原花青素对玉米赤霉烯酮诱导牦牛颗粒细胞氧化损伤的保护作用研究

doi:10.11843/j.issn.0366-6964.2022.09.017

摘要/Abstract

摘要： 旨在探究原花青素（procyanidins，PC）在玉米赤霉烯酮（zearalenone，ZEA）诱导牦牛颗粒细胞产生氧化损伤后，对颗粒细胞生长增殖、抗氧化性以及激素分泌的影响。本试验选取3～5岁的健康牦牛（n=3），完成卵巢颗粒细胞的分离培养，并通过免疫荧光染色鉴定颗粒细胞纯度。通过CCK-8法分别比较不同浓度ZEA （0（对照组）、5、10、20、40、60、80和100 μmol·mL^-1）、不同浓度PC （0（对照组）、0.05、0.5、2.5、5、10、50和100 μg·mL^-1）以及50 μmol·mL^-1 ZEA+5 μg·mL^-1 PC联合处理对牦牛颗粒细胞活性的影响。通过ELISA法检测对照组（未添加ZEA及PC）、50 μmol·mL^-1 ZEA组和50 μmol·mL^-1ZEA+5 μg·mL^-1PC联合处理组牦牛颗粒细胞活性氧（reactive oxygen species，ROS）以及雌二醇（E₂）水平。采用实时荧光定量PCR法检测不同组颗粒细胞中部分增殖生长、凋亡、抗氧化及E₂合成相关基因的表达水平。结果显示，本研究所分离培养得到的细胞表达颗粒细胞标志蛋白FSHR，具有较高的纯度，可以满足后续试验要求。添加不同浓度ZEA后，颗粒细胞活力随着ZEA浓度的升高而显著降低（P<0.05）。在一定浓度范围内（0~5 μg·mL^-1），随着浓度的上升，PC对颗粒细胞的活力有显著提高作用（P<0.05），且在浓度为5 μg·mL^-1时对细胞活力的提高作用最明显。与ZEA处理组相比，ZEA与PC联合处理后颗粒细胞的数量增加且细胞活力极显著提高（P<0.05），颗粒细胞增殖生长相关基因PCNA和IGF-Ⅱ 以及抗凋亡相关基因XIAP和BCL-2的表达显著上调（P<0.05）。相反，促凋亡相关基因BAX和CASP3的表达显著下调（P<0.05）。同时，ZEA+PC联合处理后能显著降低牦牛颗粒细胞活性氧水平并促进颗粒细胞分泌E₂（P<0.05），颗粒细胞中的抗氧化相关基因SOD2、GPX1及CAT和E₂合成相关基因STAR、CYP11A1及HSD3B的表达量显著上调（P<0.05）。上述结果表明，PC可提高经ZEA处理后颗粒细胞的生长增殖能力，上调颗粒细胞的活力，提高抗氧化能力，降低ROS水平以及提高E₂的分泌水平。综上所述，PC对ZEA诱导的牦牛颗粒细胞氧化损伤有一定保护作用。本研究为ZEA毒性的防治和畜牧业生产中PC的应用提供了一定的研究数据和理论支持。

关键词: 玉米赤霉烯酮, 原花青素, 牦牛, 颗粒细胞, 氧化损伤, 雌激素

Abstract: The aim of this study was to investigate the effects of procyanidins (PC) on the growth, proliferation, antioxidant properties and hormone secretion of yak granulosa cells after oxidative damage induced by zearalenone (ZEA). In this experiment, healthy yaks of 3-5 years old (n=3) were selected to complete the isolation and culture of ovarian granulosa cells, and immunofluorescence staining was performed to identify the purity of granulosa cells. The effects of different concentrations of ZEA (0 (Control group), 5, 10, 20, 40, 60, 80 and 100 μmol·mL^-1), different concentrations of PC (0 (Control group), 0.05, 0.5, 2.5, 5, 10, 50 and 100 μg·mL^-1) and the combined treatment of 50 μmol·mL^-1 ZEA+5 μg·mL^-1 PC on the activity of yak granulosa cells were compared by CCK-8 assay. The levels of reactive oxygen species (ROS) and estradiol (E₂) in granulosa cells of yaks Control group (without ZEA and PC), 50 μmol·mL^-1 ZEA and 50 μmol·mL^-1ZEA+5 μg·mL^-1PC groups were measured by ELISA. The expression levels of genes related to proliferation and growth, apoptosis, antioxidant and E₂ synthesis in different groups of granulosa cells were detected by real-time fluorescence quantitative PCR. The results showed that, cells isolated and cultured in this study expressed granulosa cell marker protein FSHR, which had high purity and could meet the requirements of subsequent experiments. After adding different concentrations of ZEA, the viability of granulosa cells decreased obviously with the increase of ZEA concentration (P<0.05). In a certain concentration range (0-5 μg·mL^-1), PC could significantly improve the viability of granulosa cells with the increase of concentration (P<0.05), and the effect was most obvious when the concentration was at 5 μg·mL^-1. Compared with ZEA treatment group, the combined treatment of ZEA+PC significantly increased the number of granulosa cells and cell viability(P<0.05). The expression of proliferation- and growth-related genes PCNA and IGF-II, as well as anti-apoptotic genes XIAP and BCL-2, significantly increased(P<0.05). On the contrary, the expression of pro-apoptotic genes BAX and CASP3 significantly decreased(P<0.05). Meanwhile, the combined treatment of ZEA+PC could significantly reduce the level of reactive oxygen species and promote the secretion of E₂ in yak granulosa cells(P<0.05). The expression of antioxidant-related genes SOD2, GPX1 and CAT, as well as E₂ synthesis-related genes STAR, CYP11A1 and HSD3B significantly up-regulated in granulosa cells(P<0.05). The above results suggest that PC promotes the growth and proliferation of granulosa cells after ZEA treatment, up-regulates the viability of granulosa cells, improves the antioxidant capacity, reduces the level of ROS, and increases the secretion level of E₂. In conclusion, PC has a protective effect on ZEA-induced oxidative damage in yak granulosa cells. This study provides some research data and theoretical support for the prevention and treatment of ZEA toxicity and the application of PC in animal husbandry.

Key words: zearalenone, proanthocyanidins, yak, granulosa cells, oxidative damage, estrogen

中图分类号:

S823.85

唐紫雯, 程华琴, 刘冬菊, 彭毛卓玛, 杨雪, 李键, 殷实. 原花青素对玉米赤霉烯酮诱导牦牛颗粒细胞氧化损伤的保护作用研究[J]. 畜牧兽医学报, 2022, 53(9): 3006-3017.

TANG Ziwen, CHENG Huaqin, LIU Dongju, PHAGMO Droma, YANG Xue, LI Jian, YIN Shi. The Protective Effect of Proanthocyanidins on Oxidative Damage of Yak Granulosa Cells Induced by Zearalenone[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3006-3017.

参考文献 48

[1]	韦唯, 吴奇强.浅谈牦牛资源研究与利用进展[J].农家参谋, 2019(17):111-112.WEI W, WU Q Q.Progress in research and utilization of yak resources[J].Nongjiacanmou, 2019(17):111-112.(in Chinese)
[2]	XIONG X R, LAN D L, LI J, et al.Supplementation of cilostazol during in vitro maturation enhances the meiosis and developmental competence of yak oocytes by influencing cAMP content and mRNA expression[J].Anim Reprod Sci, 2017, 186:21-30.
[3]	HOQUE S A M, UMEHARA T, KAWAI T, et al.Adverse effect of superoxide-induced mitochondrial damage in granulosa cells on follicular development in mouse ovaries[J].Free Radic Biol Med, 2021, 163:344-355.
[4]	YOSHIMURA Y, BARUA A.Female reproductive system and immunology[J].Adv Exp Med Biol, 2017, 1001:33-57.
[5]	HEWITT S C, WINUTHAYANON W, KORACH K S.What's new in estrogen receptor action in the female reproductive tract[J].J Mol Endocrinol, 2016, 56(2):R55-R71.
[6]	HENRÍQUEZ S, KOHEN P, XU X, et al.Significance of pro-angiogenic estrogen metabolites in normal follicular development and follicular growth arrest in polycystic ovary syndrome[J].Hum Reprod, 2020, 35(7):1655-1665.
[7]	SHI J X, LIU C Y, CHEN M Y, et al.The interference effects of bisphenol A on the synthesis of steroid hormones in human ovarian granulosa cells[J].Environ Toxicol, 2021, 36(4):665-674.
[8]	YUAN X H, YANG C R, WANG X N, et al.Progesterone maintains the status of granulosa cells and slows follicle development partly through PGRMC1[J].J Cell Physiol, 2019, 234(1):709-720.
[9]	何莉娜.miR-495-3p调节山羊卵巢颗粒细胞功能的机制研究[D].重庆:西南大学, 2021.HE L N.The mechanism of miR-495-3p regulating the function of goat ovarian granulosa cells[D].Chongqing:Southwest University, 2021.(in Chinese)
[10]	于阳洋, 孙振高.卵母细胞衰老的氧化应激机制研究进展[J].中国性科学, 2019, 28(8):113-116.YU Y Y, SUN Z G.Research progressin the oxidative stress mechanism of oocyte senescence[J].Chinese Journal of Human Sexuality, 2019, 28(8):113-116.(in Chinese)
[11]	ZHENG W L, WANG B J, WANG L, et al.ROS-mediated cell cycle arrest and apoptosis induced by zearalenone in mouse sertoli cells via ER stress and the ATP/AMPK pathway[J].Toxins (Basel), 2018, 10(1):24.
[12]	LONG M, YANG S H, ZHANG Y, et al.Proanthocyanidin protects against acute zearalenone-induced testicular oxidative damage in male mice[J].Environ Sci Pollut Res, 2017, 24(1):938-946.
[13]	TERAO H, WADA-HIRAIKE O, NAGUMO A, et al.Role of oxidative stress in follicular fluid on embryos of patients undergoing assisted reproductive technology treatment[J].J Obstet Gynaecol Res, 2019, 45(9):1884-1891.
[14]	SARKAR A, MAHENDRAN T S, MEENAKSHISUNDARAM A, et al.Role of cerium oxide nanoparticles in improving oxidative stress and developmental delays in Drosophila melanogaster as an in-vivo model for bisphenol a toxicity[J].Chemosphere, 2021, 284:131363.
[15]	VENTO M.Oxidative stress in the perinatal period[J].Free Radic Biol Med, 2019, 142:1-2.
[16]	DE CASTRO CAVALLARI F, LEAL C L V, ZVI R, et al.Effects of melatonin on production of reactive oxygen species and developmental competence of bovine oocytes exposed to heat shock and oxidative stress during in vitro maturation[J].Zygote, 2019, 27(3):180-186.
[17]	TSOULOUFI T K, TSAKMAKIDIS I A, TSOUSIS G, et al.The effect of subchronic oral exposure to zearalenone on hematologic and biochemical analytes, and the blood redox status of adult rabbit bucks[J].Vet Clin Pathol, 2019, 48(2):328-334.
[18]	FRAEYMAN S, CROUBELS S, DEVREESE M, et al.Emerging Fusarium and Alternaria mycotoxins:occurrence, toxicity and toxicokinetics[J].Toxins (Basel), 2017, 9(7):228.
[19]	SALAH-ABBōS J B, BELGACEM H, EZZDINI K, et al.Zearalenone nephrotoxicity:DNA fragmentation, apoptotic gene expression and oxidative stress protected by Lactobacillus plantarum MON03[J].Toxicon, 2020, 175:28-35.
[20]	MARIN D E, PISTOL G C, BULGARU C V, et al.Cytotoxic and inflammatory effects of individual and combined exposure of HepG2 cells to zearalenone and its metabolites[J].Naunyn-Schmiedeberg's Arch Pharmacol, 2019, 392(8):937-947.
[21]	PISTOL G C, BRAICU C, MOTIU M, et al.Zearalenone mycotoxin affects immune mediators, MAPK signalling molecules, nuclear receptors and genome-wide gene expression in pig spleen[J].PLoS One, 2015, 10(5):e0127503.
[22]	ZHANG K Z, TAN X T, LI Y, et al.Transcriptional profiling analysis of Zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1[J].Ecotoxicol Environ Saf, 2018, 153:135-141.
[23]	BELGACEM H, SALAH-ABBōS J B, EZZDINI K, et al.Lactobacillus plantarum MON03 counteracts zearalenone génotoxicty in mice:Chromosome aberrations, micronuclei, DNA fragmentation and apoptotique gene expression[J].Mutat Res/Genet Toxicol Environ Mutagen, 2019, 840:11-19.
[24]	卢春亭, 肖仕鑫, 钟佩云, 等.姜黄素对玉米赤霉烯酮诱导的猪肾上皮细胞凋亡的保护作用[J].应用与环境生物学报, 2022, 28(6):1-10.LU C T, XIAO S X, ZHONG P Y, et al.Protective effect of curcumin on zearalenone-induced apoptosis in porcine renal epithelial cells[J].Chinese Journal of Applied and Environmental Biology, 2022, 28(6):1-10.(in Chinese)
[25]	GHAJARI G, NABIUNI M, AMINI E.The association between testicular toxicity induced by Li2Co3 and protective effect of Ganoderma lucidum:Alteration of Bax & c-Kit genes expression[J].Tissue Cell, 2021, 72:101552.
[26]	PALLARÉS N, SEBASTIÀ A, MARTÍNEZ-LUCAS V, et al.High pressure processing impact on alternariol and aflatoxins of grape juice and fruit juice-milk based beverages[J].Molecules, 2021, 26(12):3769.
[27]	姜薇, 时晓丹, 赵纯, 等.氧化应激对植入前胚胎发育的影响[J].中华生殖与避孕杂志, 2020, 40(1):50-54.JIANG W, SHI X D, ZHAO C, et al.Effect of oxidative stress on pre-implantation embryo development[J].Chinese Journal of Reproduction and Contraception, 2020, 40(1):50-54.(in Chinese)
[28]	BAGCHI D, SWAROOP A, PREUSS H G, et al.Free radical scavenging, antioxidant and cancer chemoprevention by grape seed proanthocyanidin:an overview[J].Mutat Res, 2014, 768:69-73.
[29]	FIDELIS M, DE OLIVEIRA S M, SANTOS J S, et al.From byproduct to a functional ingredient:Camu-camu (Myrciaria dubia) seed extract as an antioxidant agent in a yogurt model[J].J Dairy Sci, 2020, 103(2):1131-1140.
[30]	GAO W L, LI X H, DUN X P, et al.Grape seed proanthocyanidin extract ameliorates streptozotocin-induced cognitive and synaptic plasticity deficits by inhibiting oxidative stress and preserving AKT and ERK activities[J].Curr Med Sci, 2020, 40(3):434-443.
[31]	AMANKWAAH C, LI J R, LEE J, et al.Development of antiviral and bacteriostatic chitosan-based food packaging material with grape seed extract for murine norovirus, Escherichia coli and Listeria innocua control[J].Food Sci Nutr, 2020, 8(11):6174-6181.
[32]	季乃尧.原花青素对沙门氏菌LPS诱导的鸡卵巢炎性损伤的缓解作用[D].杭州:浙江大学, 2020.JI N Y.Attenuating effect of GSPE on Salmonella LPS-induced inflammatory injury of chicken ovary[D].Hangzhou:Zhejiang University, 2020.(in Chinese)
[33]	王友良, 甘功友, 张冬初, 等.原花青素对反式脂肪酸致雄性小鼠生殖损伤的保护作用[J].食品安全质量检测学报, 2018, 9(20):5365-5369.WANG Y L, GAN G Y, ZHANG D C, et al.Protective effect of proanthocyanidins on reproductive damage induced by trans fatty acids in male mice[J].Journal of Food Safety & Quality, 2018, 9(20):5365-5369.(in Chinese)
[34]	何刘.原花青素对镉致Wistar大鼠雄性生殖系统损伤干预作用的研究[D].沈阳:沈阳农业大学, 2018.HE L.Study on the intervention of proanthocyanidins on reproductive system damage in male Wistar rats caused by cadmium[D].Shenyang:Shenyang Agricultural University, 2018.(in Chinese)
[35]	SMITH O E, ROUSSEL V, MORIN F, et al.Steroidogenic factor 1 regulation of the hypothalamic-pituitary-ovarian axis of adult female mice[J].Endocrinology, 2022, 163(4):bqac028.
[36]	高鑫.玉米赤霉烯酮对大鼠的生殖毒性及水飞蓟素缓解其危害的机制[D].武汉:华中农业大学, 2018.GAO X.Study of reproductive toxicity of zearalenone and ameliorative effects of silymarin in rats[D].Wuhan:Huazhong Agricultural University Library, 2018.(in Chinese)
[37]	ARNOLD C.Tracking zearalenone:placental transfer of a fungal toxin[J].Environ Health Perspect, 2020, 128(7):074001.
[38]	KAŹMIERCZAK-BARAŃSKA J, BOGUSZEWSKA K, ADAMUS-GRABICKA A, et al.Two faces of vitamin C-antioxidative and pro-oxidative agent[J].Nutrients, 2020, 12(5):1501.
[39]	DUDYLINA A L, IVANOVA M V, SHUMAEV K B, et al.Superoxide formation in cardiac mitochondria and effect of phenolic antioxidants[J].Cell Biochem Biophys, 2019, 77(1):99-107.
[40]	马爱国, 刘四朝.不同剂量维生素C对DNA氧化损伤影响的研究[J].营养学报, 2001, 23(1):12-15.MA A G, LIU S C.Effect of different levels of ascorbic acid on DNA damage[J].Acta Nutrimenta Sinica, 2001, 23(1):12-15.(in Chinese)
[41]	范潇予, 张倩倩, 李超, 等.抗氧化剂抗氧化活性到促氧化作用的转变[J].食品与药品, 2016, 18(5):364-369.FAN X Y, ZHANG Q Q, LI C, et al.The transformation from anti-oxidant to oxidant[J].Food and Drug, 2016, 18(5):364-369.(in Chinese)
[42]	ONUMAH O E, JULES G E, ZHAO Y F, et al.Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells[J].Free Radic Biol Med, 2009, 46(12):1658-1667.
[43]	WANG X, ZHANG W, CHEN H L, et al.High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS[J]. Toxicol Lett, 2014, 224(1):16-23.
[44]	王丽雪, 解玉怀, 张桂国.植物源性抗氧化剂的应用及其作用机制[J].动物营养学报, 2017, 29(5):1481-1488.WANG L X, XIE Y H, ZHANG G G.Application of phytogenic antioxidants and its mechanisms[J].Chinese Journal of Animal Nutrition, 2017, 29(5):1481-1488.(in Chinese)
[45]	邢宝松, 张家庆, 任巧玲, 等.原花青素B2对猪颗粒细胞氧化损伤的保护作用及机制研究[J].河南农业科学, 2022, 51(1):146-153.XING B S, ZHANG J Q, REN Q L, et al.Protective effect and mechanism of proanthocyanidins B2 on oxidative damage in porcine granulosa cells[J].Journal of Henan Agricultural Sciences, 2022, 51(1):146-153.(in Chinese)
[46]	GAO W, JIN Y X, HAO J D, et al.Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress[J]. Mol Reprod Dev, 2021, 88(1):55-66.
[47]	PIZZO F, CALONI F, SCHREIBER N B, et al.In vitro effects of deoxynivalenol and zearalenone major metabolites alone and combined, on cell proliferation, steroid production and gene expression in bovine small-follicle granulosa cells[J].Toxicon, 2016, 109:70-83.
[48]	BARBE A, RAMÉ C, MELLOUK N, et al.Effects of grape seed extract and proanthocyanidin B2 on in vitro proliferation, viability, steroidogenesis, oxidative stress, and cell signaling in human granulosa cells[J].Int J Mol Sci, 2019, 20(17):4215.