玉米赤霉烯酮吸附剂对后备母猪子宫中LC3、PCNA分布和表达的影响

doi:10.3864/j.issn.0578-1752.2021.18.018

摘要/Abstract

摘要：

【目的】已有研究证实玉米赤霉烯酮（ZEA）在动物体内可通过雌激素受体激活雌激素敏感基因,产生生殖毒性,影响子宫内膜细胞的生长、卵母细胞的成熟及卵泡颗粒细胞的增殖。探讨玉米赤霉烯酮吸附剂（沸石+蒙脱石等复合制剂）对后备母猪子宫的LC3与PCNA的分布和蛋白表达量的影响, 从组织化学角度探讨该ZEA新型吸附剂的脱毒效果。【方法】选择体重30±2.11kg左右的（杜×长×大）健康三元杂交后备母猪48头,随机分为 6 组,每组8头,对照组饲喂基础饲粮、玉米赤霉烯酮组（ZEA）饲喂含有1.008 mg·kg^-1玉米赤霉烯酮的饲粮、0.1%新型吸附剂组（ZEA0.1）饲喂含有1.0 g·kg^-1吸附剂的ZEA组日粮、0.25%新型吸附剂组（ZEA0.25）饲喂含有2.5 g·kg^-1吸附剂的ZEA组日粮、0.5%吸附剂组（ZEA0.5）饲喂含有5.0 g·kg^-1吸附剂的ZEA组日粮、蒙脱石组（ZEA-M）饲喂含有2.5 g·kg^-1蒙脱石的ZEA组日粮。预试期 7 d,正试期21 d,试验结束进行屠宰取样。【结果】ZEA引起子宫器官指数增加,添加0.25%与0.5%新型吸附剂降低子宫器官指数效果明显。免疫组化结果显示子宫中 LC3 和PCNA阳性细胞主要分布在子宫腺上皮细胞内,腔上皮细胞的LC3免疫阳性反应弱于腺上皮细胞。LC3阳性反应在对照组的子宫腺上皮细胞和腔上皮细胞胞质内呈强阳性表达;ZEA组明显弱于对照组,新型吸附剂组的LC3的免疫阳性反应及阳性细胞数量明显较ZEA组增加,且有剂量依赖趋势,但ZEA-M组效果不明显,略多于ZEA组。各组的子宫腔上皮和腺上皮中PCNA的观察结果与LC3变化趋势相反。WB和qRT-PCR结果也显示添加0.25%与0.5%新型吸附剂可促进LC3的表达,增强了自噬,降低了PCNA表达。这些结果提示ZEA抑制了LC3的表达,抑制细胞自噬,破坏子宫内膜细胞和腺上皮细胞的稳态。但新型吸附剂使LC3蛋白表达上调,PCNA表达降低,可以促进细胞自噬,并抵抗由ZEA引起的子宫内膜细胞异常增殖（PCNA表达增加）,通过双向调节对细胞起到很好的保护作用。该结果为新型吸附剂的应用提供了理论依据。【结论】本试验条件下,ZEA可引起后备母猪子宫发生增殖反应,新型吸附剂一定限度内能够对抗ZEA对子宫正常生理机能的副作用。以添加0.25%与0.5%剂量的新型吸附剂较为合适。

关键词: 玉米赤霉烯酮, 子宫, LC3, PCNA, 后备母猪, 吸附剂

Abstract:

【Objective】 It has been proved that zearalenone (ZEA) can activate estrogen sensitive gene through estrogen receptor in animals, resulting in reproductive toxicity, and affecting endometrial cell growth, oocyte maturation and follicular granulosa cell proliferation. The purpose of this experiment was to investigate the effects of ZEA adsorbent (zeolite + montmorillonite combinations) on distribution and expression of LC3 and PCNA in the uterus of gilts, and to discuss the detoxification effect of the new ZEA adsorbent from the perspective of histochemistry. 【Method】 A total of 48 healthy gilts with a body weight of 30±2.11 kg were randomly divided into 6 groups (n = 8): control group with basal diet, the ZEA group with basal diet + 1.008 mg∙kg^-1ZEA, the 0.1% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 1.0 g∙kg^-1 new adsorbent, the 0.25% ZEA adsorbent (ZEA 0.25) group with ZEA diet + 2.5 g∙kg^-1 new adsorbent, the 0.5% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 5.0 g∙kg^-1 new adsorbent, and the ZEA montmorillonite (ZEA+M) group with ZEA diet + 2.5 g∙kg^-1 montmorillonite. The preliminary trail period was 7 d, and the trial period was 21 d. 【Result】 ZEA increased the uterine organ index, and adding 0.25% and 0.5% adsorbents in the diet decreased the index of uterine organs obviously. The LC3 and PCNA positive cells were mainly distributed in the glandular epithelial cells, and the LC3 immunoreactivity of lumen epithelial cells were weaker than that of glandular epithelial cells. The immunoreactivity of LC3 in control group was stronger than that of ZEA group. The immunoreactivity and number of positive cells of LC3 in the ZEA new adsorbent group were significantly higher than those in the ZEA group, and there was a dose-dependent trend, but the effect of the ZEA-M group was increased slightly more than that of ZEA group. The results of PCNA in the luminal and glandular epithelium were contrary to that of LC3. The WB and qRT-PCR results also showed that 0.25% and 0.5% new adsorbents could promote the expression of LC3, increase autophagy, and decrease the PCNA immunoreactive reaction. These results indicated that ZEA inhibited the expression of LC3, inhibited autophagy and destroyed the homeostasis of endometrial cells and glandular epithelial cells. However, the new adsorbent could increase the expression of LC3 protein and decrease the expression of PCNA, which could promote autophagy and resist the abnormal proliferation of endometrial cells caused by ZEA. The new adsorbent had a good protective effect on uterine cells through bidirectional regulation.The experimental results provided theoretical basis for the further application of the new adsorbent. 【Conclusion】In this experiment, ZEA induced the uterus proliferation reaction, the new adsorbent (zeolite + montmorillonite combinations) resisted the negative effect of ZEA on the normal physiological function of uterus within a certain limits. As a result, 0.25% and 0.5% doses of this newadsorbents was suitable.

Key words: zearalenone, uterus, LC3, PCNA, gilts, adsorbent

黄丽波,王金全,高文博,陈红菊,侯衍猛,袁学军,王春阳. 玉米赤霉烯酮吸附剂对后备母猪子宫中LC3、PCNA分布和表达的影响[J]. 中国农业科学, 2021, 54(18): 4008-4017.

HUANG LiBo,WANG JinQuan,GAO WenBo,CHEN HongJu,HOU YanMeng,YUAN XueJun,WANG ChunYang. Effects of Dietary Zearalenone Adsorbent on the Distribution and Expression of LC3 and PCNA in the Uterus of Gilts[J]. Scientia Agricultura Sinica, 2021, 54(18): 4008-4017.

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[7]	GAO X, SUN L, ZHANG N, LI C, ZHANG J, XIAO Z, QI D. Gestational zearalenone exposure causes reproductive and developmental toxicityin pregnant ratsand female offspring. Toxins (Basel), 2017, 9(1):21. doi: 10.3390/toxins9010021
[8]	GREEN M L, DIEKMAN M A, MALAYER J R, SCHEIDT A B, LONG G G. Effect of prepubertal consumption of Zearalenone on puberty and subsequent reproduction of gilts. Journal of Animal Science, 1990, 68(1):171-178. doi: 10.2527/1990.681171x
[9]	YANG L J, ZHOU M, HUANG L B, YANG W R, YANG Z B, JIANG S Z, GE J S. Zearalenone-promoted follicle growth through modulation of wnt-1/β-catenin signaling pathway and expression of estrogen receptor genes in ovaries of postweaning piglets. Journal of Agricultural and Food Chemistry, 2018, 66(30):7899-7906. doi: 10.1021/acs.jafc.8b02101
[10]	ZHOU M, YANG L J, SHAO M H, WANG Y X, YANG W R, HUANG L B, ZHOU X M, JIANG S Z, YANG Z B. Effects of zearalenone exposure on the TGF-β1/Smad3 signaling pathway and the expression of proliferation or apoptosis related genes of post-weaning gilts. Toxins, 2018, 10(2):49-61. doi: 10.3390/toxins10020049
[11]	DAI M L, JIANG S Z, YUAN X J, YANG W R, YANG Z B, HUANG L B. Effects of Zearalenone-diet on expression of ghrelin and PCNA genes in ovaries of post-weaning piglets. Animal Reproduction Science, 2016, 168:126-137. doi: 10.1016/j.anireprosci.2016.03.006
[12]	YOUNG L G, KING G J. Low concentrations of Zearalenone in diets of mature gilts. Journal of Animal Science, 1986, 63(4):1191-1196. doi: 10.2527/jas1986.6341191x
[13]	MINERVINI F, DELL'AQUILA M E. Zearalenone and reproductive function in farm animals. International Journal of Molecular Sciences, 2008, 9(12):2570-2584. doi: 10.3390/ijms9122570
[14]	RAINEY M R, TUBBS R C, BENNETT L W, COXN M. Prepubertal exposure to dietary zearalenone alters hypothalamo-hypophysial function but does not impair post pubertal reproductive function of gilts. Journal of Animal Science, 1990, 68(7):2015-2022. doi: 10.2527/1990.6872015x
[15]	EDWARDS S, CANTLEY T C, DAY B N. The effects of Zearalenone on reproduction in swine. II. The effect on puberty attainment and postweaning rebreeding performance. Theriogenology, 1987, 28(1):51-58. doi: 10.1016/0093-691X(87)90185-3
[16]	YANG J Y, ZHANG Y F, WANG Y Q, CUI S. Toxic effects of Zearalenone and alpha-Zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells. Toxicology in Vitro, 2007, 21(4):558-565. doi: 10.1016/j.tiv.2006.10.013
[17]	GAO Y S, ZHAO Y, ZHANG H F, ZHANG P F, LIU J, FENG Y N, MEN Y H, LI L, SHEN W, SUN Z Y, MIN L J. Pubertal exposure to low doses of Zearalenone disrupting spermatogenesis through ERα related genetic and epigenetic pathways. Toxicology Letters, 2019, 315:31-38. doi: 10.1016/j.toxlet.2019.08.007
[18]	ABID-ESSEFI S, BAUDRIMONT I, HASSEN W, OUANES Z, MOBIO T A, ANANE R, CREPPY E E, BACHA H. DNA fragmentation, apoptosis and cell cycle arrest induced by Zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology, 2003, 192(2/3):237-248. doi: 10.1016/S0300-483X(03)00329-9
[1]	ZINEDINE A, SORIANO J M, MOLTO J C, MAñES J. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: An oestrogenic mycotoxin. Food and Chemical Toxicology, 2007, 45(1):1-18. doi: 10.1016/j.fct.2006.07.030
[2]	ITO Y, OHTSUBO K. Effects of neonatal administration of zearalenone on the reproductive physiology of female mice. Journal of Veterinary Medical Science, 1994, 56(6):1155-1159. doi: 10.1292/jvms.56.1155
[19]	ABID-ESSEFI S, OUANES Z, HASSEN W, BAUDRIMONT I, CREPPY E, BACHA H. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to Zearalenone. Toxicology in Vitro, 2004, 18(4):467-474. doi: 10.1016/j.tiv.2003.12.011
[20]	CHOI S, SHIN H, SONG H, LIM H J. Suppression of autophagic activation in the mouse uterus by estrogen and progesterone. The Journal of Endocrinology, 2014, 221(1):39-50. doi: 10.1530/JOE-13-0449
[21]	WEAVER A C, SEE M T, KIM S W. Protective effect of two yeast based feed additives on pigs chronically exposed to deoxynivalenol and Zearalenone. Toxins, 2014, 6(12):3336-3353. doi: 10.3390/toxins6123336
[22]	ZHANG Y, GAO R, LIU M, SHI B, SHAN A, CHENG B. Use of modified halloysite nanotubes in the feed reduces the toxic effects of zearalenone on sow reproduction and piglet development. Theriogenology, 2015, 83(5):932-941. doi: 10.1016/j.theriogenology.2014.11.027
[23]	MIZUSHIMA N, KOMATSU M. Autophagy: renovation of cells and tissues. Cell, 2011, 147(4):728-741. doi: 10.1016/j.cell.2011.10.026
[24]	RUNWAL G, STAMATAKOU E, SIDDIQI F H, PURI C, ZHU Y, RUBINSZTEIN D C. LC3-positive structures are prominent in autophagy-deficient cells. Scientific Reports, 2019, 9(1):10147. doi: 10.1038/s41598-019-46657-z
[25]	HECKMANN B L, GREEN D R. LC3-associated phagocytosis at a glance. Journal Cell Science, 2019, 132(5). pii: jcs222984. doi: 10.1242/jcs.222984. doi: 10.1242/jcs.222984
[26]	AMÂLINEIC, PÂVÂLEANU I, GRIGORAS A, CÂRUNTU I D, GIUSCÂ S E, AVÂDÂNEI E R, LOZNEANU L, BALAN R A. The endometrial regeneration frontiers: from mechanisms to applications in regenerative medicine. Romanian Journal of Morphology and Embryology, 2018, 59(2):407-425.
[27]	ZHOU S T, ZHAO L J, YI T, WEI Y Q, ZHAO X. Menopause- induced uterine epithelium atrophy results from arachidonic acid/ prostaglandin E2 axis inhibition-mediated autophagic cell death. Scientific Reports, 2016, 6:31408. doi: 10.1038/srep31408
[28]	CHOI J, JO M, LEE E, OH Y K, CHOI D. The role of autophagy in human endometrium. Biology of Reproduction, 2012, 86(3):70.
[29]	LIU Y, CHANG J A, WANG P, YIN Q Q, HUANG W W, LIU C Q, BAI X X, ZHU Q, GAO T Z, ZHOU P. Effects of Saccharomyces cerevisiae on alleviating cytotoxicity of porcine jejunal epithelia cells induced by deoxynivalenol. AMB Express, 2019, 9(1):137. doi: 10.1186/s13568-019-0863-9
[30]	JAKOPOVIĆ Ž, HANOUSEK ČIČA K, MRVČIĆ J, PUCIĆ I, ČANAK I, JADRANKA F, PLEADIN J, STANZER D, ZJALIĆ S, MARKOV K, JAKOPOVIĆ Ž., MRVČIĆ J. Properties and fermentation activity of industrial yeasts saccharomyces cerevisiae, S.uvarum, Candida Utilis and Kluyveromyces marxianus exposed to AFB₁, OTA and ZEA. Food. Technology Biotechnology, 2018, 56(2):208-217.
[31]	JIANG S Z, YANG Z B, YANG W R, WANG S J, WANG Y, BROOMHEAD J, JOHNSTON S L, CHI F. Effect on hepatonephric organs, serum metabolites and oxidative stress in post-weaning piglets fed purified Zearalenone-contaminated diets with or without Calibrin-Z. Journal of Animal Physiology and Animal Nutrition, 2012, 96(6):1147-1156. doi: 10.1111/jpn.2012.96.issue-6
[32]	LIU Y, QIN X Y, LU X F. Crocin improves endometriosis by inhibiting cell proliferation and the release of inflammatory factors. Biomedicine & Pharmacotherapy, 2018, 106:1678-1685. doi: 10.1016/j.biopha.2018.07.108
[3]	PARVEEN M, ZHU Y, KIYAMA R. Expression profiling of the genes responding to Zearalenone and its analogues using estrogen- responsive genes. FEBS Letters, 2009, 583(14):2377-2384. doi: 10.1016/j.febslet.2009.06.035
[4]	ZHENG W L, WANG B J, SI M X, ZOU H, SONG R L, GU J H, YUAN Y, LIU X Z, ZHU G Q, BAI J F, BIAN J C, LIU Z P. Zearalenone altered the cytoskeletal structure via ER stress- autophagy-oxidative stress pathway in mouse TM4 Sertoli cells. Scientific Reports, 2018, 8(1):3320. doi: 10.1038/s41598-018-21567-8
[5]	TIEMANN U, TOMEK W, SCHNEIDERF, VANSELOW J. Effects of the mycotoxins alpha- and beta-zearalenol on regulation of progesterone synthesis in cultured granulose cells from porcine ovaries. Reproductive Toxicology, 2003, 17(6):673-681. doi: 10.1016/j.reprotox.2003.07.001
[6]	WILEY A A, KAUFFOLD J, WäHNER M, CREAN-HARRIS B, MILLER D J, BAGNELL C A, BARTOL F F. Laser microdissection of neonatal porcine endometrium for tissue-specific valuation of relaxin receptor (RXFP1) expression in response to perinatal zearalenone exposure. Annals of the New York Academy of Sciences, 2009, 1160:190-191. doi: 10.1111/j.1749-6632.2008.03821.x

日粮组成 Ingredients	含量 Content(%)	营养水平¹Nutrient levels
玉米Corn (%)	66.00	代谢能 Metabolic energy (MJ·kg^-1)	12.79
豆粕 Soybean meal (%)	21.00	粗蛋白 Crude proetin (%)	17.40
麸皮 Wheat bran (%)	5.00	钙 Calcium (%)	0.78
膨化大豆 Expanded soybean (%)	4.00	有效磷 Phosphorus (%)	0.30
预混料²Premix (%)	4.00	赖氨酸 Lysine (%)	0.96
合计 Total	100	蛋+胱 Sulphur amino acid (%)	0.60

目的基因 Target gene	引物序列(5' -3') Primer sequence	产物大小 Product size (bp)	登录号 Accession No.
GAPDH	F: ATGGTGAAGGTCGGAGTGAA	154	NM_001206359.1
GAPDH	R:CGTGGGTGGAATCATACTGG	154	NM_001206359.1
LC3	F: AACGAAATTCCTGGTGCCTGA	90	NM_100322893
LC3	R: :AAGGCTTGGTTAGCATTGAGCTG	90	NM_100322893
PCNA	F: GTGATTCCACCACCATGTTC	145	NM_001291925.1
PCNA	R: TGAGACGAGTCCATGCTCTG	145	NM_001291925.1

项目 Items	对照组Control	ZEA	ZEA0.1	ZEA0.25	ZEA0.5	ZEA-M
子宫器官指数 Organ index of uterus (g∙kg^-1)	1.91±0.31a	2.72±0.52b	2.05± 0.35a	1.93± 0.27a	1.84± 0.29a	1.81± 0.15a
数值标不同小写字母者表示差异显著(P<0.05)。n=8 Values with different superscripts differ significantly at P<0.05. n=8