Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 5: 3317 - 3328

DOI: https://doi.org/10.12418/CJAN2024.284

RESEARCH PAPER

Effects of Resveratrol on Uterine and Ovarian Injury of Zearalenone-Induced Female Rats

  • YAO Dan , 1, 2 ,
  • LI Yina 1, 2 ,
  • CHENG Xiaoli 1, 2 ,
  • HE Zengwen 1 ,
  • WANG Yue 1, 2 ,
  • MA Xiaojun , 1, * ,
  • ZHANG Yong , 1, 2, *
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  • 1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
  • 2 Gansu Provincial Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation, Lanzhou 730070, China
* MA Xiaojun, associate professor, E-mail: ;
ZHANG Yong, professor, E-mail:

Received date: 2023-12-08

  Online published: 2024-05-15

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Abstract

This experiment was conducted to investigate the effects of resveratrol on uterine and ovarian injury of zearalenone-induced female rats by establishing an effective toxicity model of zearalenone-induced female rats. 1) Thirty-two 2-month-old female rats were randomly divided into 4 groups with 8 replicates per group and 1 rat per replicate. Rats in the control group were injected intraperitoneally with corn oil per day, and rats in the low, medium and high concentration groups were injected intraperitoneally with 1.5, 3.0 and 5.0 mg/kg of zearalenone per day, respectively, at a daily dose of 0.5 mL for a period of 5 days. 2) Thirty-two 2-month-old female rats were randomly divided into 4 groups with 8 replicates per group and 1 rat per replicate. During days 1 to 21, rats in the RSV group and RSV+ZEA group were given with 40 mg/kg resveratrol per day, and rats in the blank group and ZEA group were given with sodium carboxymethylcellulose solution per day, at a volume of 1 mL per day; during days 22 to 26, the animal models of zearalenone-induced apoptosis in the reproductive system of female rats was constructed for the female rats in ZEA group and RSV+ZEA group according to the above method, and the blank group and RSV group were injected intraperitoneally with corn oil. The results showed as follows: 1) compared with the control group, the medium and high concentration groups showed thinner endometrium, more uterine glands, increased of atretic follicles number, enlarged and severely congested corpus luteum, the relative expression levels of B lymphoblastoma-2-related X protein (Bax) and cysteine aspartate protease-3 (Caspase-3) mRNA and protein in uterus and ovary were significantly increased (P<0.05), and the relative expression levels of B lymphoblastoma-2 (Bcl-2) mRNA and protein were significantly decreased (P<0.05). 2) Compared with ZEA group, the RSV+ZEA group had a thickened endometrium with aligned epithelial cells and fewer uterine glands; developing follicles were seen in the ovary, the number of atretic follicles was reduced, and there was reduced corpus luteum congestion. Compared with blank and RSV+ZEA groups, the positive expression of Bax and Caspase-3 was enhanced in uterus and ovary of ZEA group, while the positive expression of Bcl-2 was attenuated. Compared with ZEA group, the relative expression levels of Bax and Caspase-3 mRNA and protein in uterus and ovary of RSV+ZEA group were significantly decreased (P<0.05), and the relative expression levels of Bcl-2 mRNA and protein were significantly increased (P<0.05). In conclusion, the zearalenone toxicity animal model can be successfully established in female rats by continuous intraperitoneal injection of zearalenone for 5 d in 2-month-old female rats (3.0 mg/kg per day, with corn oil as solvent). The zearalenone can damage the uterus and ovary of female rats, and resveratrol has a significant protective effect against zearalenone-induced reproductive toxicity.

Key words： zearalenone; resveratrol; apoptosis; reproductive

Cite this article

YAO Dan , LI Yina , CHENG Xiaoli , HE Zengwen , WANG Yue , MA Xiaojun , ZHANG Yong . Effects of Resveratrol on Uterine and Ovarian Injury of Zearalenone-Induced Female Rats[J]. Chinese Journal of Animal Nutrition, 2024 , 36(5) : 3317 -3328 . DOI: 10.12418/CJAN2024.284

玉米赤霉烯酮(zearalenone,ZEA)是镰刀菌属细菌污染玉米所产生的一种霉菌毒素,主要存在于玉米、大麦、小麦及以此类谷物为原料的饲粮中,具有热稳定性[1]。人和动物摄入被镰刀菌污染的谷物及其加工产品[2],或者摄入被污染的动物源性食品,如猪肉、牛奶等都可能导致中毒[3]。玉米赤霉烯酮结构与内源性雌激素相似,进入体内与雌激素受体竞争性结合,从而引发一系列类雌激素作用[4],主要扰乱动物生殖激素的分泌,诱导卵巢颗粒细胞氧化应激和凋亡[5],还会造成肝脏等消化系统的损伤[6]、引发肿瘤[7]等。玉米赤霉烯酮的毒性作用广泛且不易去除,关于其毒性作用的研究和防控就显得尤为重要。
白藜芦醇(resveratrol,RSV)是一种天然雌激素,加拿大和日本等国药品与食品监督管理局已将其列入保健品原料[8]。白藜芦醇有抗凋亡、抗氧化、抗炎、抗癌和心血管保护等作用。体内外研究表明,白藜芦醇可以通过抑制卵巢颗粒细胞凋亡来减轻原发性卵巢功能不全[9],可以有效抑制小鼠卵巢细胞的凋亡[10],通过抑制氧化应激来提高细胞活力[11];也可以通过影响细胞的生长分裂、凋亡、转移和血管生成等多种信号转导途径来影响癌症进展[12]。
玉米赤霉烯酮在动物饲料原料中污染范围广且不易失活,但目前尚未发现特效解毒剂和适合生产实际的吸附剂,给畜牧业的发展及人类健康带来了巨大影响。玉米赤霉烯酮对动物的生殖系统影响显著,而白藜芦醇作为一种天然雌激素,目前已有研究证明白藜芦醇对玉米赤霉烯酮所致的小鼠肝脏损伤有一定保护作用[13]。因此,本试验通过建立有效的大鼠模型,探讨白藜芦醇对玉米赤霉烯酮所致大鼠生殖系统凋亡是否有一定的影响,为以后研究白藜芦醇对玉米赤霉烯酮毒性影响提供基础,以期在未来的生产实践中提供理论参考。

1 材料与方法

1.1 试验材料

试验动物由中国农业科学院兰州兽医研究所提供,选择体重(200±10) g的2月龄无特定病原体(SPF)雌性大鼠。
玉米赤霉烯酮,纯度≥98%,购自成都乐美天医药科技有限公司;反式白藜芦醇,纯度≥98%,购自北京索莱宝科技有限公司;免疫组化试剂盒,购自北武汉博士德生物工程有限公司;β-肌动蛋白(β-actin)、B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)抗体,购自武汉三鹰生物技术有限公司;反转录试剂盒和实时荧光定量PCR(qPCR)试剂盒,购自湖南艾科瑞生物工程有限公司;其他常规试剂均为国药产品分析纯。

1.2 试验设计

1.2.1 玉米赤霉烯酮所致大鼠毒性模型的建立

将32只2月龄雌性大鼠随机分为4组,每组8个重复,每个重复1只。对照组大鼠每天腹腔注射玉米油,低、中、高浓度组大鼠分别每天腹腔注射1.5、3.0和5.0 mg/kg玉米赤霉烯酮,每天注射量均为0.5 mL,玉米赤霉烯酮以玉米油为溶剂按体重配比,试验期5 d。大鼠末次给药12 h后处死,采取子宫和卵巢,一部分用多聚甲醛固定,剩余组织脏器放入冻存管于-80 ℃保存备用。

1.2.2 白藜芦醇对玉米赤霉烯酮所致雌性大鼠子宫和卵巢损伤的影响

将32只2月龄雌性大鼠随机分为4组,每组8个重复,每个重复1只。第1~21天,RSV组和RSV+ZEA组大鼠每天灌胃40 mg/kg白藜芦醇,空白组和ZEA组大鼠每天灌胃羧甲基纤维素钠溶液,每天灌胃量均为1 mL,白藜芦醇以1%羧甲基纤维素钠溶液为溶剂按体重配比;第22~26天,ZEA组和RSV+ZEA组大鼠连续5 d腹腔注射玉米赤霉烯酮(3.0 mg/kg,玉米油为溶剂,5 d),空白组和RSV组大鼠连续5 d腹腔注射玉米油,每次注射量为0.5 mL。大鼠末次给药12 h后处死,采取子宫和卵巢,一部分用多聚甲醛固定,剩余组织脏器放入冻存管于-80 ℃保存备用。

1.3 苏木精-伊红(HE)染色观察组织脏器病理变化

将多聚甲醛固定组织样品脱水、石蜡包埋、切片,将石蜡切片脱蜡至水,蒸馏水洗5 min,苏木精染色3~7 min,流水冲洗5 min,1%盐酸乙醇30 s,蒸馏水洗30 s,伊红染色3 min,蒸馏水洗30 s,梯度酒精脱水,二甲苯透明,树脂封片,显微镜观察组织病理学变化。

1.4 免疫组织化学方法检测相关蛋白的表达情况

组织石蜡切片脱蜡至水,磷酸盐缓冲液(PBS)洗3次,过氧化氢(H2O2)孵育10 min,PBS洗3次,柠檬酸钠抗原修复,封闭液孵育30 min,Bax、Bcl-2和Caspase-3抗体稀释后4 ℃过夜(Bax抗体稀释比例1∶150,Bcl-2抗体稀释比例1∶60,Caspase-3抗体稀释比例1∶60),PBS洗3次,生物素标记二抗37 ℃孵育30 min,PBS洗3次,生物素-链霉亲和素-过氧化物酶复合物(SABC)孵育30 min,PBS洗3次,二氨基联苯胺(DAB)试剂显色,终止后苏木素复染,自来水冲洗30 min,梯度酒精脱水,二甲苯透明,树脂封片,显微镜下观察。

1.5 qPCR方法检测相关基因的表达情况

提取大鼠子宫和卵巢总RNA,反转录后按试剂盒说明书加入反应试剂,检测Bax、Bcl-2和Caspase-3的mRNA相对表达量,引物序列信息见表1。采用2-△△Ct法处理后进行数据分析。
表1 引物序列信息

Table 1 Information of primer sequences

基因
Genes		 上游引物序列
Upstream primer
sequence (5'—3')		 下游引物序列
Downstream primer
sequence (5'—3')		 登录号
Accession number
β-肌动蛋白β-actin CTATGAGGGTTACGCGCTCC ATGTCACGCACGATTTCCCT NM_031144.3
B淋巴细胞瘤-2相关X蛋白
Bax		 TCACGTGACCGGGGCG ATGATCTGTTCAGAGCTGGTGG NM_017059.2
B淋巴细胞瘤-2 Bcl-2 GGTGAACTGGGGGAGGATTG AGAGCGATGTTGTCCACCAG NM_016993.2
半胱氨酸天冬氨酸蛋白酶-3
Caspase-3		 GGACAACAACGAAACCTCCG ACACAAGCCCATTTCAGGGT XM_039094205.1

1.6 Western Blotting检测相关蛋白的表达

组织样品研磨提取蛋白,部分蛋白变性,另一部分放入-80 ℃保存。变性蛋白电泳,转膜,封闭,加入稀释的β-actin、Bax、Bcl-2和Caspase-3抗体于4 ℃过夜(Bax抗体稀释比例1∶600,Bcl-2抗体稀释比例1∶600,Caspase-3抗体稀释比例1∶500),磷酸缓冲液加吐温20(PBST)洗3次,二抗37 ℃孵育1 h,PBST洗3次,曝光。Image J扫描灰度值后进行数据分析。

1.7 数据统计分析

试验数据用SPSS 26.0软件进行统计分析,采用单因素方差分析(one-way ANOVA)和LSD检验,结果以平均值±标准差表示,P>0.05表示差异不显著,P<0.05表示差异显著。用Origin 86(2021)进行绘图。

2 结果

2.1 玉米赤霉烯酮所致大鼠毒性模型的建立

2.1.1 大鼠子宫和卵巢组织病理结构

如图1所示,大鼠子宫和卵巢组织病理变化显示,对照组子宫内膜上皮细胞呈单层柱状,子宫腺少,肌层与外膜界限明显;低、中、高浓度组子宫内膜变薄、肌层与对照组相比增厚,子宫腺明显增多,与玉米赤霉烯酮的浓度呈正向关;中、高浓度组子宫内膜基底部可见大量细胞核呈深蓝黑色,胞核浓染、胞质固缩。对照组卵巢皮质部可观察到原始卵泡、初级卵泡和次级卵泡,卵母细胞正常发育,黄体未见充血;低、中、高浓度组皮质部可观察到原始卵泡和初级卵泡数量明显减少,闭锁卵泡数量增多,黄体增多、增大且充血严重;中、高浓度组皮质部基本观察不到原始卵泡和初级卵泡,只有少数次级卵泡未发生闭锁。
图1 玉米赤霉烯酮对大鼠子宫和卵巢组织病理的影响

EP:黏膜上皮 mucosal epithelium;LP:固有层 lamina propria;UG:子宫腺体 uterine glands;TM:肌层 myometrium;SM:黏膜层 mucosa;PyF:初级卵泡 primary follicle;PrF:原始卵泡 primordial follicle;SF:次级卵泡 secondary follicle;AF:闭锁卵泡 atretic follicle;CL:黄体 corpus luteum;L:血管 blood vessels。图3同 the same as Fig.3。

A:子宫;B:卵巢;1~4分别为对照组、低浓度组、中浓度组、高浓度组。A: uterus; B: 卵巢 ovary; 1 to 4 were control group, low concentration group, medium concentration group and high concentration group, respectively.

Fig.1 Effects of ZEA on uterine and ovarian tissue pathology of rats (50×)

2.1.2 大鼠子宫和卵巢中Bax、Bcl-2、Caspase-3的mRNA和蛋白相对表达量

如图2所示,大鼠子宫和卵巢中Bax、Bcl-2、Caspase-3的mRNA和蛋白相对表达量显示,与对照组相比,中、高浓度组的子宫和卵巢中Bax和Caspase-3的mRNA和蛋白相对表达量显著升高(P<0.05),Bcl-2的mRNA和蛋白相对表达量显著降低(P<0.05)。
图2 大鼠子宫和卵巢中Bax、Bcl-2、Caspase-3的mRNA和蛋白相对表达量

-actin:β-肌动蛋白;Bax:B淋巴细胞瘤-2相关X蛋白:B淋巴细胞瘤-2;Caspase-3:半胱氨酸天冬氨酸蛋白酶-3。数据柱形标注不同小写字母表示差异显著(P<0.05)。图6、图7同。β-actin: beta-actin; Bax: B-cell lymphoma-2 related X protein; Bcl-2: B-cell lymphoma-2; Caspase-3: cysteinyl aspartate specific proteinase-3. Value columns with different small letters mean significant difference (P<0.05). The same as Fig.6 and Fig.7。

a:大鼠子宫和卵巢中Bax、Bcl-2和Caspase-3 mRNA相对表达量;b:大鼠子宫和卵巢中Bax、Bcl-2和Caspase-3蛋白相对表达量。a: relative expression levels of Bax, Bcl-2 and Caspase-3 mRNA in uterus and ovary of rats; b: relative expression of Bax, Bcl-2 and Caspase-3 proteins in uterus and ovary of rats.

1~4分别为对照组、低浓度组、中浓度组、高浓度组。1 to 4 were control group, low concentration group, medium concentration group and high concentration group, respectively.

Fig.2 Relative expression levels of Bax, Bcl-2 and Caspase-3 in uterus and ovary of rats

2.2 白藜芦醇对玉米赤霉烯酮所致大鼠子宫和卵巢损伤的影响

2.2.1 大鼠子宫和卵巢组织的病理结构

如图3所示,大鼠子宫和卵巢组织病理变化显示,RSV组和空白组子宫内膜上皮细胞为柱状上皮细胞,排列整齐,子宫腺固有层、肌层、外膜分界明显;卵巢观察到原始卵泡、初级卵泡和次级卵泡,黄体较小。与空白组相比,ZEA组子宫内膜明显变薄,子宫腺明显增多,内膜基底部细胞出现大量核浓染,肌层界限不明显;卵巢中闭锁卵泡增多,观察不到原始卵泡和初级卵泡,黄体增多增大。与ZEA组相比,RSV+ZEA组子宫内膜上皮变厚,子宫腺明显减少,内膜基底部细胞核浓染明显减少;卵巢中原始卵泡、初级卵泡和次级卵泡结构明显,闭锁卵泡数量减少,黄体数量减少。
图3 白藜芦醇对玉米赤霉烯酮所致大鼠子宫和卵巢组织病理的影响

A:子宫;B:卵巢;1~4分别为空白组、RSV组、RSV+ZEA组、ZEA组。

Fig.3 Effects of RSV on uterus and ovary histopathology of ZEA-induced rats (50×)

A: uterus; B: 卵巢 ovary; 1 to 4 were blank group, RSV group, RSV+ZEA group and ZEA group, respectively.

2.2.2 大鼠子宫和卵巢中Bax、Bcl-2和Caspase-3蛋白的表达

如图4、图5所示,免疫组织化学显示,子宫中Bax、Bcl-2和Caspase-3蛋白于子宫内膜上皮细胞和固有层中有阳性表达。卵巢中Bcl-2和Caspase-3在卵泡颗粒细胞、卵泡内外膜中有阳性表达,Bax在正常发育卵泡的内外膜、闭锁卵泡的颗粒细胞和间质腺中阳性表达较明显。与空白组和RSV+ZEA组相比,ZEA组子宫和卵巢的Bax和Caspase-3阳性表达明显增强,而Bcl-2阳性表达减弱;空白组与RSV+ZEA组相比无明显变化。
图4 白藜芦醇对玉米赤霉烯酮所致大鼠子宫Bax、Bcl-2和Caspase-3蛋白表达的影响

EP:黏膜上皮 mucous epithelium;LP:固有层 lamina propria。

A:空白:B:Bcl-2;C:Bax;D:Caspase-3;1~4分别为空白组、RSV组、RSV+ZEA组、ZEA组。图5同。A: blank; B: Bcl-2; C: Bax; D: Caspase-3; 1 to 4 were blank group, RSV group, RSV+ZEA group and ZEA group, respectively. The same as Fig.5.

Fig.4 Effects of RSV on Bax, Bcl-2 and Caspase-3 protein expression in uterus of ZEA-induced rats (400×)

图5 白藜芦醇对玉米赤霉烯酮所致大鼠卵巢Bax、Bcl-2和Caspase-3蛋白表达的影响

O:卵母细胞 oocyte;GC:颗粒细胞 granulosa cell;TI:内膜层 inner membrane layer;TE:外膜层 outer membrane layer。

Fig.5 Effects of RSV on Bax, Bcl-2 and Caspase-3 protein expression in ovary of ZEA-induced rats (400×)

2.2.3 大鼠子宫和卵巢中Bax、Bcl-2和Caspase-3的mRNA相对表达量

如图6所示,Bax、Bcl-2和Caspase-3的mRNA相对表达量显示,与空白组相比,ZEA组的子宫和卵巢中Bax和Caspase-3的mRNA相对表达量显著升高(P<0.05),Bcl-2的mRNA相对表达量显著降低(P<0.05);与ZEA组相比,RSV+ZEA组的子宫和卵巢中Bax和Caspase-3的mRNA相对表达量显著降低(P<0.05),Bcl-2的mRNA相对表达量显著升高(P<0.05);与空白组相比,RSV+ZEA组的子宫和卵巢中Caspase-3的mRNA相对表达量差异不显著(P>0.05)。
图6 白藜芦醇对玉米赤霉烯酮所致大鼠子宫和卵巢Bax、Bcl-2和Caspase-3的mRNA相对表达量的影响

1~4分别为空白组、RSV组、RSV+ZEA组、ZEA组。图7同。

Fig.6 Effects of RSV on relative expression levels of Bax, Bcl-2 and Caspase-3 mRNA in uterus and ovary of ZEA-induced rats

1 to 4 were blank group, RSV group, RSV+ZEA group and ZEA group, respectively. The same as Table 7.

2.2.4 大鼠子宫和卵巢中Bax、Bcl-2和Caspase-3的蛋白相对表达量

如图7所示,子宫和卵巢中Bax、Bcl-2和Caspase-3的蛋白相对表达量显示,与空白组相比,ZEA组的子宫和卵巢中Bax和Caspase-3的蛋白相对表达量显著升高(P<0.05),Bcl-2的蛋白相对表达量显著降低(P<0.05)。与ZEA组相比,RSV+ZEA组的子宫和卵巢中Bax和Caspase-3的蛋白相对表达量显著降低(P<0.05),Bcl-2的蛋白相对表达量显著升高(P<0.05);与空白组相比,RSV+ZEA组的子宫中Caspase-3的蛋白相对表达量差异不显著(P>0.05)。
图7 白藜芦醇对玉米赤霉烯酮所致大鼠子宫和卵巢Bax、Bcl-2和Caspase-3的蛋白相对表达量的影响

Fig.7 Effects of RSV on relative expression levels of Bax, Bcl-2 and Caspase-3 protein in uterus and ovary of ZEA-induced rats

3 讨论

3.1 玉米赤霉烯酮所致雌性大鼠毒性模型的建立

玉米赤霉烯酮与内源性雌激素结构相似,主要导致动物机体生殖系统功能障碍,雌性动物出现卵巢[14]和子宫[15]的细胞凋亡,卵巢颗粒细胞氧化应激,子宫增生,外阴红肿[16],进而导致繁殖障碍,严重危害生产性能[17],也会导致肝脏[18]、肾脏[19]、脾脏[20]等组织损伤,玉米赤霉烯酮还具有一定的免疫毒性[17]。目前玉米赤霉烯酮并没有特效解毒剂,生产实际中通常采用停喂饲料的方式控制毒性发展[21]。
本试验结果显示,中、高浓度组的子宫内膜变薄,肌层增厚,内膜上皮细胞基底部发现大量核浓染,肌层界限不明显;闭锁卵泡数量增多,正常发育卵泡数量减少,仅见次级卵泡,黄体数量明显增多。王丙雷等[22]在玉米赤霉烯酮对小鼠子宫影响的试验中发现,玉米赤霉烯酮及代谢产物可导致机体出现一系列雌激素亢进现象,如子宫肌层显著增厚,子宫腺增多;周敏等[23]研究玉米赤霉烯酮对断奶仔猪的影响中发现,与对照组相比,玉米赤霉烯酮组的子宫肌层增厚,子宫腺增多、腺腔增大,内膜上皮内陷变多变深并且具有剂量依赖性;刘秀凤等[24]研究不同浓度玉米赤霉烯酮对断奶仔猪卵巢发育的影响发现,低浓度组原始卵泡数量减少,可观察到闭锁卵泡;中浓度组闭锁卵泡主要为有腔次级生长卵泡;高浓度组的卵巢闭锁卵泡主要为初级生长卵泡。上述结果均与本试验结果一致。
本试验选择细胞凋亡相关因子为Bcl-2、Bax和Caspase-3。细胞凋亡机制被Bcl-2家族成员严密调控,Bcl-2/Bax可相对准确地揭示细胞未来是否会发生凋亡[25]。Miramar等[26]研究发现,Bcl-2先接受细胞内死亡信号,进而激活下游Caspase-3,准确反映细胞凋亡的发生。本试验中,与对照组相比,中、高浓度组的子宫和卵巢中Bax和Caspase-3的mRNA和蛋白相对表达量显著上升,Bcl-2的mRNA和蛋白相对表达量显著降低。有研究表明,玉米赤霉烯酮介导的细胞凋亡机制是通过上调Bax表达的同时下调Bcl-2表达[27]。俞亚玲等[28]研究发现,雌性大鼠玉米赤霉烯酮中毒模型复制成功的标志为卵泡闭锁,卵巢颗粒细胞凋亡。综上所述,雌性大鼠连续腹腔注射玉米赤霉烯酮5 d(每天3.0 mg/kg,玉米油为溶剂)即可建立雌性大鼠玉米赤霉烯酮中毒病理模型。

3.2 白藜芦醇对玉米赤霉烯酮所致雌性大鼠子宫和卵巢损伤的保护作用

白藜芦醇是一种天然多酚类化合物,具有抗癌、抗衰老和抗凋亡等多种活性[29],还有一定的免疫调节作用[30],可以降低热应激造成的淋巴细胞凋亡[31],缓解卵巢氧化应激、抑制卵巢细胞凋亡[32]。试验结果显示,与ZEA组相比,RSV+ZEA组的大鼠子宫和卵巢中Bax、Caspase-3的mRNA和蛋白相对表达量显著降低,同时Bcl-2的mRNA和蛋白相对表达量显著升高,证明白藜芦醇可以有效缓解玉米赤霉烯酮引起的细胞凋亡。此外,白藜芦醇还可以降低促黄体生成素(luteotropic hormone,LH)水平,保护卵巢内膜的正常形态[33]。本试验结果显示,与ZEA组相比,RSV+ZEA组子宫内膜上皮变厚,子宫腺减少,内膜基底部细胞核浓染减少;卵巢中原始卵泡、初级卵泡和次级卵泡结构明显,闭锁卵泡数量减少,黄体数量减少,证实白藜芦醇可有效缓解玉米赤霉烯酮生殖毒性,维持子宫正常形态和卵泡正常发育。
玉米赤霉烯酮进入体内与雌激素受体竞争性结合,导致雌性生殖生理紊乱,诱导卵巢颗粒细胞氧化应激和凋亡[34],也会影响雄性生殖功能,降低精液质量[35],导致睾丸癌等[4]。目前有研究证明白藜芦醇可以有效缓解玉米赤霉烯酮引起的肝脏损伤[13]和对大鼠的免疫毒性及DNA损伤[36]。本试验结果显示,白藜芦醇连续灌胃21 d(每天40 mg/kg)可有效保护玉米赤霉烯酮所致的雌性大鼠卵巢和子宫的毒性损伤。综上所述,白藜芦醇极有潜力成为一种缓解玉米赤霉烯酮毒性的饲料添加剂。

4 结论

① 2月龄雌性大鼠连续腹腔注射玉米赤霉烯酮5 d(每天3.0 mg/kg,玉米油为溶剂)可成功建立雌性大鼠玉米赤霉烯酮毒性动物模型。
② 白藜芦醇可以通过抗凋亡作用有效保护玉米赤霉烯酮所致的雌性大鼠卵巢和子宫的损伤,从而缓解其生殖毒性,为白藜芦醇作为保护性饲料添加剂提供参考。

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