采样前禁食24 h,自由饮水。静脉采血,3 500 r · min-1离心15 min,取上清液,置于-20 ℃冰箱保存。采血后屠宰,采集肝脏组织,部分放入4%多聚甲醛溶液固定,用于制作肝脏病理切片;部分-20 ℃冷冻保存,用于肝脏氧化应激反应指标的测定。

1.2.3 生化指标检

测肝脏于预冷生理盐水中漂洗,除去血液,滤纸拭干,称质量并计算肝脏指数(肝脏指数=肝脏质量/体质量×100%)。取肝脏左叶部位称质量,加预冷生理盐水,用匀浆机在冰浴中制备成10%的肝匀浆,4 ℃离心(6 000 r · min-1,10 min),分离上清液,按试剂盒说明书测定肝组织匀浆中MDA、H2O2、GSH含量及T-SOD、CAT活性。蛋白含量的测定采用考马斯亮蓝法。

1.2.4 肝组织病理切片制作

取公鸡右叶相同位置肝组织,4%福尔马林固定,石蜡包埋,切片为5 μm,苏木素-伊红(HE)染色,光镜下观察各组公鸡肝脏组织病理学变化。

1.3数据统计分析

数据采用Graphprism 5.0软件进行统计分析,用单因素ANOVA法分析差异显著性,若差异显著则用LSD法进行多重比较。试验数据用平均值±标准误(x±SE)表示。

2 结果与分析 2.1 SI对PNMC处理公鸡生长性能的影响

大豆异黄酮对PNMC处理公鸡生长性能的影响见表 2。与对照组相比,PNMC组在21和42 d时体质量显著降低(P<0.05);与PNMC组相比,PNMC+SI组在21和42 d时体质量显著增加,且达到对照组水平(P<0.05)。与对照组相比,PNMC组和PNMC+SI组在1~21 d,22~42 d和1~42 d时平均日采食量和料重比均极显著增加(P<0.01);在22~42 d时平均日增体质量极显著降低(P<0.01),在1~21d和1~42 d时无显著性差异(P>0.05)。与PNMC组相比,PNMC+SI组在1~21 d和22~42 d时平均日增体质量显著升高(P<0.05);在22~42 d和1~42 d时平均日采食量极显著下降(P<0.01),而在1~21 d时平均日采食量极显著升高(P<0.01);料重比在不同时期和全期均极显著降低(P<0.01)。

表 2 大豆异黄酮对3-甲基-4-硝基酚(PNMC)处理公鸡生长性能的影响 Table 2 Effects of soybean isoflavone(SI)on growth performance of 3-methyl-4-nitrophenol(PNMC)exposed roosters
项目
Items
日龄
Day-old
处理Treatment
对照组
Control group
PNMC组
PNMC group
PNMC+SI组
PNMC+SI group
体质量/g
Body weight
21673.20±8.77a632.80±7.40b706.20±19.85c
422 089.12±41.42a1 897.00±38.16b2 025.00±38.49a
平均日增体质量/g
Average daily gain
1~2129.84±0.42ab27.92±0.35a31.41±0.95b
22~4267.42±0.42A60.20±0.71B63.80±0.20C
1~4248.62±0.98A44.06±0.91B46.52±0.87AB
平均日采食量/g
Average daily feed intake
1~2163.44±0.09A69.79±0.34B71.42±0.44C
22~42138.50±4.11A182.40±1.79B165.50±3.38C
1~42101.10±2.06A126.10±1.75B118.50±2.83C
料重比
Feed/gain
1~212.13±0.03A2.50±0.03B2.29±0.07C
22~422.06±0.01A3.03±0.02B2.64±0.04C
1~422.09±0.04A2.87±0.06B2.56±0.05C
注:同行数据肩标大、小写字母不同分别表示差异极显著(P<0.01)和差异显著(P<0.05)。 Note: In the same row,values with different capital or small letter superscripts mean significant difference at 0.01 or 0.05 levels. The same as follows.
2.2 SI对PNMC处理公鸡肝脏氧化和抗氧化指标的影响

表 3可知:与对照组相比,PNMC组和PNMC+SI组在42 d时肝脏质量显著减少(P<0.05);与PNMC组相比,PNMC+SI组肝脏质量和肝脏指数无显著性差异(P>0.05)。

表 3 大豆异黄酮对PNMC处理公鸡肝脏质量及肝脏指数的影响 Table 3 Effects of SI on the liver weight and liver index of PNMC exposed roosters
项目
Items
日龄
Day-old
处理Treatment
对照组Control groupPNMC组PNMC groupPNMC+SI组PNMC+SI group
肝脏质量/g2115.07±0.9614.29±0.4314.24±0.61
Liver weight4238.22±0.83a34.27±1.22b34.29±1.03b
肝脏指数/%212.24±0.142.26±0.072.02±0.09
Liver index421.83±0.041.81±0.061.69±0.05

表 4可知:饮水中添加10 mg · L-1 PNMC在42 d时显著降低肝脏GSH含量(P<0.05),CAT和T-SOD活性也下降,但差异不显著(P>0.05),MDA和H2O2含量无明显变化(P>0.05)。与PNMC组相比,PNMC+SI组在42 d时GSH含量恢复到正常水平,且2组间差异显著(P<0.05),CAT和T-SOD活性呈上升的趋势,但没达到显著水平(P>0.05)。

表 4 大豆异黄酮对PNMC处理公鸡肝脏氧化和抗氧化指标的影响 Table 4 Effects of SI on liver oxidant and antioxidant action of PNMC exposed roosters
项目
Items
日龄
Day-old
处理Treatment
对照组
Control group
PNMC组
PNMC group
PNMC+SI组
PNMC+SI group
丙二醛含量/(mmol·g-1)210.51±0.040.52±0.020.39±0.04
MDA content420.62±0.030.60±0.020.57±0.03
过氧化氢酶活性/(U·g-1)2118.73±0.9618.24±1.0417.82±1.21
CAT activity4215.46±0.8714.29±1.1318.29±0.57
总超氧化物歧化酶活性/(U·mg-1)2129.54±2.1326.37±2.6529.63±3.32
T-SOD activity4229.28±2.0427.83±1.2933.25±1.31
谷胱甘肽含量/(mg·g-1)212.24±0.212.78±0.842.67±0.53
GSH content427.83±1.06a5.72±0.63b7.79±0.85a
过氧化氢含量/(mmol·g-1)2118.49±1.2215.26±1.3915.58±1.49
H2O2 content4216.28±2.0314.87±1.6816.29±1.92
2.3 SI对PNMC处理公鸡肝脏组织结构的影响

在正常生理状态下,肝组织切片HE染色图显示肝细胞排列整齐,以中央静脉为中心呈放射状排列,肝细胞形状规则,细胞分界清,排列致密而均一,无细胞变性和异常[19]。由本试验肝组织HE染色切片图可见,在21 d时,对照组公鸡的肝组织结构正常,肝细胞没有变性、坏死等病理性变化(图 1-A)。PNMC组公鸡的肝组织结构明显被破坏,肝细胞核着色更深,脏组织间隙较大,肝血窦变宽,细胞整体排列松散不紧凑,大部分肝细胞出现浑浊肿胀(图 1-B)。PNMC+SI组肝细胞坏死、变性等病变得到了很大程度的减轻,公鸡肝脏组织结构和细胞形态基本恢复正常,表明大豆异黄酮具有较好的保护肝细胞作用(图 1-C)。

图 1 21日龄(A~C)和42日龄(D~F)公鸡肝脏组织学图片 Fig. 1 Histomorphology in rooster liver tissue on 21 day-old(A-C)and 42 day-old(D-F) A,D:对照组Control group;B,E:PNMC组PNMC group;C,F:PNMC+SI组PNMC+SI group

在42 d时,对照组肝小叶结构完整,肝索排列整齐,肝细胞大小均匀,无明显病理改变,无炎症细胞浸润(图 1-D)。PNMC组肝小叶结构被破坏,肝细胞索排列紊乱,可见肝细胞坏死,肝细胞核明显大小不等,呈不同程度的固缩(图 1-E)。与PNMC组比较,PNMC+SI组肝小叶结构清楚,肝细胞排列较整齐,细胞核染色明显均匀,坏死减少,肝组织炎症明显减轻,上述肝组织病理学损伤有所缓解(图 1-F)。

3 讨论

饮水中添加10 mg · L-1 PNMC和饲料中添加100 mg · kg-1 SI不会引起急性中毒症状[20, 21]。本试验表明,PNMC显著降低了公鸡的日增质量、体质量和肝脏质量,增加了料重比,显著影响了公鸡的生长性能。这一结果与岳卓等[22]、Li等[23, 24]的研究类似。分析其原因,当10 mg · L-1 PNMC进入动物消化道后,可能改变消化道内环境,降低了动物对养分的消化吸收能力,进一步影响机体的代谢,从而导致生长受到抑制;饲料中添加大豆异黄酮能显著地改善PNMC对公鸡生长性能造成的影响,显著降低了料重比,同时增加了日增质量和体质量。

氧化和抗氧化指标的变化是机体是否受到氧化应激的直观表现[25, 26]。布同良等[27]研究表明氧化应激在PNMC引起生殖毒性中发挥了重要作用,PNMC能使细胞活性氧增加,破坏机体抗氧化防御体系,改变抗氧化酶活性及脂质过氧化水平,从而导致细胞氧化损伤。在正常情况下,机体内GSH、T-SOD、CAT等抗氧化物质可有效清除自由基,维持细胞氧化/抗氧化动态平衡;MDA是细胞膜受到过氧化攻击后脂质过氧化的终产物,是细胞发生脂质过氧化反应的产物中具有代表性的产物,能够间接反映机体组织细胞内脂质过氧化损伤的程度[28]。布同良等[29]研究发现100 mg · kg-1的PNMC腹腔注射小鼠,可导致睾丸活性氧生成及脂质过氧化程度增加,同时显著降低GSH含量和SOD、GSH-Px的活性。在本试验中,PNMC组公鸡肝脏GSH含量显著降低,CAT和T-SOD活性也呈现下降趋势,表明PNMC处理消耗了肝脏内大量抗氧化酶及非酶物质,而饲料中添加SI后可增高肝损伤公鸡抗氧化酶活性,保护肝脏功能。提示SI可抑制氧自由基反应,增强机体抗氧化能力,从而加速自由基的清除,维持机体的氧化和抗氧化平衡。目前,大豆异黄酮对于肝损伤方面的研究还较少,研究发现,三羟异黄酮对扑热息痛导致的肝损伤具有防治作用,其机制是通过增高抗氧化酶活性、增强对氧化应激的抵抗能力而实现的[30];同时,体外试验表明,大豆异黄酮及黄豆苷元给予肝细胞HepG2和Huh-7时,能增强其过氧化氢酶活性[31]。我们的研究结果也发现大豆异黄酮能降低脂质过氧化产物的含量,提高抗氧化酶的活性,但是,大豆异黄酮处理并没有显示出显著的抗氧化活性。

饮水中添加10 mg · L-1 PNMC对公鸡肝脏组织学形态有一定程度的损伤,通过饲料中添加100 mg · kg-1 SI可以起到修复的作用。这与前人报道类似,如宋美艳等[32]研究表明10 mg · kg-1的PNP皮下注射可引起大鼠肝脏的局部严重损伤,表现为肝脏细胞核固缩凝聚,汇管区和中央静脉有大量淋巴细胞浸润等。史小琴等[33]通过家兔肝缺血/再灌注30和60 min时,静脉注射黄酮类物质灯盏花乙素可使肝组织细胞形态学变化明显减轻。李涛等[34]在CCL4所致的大鼠急性化学性肝损伤试验中,发现异黄酮类物质葛根素能明显减轻肝组织变性、坏死程度,缓解肝组织的病理改变。其可能机制是SI通过抑制氧自由基的生成,增强氧自由基的清除,降低PNMC引起的肝脏氧化损伤,从而使肝脏恢复到正常状态。

在本试验中,饮水中添加10 mg · L-1 PNMC显著降低了公鸡日增质量和肝质量,组织切片也显示了一定的损伤,肝组织GSH含量下降,同时CAT、T-SOD等抗氧化酶活性均有降低的趋势,而在饲料中添加100 mg · kg-1 SI可以显著增加公鸡体质量和日增质量,减轻肝组织病理损伤,肝组织GSH含量升高,CAT、T-SOD等抗氧化酶活性有上升的趋势,MDA含量呈下降的趋势。此结果表明:大豆异黄酮能够缓解PNMC引起的公鸡肝损伤,增强公鸡肝脏抗氧化能力。其机制可能与提高机体抗氧化酶活性,加速自由基的清除,减少脂质过氧化有关。然而,大豆异黄酮由多种结构的黄酮组成,成分复杂,对肝损伤保护作用的机制可能存在诸多靶点,其药理效应和具体作用机制还需进一步研究。

参考文献(References)
[1] 梁增辉,何世华,孙成均,等. 引起青蛙畸形的环境内分泌干扰物的初步研究[J]. 环境与健康杂志,2002,19(6):419-421. Liang Z H,He S H,Sun C J,et al. Study on endocrine disruptor chemicals causing malformed frogs[J]. Journal of Environment and Health,2002,19(6):419-421(in Chinese).
[2] Mori Y,Kamata K,Toda N,et al. Isolation of nitrophenols from diesel exhaust particles(DEP)asvasodilatation compounds[J]. Biological and Pharmaceutical Bulletin,2003,28(3):394-395.
[3] Shinji T,Kazuyuki K,Hideyuki H,et al. Investigation of vasodilatory substances in diesel exhaust particles(DEP):isolation and identification of nitrophen old erivatives[J]. Journal of Health Science,2004,50(2):133-141.
[4] Zayed S M,Mahdy F. Decomposition of 14C-fenitrothion under the influence of UV and sunlight under tropical and subtropical conditions[J]. Chemosphere,2008,7(9):1653-1659.
[5] Boucher B A,Cotterchio M,Anderson L N,et al. Use of isoflavone supplements is associated with reduced postmenopausal breast cancer risk[J]. International Journal of Cancer,2013,132(6):1439-1450.
[6] Kurahashi N,Iwasaki M,Sasazuki S,et al. Soy product and isoflavone consumption in relation to prostate cancer in Japanese men[J]. Cancer Epidemiology,Biomarkers and Prevention,2007,16(3):538-545.
[7] 戴雨,杨林,牛建昭,等. 大豆异黄酮对更年期妇女症状及性激素的影响[J]. 北京中医药大学学报,2004,27(1):80-82. Dai Y,Yang L,Niu J Z,et al. Effects of soybean isoflavones on the menopausal symptoms and the secretion of sex hormones[J]. Journal of Beijing University of Traditional Chinese Medicine,2004,27(1):80-82(in Chinese with English abstract).
[8] 汪远金,许金林,张杰,等. 大豆异黄酮对去势大鼠骨密度及骨病理形态学作用的初步研究[J]. 中国中医药科技,2003,10(5):284-286. Wang Y J,Xu J L,Zhang J,et al. Experimental study of rats with intestinal irritable syndrome treated with ningchangdecoction[J]. Chinese Journal of Traditional Medical Science and Technology,2003,10(5):284-286(in Chinese).
[9] Yuan X X,Zhang B,Li L L,et al. Effects of soybean isoflavones on reproductive parameters in Chinese mini-pig boars[J]. Journal of Animal Science Biotechnology,2012,3(4):1-8.
[10] Hirohata M,Ono K,Takasaki J,et al. Anti-amyloidogenic effects of soybean isoflavones in vitro:fluoreseenee spectroscopy demonstrating direct binding to Aβ monomers,oligomers and fibrils[J]. Biochimical and Biophysical Acta-General Subjects,2012,1822(8):1316-1324.
[11] Masilamani M,Wei J,Sampson H A. Regulation of the immune response by soybean isoflavones[J]. Immune Research,2012,54(1/2/3):95-110.
[12] Masilamani M. Insights gained from 20 years of soy research[J]. Journal of Nutrition,2010,140(12):2289-2295.
[13] 方士英,徐茂红,赵克霞,等. 葛根总黄酮对刀豆蛋白A诱导的小鼠免疫性肝损伤保护作用及其机制的初步研究[J]. 中国药理学通报,2012,28(7):1033-1034. Fang S Y,Xu M H,Zhao K X,et al. Study on protective effects of total puerariaflavonoidson immunological liver injury in mice induced by concanavalin A and its mechanism[J]. Chinese Pharmacological Bulletin,2012,28(7):1033-1034(in Chinese with English abstract).
[14] 张韬,金静君,赖力,等. 银杏黄酮对四氯化碳诱导的小鼠肝损伤的保护作用[J]. 时珍国医国药,2012,23(7):1704-1706. Zhang T,Jin J J,Lai L,et al. Protective effects of ginkgetin against carbon tetrachloride-induced hepatic injury in mice[J]. Lishizhen Medicine and Materia Medical Research,2012,23(7):1704-1706(in Chinese with English abstract).
[15] 肖凤霞,邓韬,邓少东,等. 毛菊红总黄酮对酒精性肝损伤大鼠肝细胞凋亡的影响[J]. 广东医学学报,2012,28(3):316-318. Xiao F X,Deng T,Deng S D,et al. Effects of total flavonoids of Exocarpium Citri Grandis on hepatocyte apoptosis in rats with alcoholic hepatic injury[J]. Journal of Guangdong Pharmaceutical University,2012,28(3):316-318(in Chinese with English abstract).
[16] 杨丽娜,郭英,陈秋丽,等. 大豆复合物对四氯化碳致大鼠肝损伤的防护作用[J]. 中国老年学杂志,2009,29(3):559-560. Yang L N,Guo Y,Chen Q L,et al. Protective effect of soybean complex on hepatic injury induced by carbon tetrachloride in rats[J]. Chinese Journal of Gerontology,2009,29(3):559-560(in Chinese with English abstract).
[17] 李建芳,陈必成,余震,等. 染料木素对硫代乙酰胺诱导的肝纤维化大鼠PDGF-BB表达的影响[J]. 肝胆胰外科杂志,2009,21(2):118-121,125. Li J F,Chen B C,Yu Z,et al. Effect of genistein on the expression of PDGF-BB in hepatic fibrosis of rats induced by thioacetamide[J]. Journal of Hepatopanereatobiliary Surgery,2009,21(2):118-121,125(in Chinese with English abstract).
[18] 杨修仕. 大豆皂苷对急性酒精性肝损伤的保护作用研究[D]. 太原:山西大学,2011. Yang X S. Protective effects of soyasaponins on acute alcohol-induced liver damage[D]. Taiyuan:Shanxi University,2011(in Chinese with English abstract).
[19] Xiang Q,Liu G,Wang Y,et al. Carnosicacid attenuates lipopolysaccharide-induced liver injury in rats via fortifying cellular antioxidant defense system[J]. Food and Chemical Toxicology,2013,53:1-9.
[20] 徐伟风,朴元国,李延森,等. 3-甲基-4-硝基酚对公鸡睾丸早期发育的影响[J]. 生态毒理学报,2015,10(4):900-905. Xu W F,Piao Y G,Li Y S,et al. Effects of 3-methyl-4-nitrophenol on the early development of rooster testes[J]. Asian Journal of Ecotoxicology,2015,10(4):900-905(in Chinese with English abstract).
[21] 何学军,张妮娅,徐雪梅,等. 大豆异黄酮抗霉变饲料对肉鸡的影响及机制[J]. 中国兽医学报,2008,28(12):1468-1473. He X J,Zhang N Y,Xu X M,et al. Effect and mechanism of soybean isoflavones on avian broilers fed mold contaminated corn[J]. Chinese Journal of Veterinary Science,2008,28(12):1468-1473(in Chinese with English abstract).
[22] 岳卓,佘锐萍,田纪景,等. 3-甲基-4-硝基酚对大鼠睾丸组织的损伤作用[J]. 动物医学进展,2010,31(增刊):66-69. Yue Z,She R P,Tian J J,et al. Study of toxic effect of 3-methly-4-nitrophenol on SD rat testis[J]. Progress in Veterinary Medicine,2010,31(Suppl):66-69(in Chinese with abstract).
[23] Li C M,Takahashi S,Taneda S,et al. Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of reproductive function in immature female Japanese quail(Coturnix japonica)[J]. Journal of Reproduction and Development,2007,53:673-678.
[24] Li C M,Taneda S,Suzuki A,et al. Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of testicular function in immature male rats[J]. Journal of Andrology,2007,28:252-258.
[25] Alpsoy L,Yildirim A,Agar G. The antioxidant effects of vitamin A,C and E on aflatoxin B1-induced oxidative stress in human lymphocytes[J]. Toxicology and Industrial Health,2009,25(2):121-127.
[26] Kotan E,Alpsoy L,Anar M,et al. Protective role of methanol extract of Cetrariais landica(L.)against oxidative stress and genotoxic effects of AFB1 in human lymphocytes in vitro[J]. Toxicology and Industrial Health,2011,27(7):599-605.
[27] 布同良,米玉玲,曾卫东,等. 3-甲基-4-硝基酚引起的睾丸氧化损伤及其修复的研究[J]. 畜牧与兽医,2012,12(4):48-51. Bu T L,Mi Y L,Zeng W D,et al. Oxidative damage induced by 3-methyl-4-nitrophenol and its repair in testis[J]. Animal Husbandry and Veterinary Medicine,2012,12(4):48-51(in Chinese).
[28] 汪纪仓,杨自军,朱华丽,等. 铅对大鼠睾丸的氧化损伤及柚皮素的保护效应[J]. 饲料工业,2012,33(19):32-33. Wang J C,Yang Z J,Zhu H L,et al. Lead-induced oxidative stress and protective effect of naringenin on testis of rats[J]. Feed Industry,2012,33(19):32-33(in Chinese).
[29] 布同良. 槲皮素缓解镉和硝基酚诱导的生精细胞毒性作用的研究[D]. 杭州:浙江大学,2011. Bu T L. Study on the effects of quercetin on the toxicity of spermatogenic cells induced by cadmium and nitrophenol[D]. Hangzhou:Zhejiang University,2011(in Chinese with English abstract).
[30] Fan Y,Rong Y,Li P,et al. Genistein protection against acetaminophen-induced liver injury via its potential impact on the activation of UDP-glucuronosyl transferase and antioxidant enzymes[J]. Food and Chemical Toxicology,2013,55:172-181.
[31] Kampkotter A,Wiegand C,Timpel C,et al. Increased expression of catalase in human hepatoma cells by the soy isoflavone,daidzein[J]. Basical Clinical Pharmacological and Toxicology,2008,102(5):437-442.
[32] 宋美艳,张永辉,朴元国,等. 4-硝基酚对大鼠肝脏的毒性及氧化损伤[J]. 生态毒理学报,2014,9(3):495-502. Song M Y,Zhang Y H,Piao Y G,et al. Toxic effects and oxidative damage of 4-nitrophenol on rat liver[J]. Asian Journal of Ecotoxicology,2014,9(3):495-502(in Chinese with English abstract).
[33] 史小琴,张晶晶,刘秀平,等. 灯盏花素对肝缺血再灌注损伤生化指标的影响[J]. 军医进修学院学报,2005,26(5):362-363. Shi X Q,Zhang J J,Liu X P,et al. Effect of breviscapine on biochemistry in hepatic ischemia-reperfusion injury[J]. Journal of Chinese PLA Postgraduate Medical School,2005,26(5):362-363(in Chinese with English abstract).
[34] 李涛,邹志森. 葛根素对大鼠肝缺血再灌注损伤的保护作用[J]. 中药药理与临床,2004,20(4):15-16. Li T,Zou Z S. The protective effect of puerarin on hepatic injury induced by ischemia-reperfusion in rat[J]. Pharmacology and Clinics of Chinese Materia Medica,2004,20(4):15-16(in Chinese).