大麻素受体1在邻苯二甲酸二丁酯暴露致雄性大鼠生殖损伤中的作用及机制研究
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摘要
目的探讨大麻素受体1(cannabinoid receptor 1, CBR1)在邻苯二甲酸二丁酯(dibutyl phthalate,DBP)暴露致雄性大鼠生殖损伤中的表达情况及作用机制。方法 60只健康雄性SD大鼠按随机数字表法分为4组,每组15只,分别为溶剂对照组、低剂量组(100 mg/kg)、中剂量组(250 mg/kg)和高剂量组(500 mg/kg),每日灌胃染毒1次,持续28 d建立动物模型。采用精子分析仪检测精子活力,密度,精子畸形率;HE染色法观察睾丸组织结构变化;采用ELISA检测血清睾酮(T)、雌二醇(E_2)、黄体生成素(LH)、促卵泡激素(FSH)和性激素结合蛋白(SHBG)水平;Western blot检测睾丸组织中p38、ERK、AKT和CBR1等基因的蛋白表达;RT-PCR检测相关基因mRNA的表达。结果与对照组相比,低、中、高剂量组大鼠前向运动精子(20.1±9.2),(22.3±6.2),(21.0±14.2)和精子总活力(73.6±3.2),(40.7±24.4),(41.2±4.6)显著降低,高剂量组精子密度(152.0±67.0)显著降低(P<0.05);与对照组相比,中、高剂量组大鼠血清T水平显著降低[(19.65±2.65),(17.40±3.50),P<0.05];与对照组相比,低、中、高剂量组大鼠血清中E_2(2.09±0.08),(2.11±0.77),(2.05±0.02)和LH(7.80±1.42),(7.68±2.28),(6.43±0.90)均显著降低(P<0.05),各暴露组大鼠FSH水平则显著上升[(2.88±0.37),(2.89±0.29),(3.37±0.30),P<0.05];与对照组相比,染毒组大鼠睾丸组织中CBR1的蛋白和mRNA水平均显著增加,p-p38、p-ERK水平均显著降低,差异有统计学意义(P<0.05)。结论邻苯二甲酸二丁酯暴露导致大鼠睾丸组织结构损伤和精子活力下降的机制,可能与DBP暴露致CBR1表达上调,抑制了ERK/p38MAPK的正常磷酸化有关。
Objective To explore the expression pattern of cannabinoid receptor 1(CBR1) in reproductive toxicity induced by dibutyl phthalate(DBP) exposure in male rats, and investigate the underlying mechanism. Methods Sixty healthy male Sprague-Dawley rats were randomly divided into 4 groups(15 animals in each group), that is, control group, and low-, medium-and high-dose groups, exposure to 100, 250 and 500 mg/kg DBP, respectively, by intragastric injection daily for 28 consecutive days. Computer-aided sperm analysis system(CASA) was used to detect sperm vitality, concentration, and rate of deformity. HE staining was employed to observe the changes in testicular tissue. The serum levels of testosterone(T), estradiol(E_2), luteinizing hormone(LH), follicle stimulating hormone(FSH) and sex hormone binding protein(SHBG) were measured by ELISA. The expression of p38, ERK, AKT and CBR1 at mRNA and protein levels was measured by RT-PCR and Western blotting. Results Compared with the control rats, the sperm motility and total sperm motility were reduced in low-(20.1±9.2 and 73.6±3.2), middle-(22.3±6.2 and 40.7±24.4) and high-dose(21.0±14.2 and 41.2±4.6) exposure groups(P<0.05). Sperm concentration was significantly lower in the high-dose group(152.0±67.0, P<0.05). Compared with the control group, serum T levels were significantly lower in the middle-(19.65±2.65) and high-dose groups(17.40±3.50, both P<0.05). The serum level of E_2 was significantly decreased in each exposure group(2.09±0.08, 2.11±0.77, 2.05±0.02) when compared to the control group. So were the serum LH levels(7.80±1.42, 7.68±2.28, 6.43±0.90)(all P<0.05). While the serum level of FSH was all significantly increased in each exposure group(2.88±0.37, 2.89±0.29, 3.37±0.30), when compared to the control group(P<0.05). In the testis tissue, the expression of CBR1 was increased at mRNA and protein levels than that of the control group, and the expression of p-p38 and p-ERK was significantly decreased than the control group(P<0.05). Conclusion DBP exposure results in impairment of testicular tissue structure and decrease of sperm motility in male rats, which may be due to the up-regulation of CBR1 and the subsequent inhibition of ERK/p38 MAPK phosphorylation.
Objective To explore the expression pattern of cannabinoid receptor 1(CBR1) in reproductive toxicity induced by dibutyl phthalate(DBP) exposure in male rats, and investigate the underlying mechanism. Methods Sixty healthy male Sprague-Dawley rats were randomly divided into 4 groups(15 animals in each group), that is, control group, and low-, medium-and high-dose groups, exposure to 100, 250 and 500 mg/kg DBP, respectively, by intragastric injection daily for 28 consecutive days. Computer-aided sperm analysis system(CASA) was used to detect sperm vitality, concentration, and rate of deformity. HE staining was employed to observe the changes in testicular tissue. The serum levels of testosterone(T), estradiol(E_2), luteinizing hormone(LH), follicle stimulating hormone(FSH) and sex hormone binding protein(SHBG) were measured by ELISA. The expression of p38, ERK, AKT and CBR1 at mRNA and protein levels was measured by RT-PCR and Western blotting. Results Compared with the control rats, the sperm motility and total sperm motility were reduced in low-(20.1±9.2 and 73.6±3.2), middle-(22.3±6.2 and 40.7±24.4) and high-dose(21.0±14.2 and 41.2±4.6) exposure groups(P<0.05). Sperm concentration was significantly lower in the high-dose group(152.0±67.0, P<0.05). Compared with the control group, serum T levels were significantly lower in the middle-(19.65±2.65) and high-dose groups(17.40±3.50, both P<0.05). The serum level of E_2 was significantly decreased in each exposure group(2.09±0.08, 2.11±0.77, 2.05±0.02) when compared to the control group. So were the serum LH levels(7.80±1.42, 7.68±2.28, 6.43±0.90)(all P<0.05). While the serum level of FSH was all significantly increased in each exposure group(2.88±0.37, 2.89±0.29, 3.37±0.30), when compared to the control group(P<0.05). In the testis tissue, the expression of CBR1 was increased at mRNA and protein levels than that of the control group, and the expression of p-p38 and p-ERK was significantly decreased than the control group(P<0.05). Conclusion DBP exposure results in impairment of testicular tissue structure and decrease of sperm motility in male rats, which may be due to the up-regulation of CBR1 and the subsequent inhibition of ERK/p38 MAPK phosphorylation.
引文
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[18] ALMOG T,NAOR Z.Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions[J].Mol Cell Endocrinol,2008,282(1/2):39-44.DOI:10.1016/j.mce.2007.11.011.
[19] 钱晓菁,常青,许增禄,等.磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达[J].解剖学报,2008,39(2):214-218.DOI:10.3321/j.issn:0529-1356.2008.02.015.QIAN X J,CHANG Q,XU Z L,et al.The expression of phospho-MAPKs in the adult rat testis[J].Acta Anat Sin,2008,39(2):214-218.DOI:10.3321/j.issn:0529- 1356.2008.02.015.
[20] SUN Y H,ZHANG D J,MAO M,et al.Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line[J].Exp Ther Med,2017,14(2):1809-1817.DOI:10.3892/etm.2017.4704.
[21] LEE H L,LIN C S,KAO S H,et al.Gallic acid induces G1 phase arrest and apoptosis of triple-negative breast cancer cell MDA-MB-231 via p38 mitogen-activated protein kinase/p21/p27 axis[J].Anticancer Drugs,2017,28(10):1150-1156.DOI:10.1097/CAD.0000000000000565.
[22] 赵金昌,靳曙光,徐一波,等.ERK-MAPK通路在DBP致大鼠睾丸缝隙连接损伤中作用[J].中国公共卫生,2017,33(2):206-210.DOI:10.11847/zgggws2017-33-02-09.ZHAO J C,JIN S G,XU Y B,et al.Role of ERK-MAPK signaling pathway in dibutyl phthalate induced gap junction injury in testis of rat[J].Chin J Public Health,2017,33(2):206-210.DOI:10.11847/zgggws2017-33-02-09.
[23] LóPEZ-CARDONA A P,PéREZ-CEREZALES S,FERNáNDEZ-GONZáLEZ R,et al.CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways[J].FASEB J,2017,31(8):3372-3382.DOI:10.1096/fj.201601382RR.
[24] HOWLETT A C,BREIVOGEL C S,CHILDERS S R,et al.Cannabinoid physiology and pharmacology:30 years of progress[J].Neuropharmacology,2004,47(Suppl 1):345-358.DOI:10.1016/j.neuropharm.2004.07.030.
[25] MOLINA-HOLGADO E,VELA J M,ARéVALO-MARTíN A,et al.Cannabinoids promote oligodendrocyte progenitor survival:involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Aktsignaling[J].J Neurosci,2002,22(22):9742-9753.
[2] DESDOITS-LETHIMONIER C,ALBERT O,LE BIZEC B,et al.Human testis steroidogenesis is inhibited by phthalates[J].Human Reproduction,2012,27(5):1451-1459.DOI:10.1093/humrep/des069.
[3] RAPINO C,BATTISTA N,BARI M,et al.Endocannabinoids as biomarkers of human reproduction[J].Hum Reprod Update,2014,20(4):501-516.DOI:10.1093/humupd/dmu004.
[4] HOWLETT A C,BARTH F,BONNER T I,et al.International union of pharmacology.XXVII.classification of cannabinoid receptors[J].Pharmacol Rev,2002,54(2):161-202.
[5] MACCARRONE M.Endocannabinoids:Friends and foes of reproduction[J].Prog Lipid Res,2009,48(6):344-354.DOI:10.1016/j.plipres.2009.07.001.
[6] FRANCAVILLA F,BATTISTA N,BARBONETTI A,et al.Characterization of the endocannabinoid system in human spermatozoa and involvement of transient receptor potential vanilloid 1 receptor in their fertilizing ability[J].Endocrinology,2009,150(10):4692-4700.DOI:10.1210/ en.2009-0057.
[7] BARBONETTI A,VASSALLO M R,FORTUNATO D,et al.Energetic metabolism and human sperm motility:impact of CB1 receptor activation[J].Endocrinology,2010,151(12):5882-5892.DOI:10.1210/en.2010-0484.
[8] POMATTO V,PALERMO F,MOSCONI G,et al.Xenoestrogens elicit a modulation of endocannabinoid system and estrogen receptors in 4NP treated goldfish,carassiusauratus[J].Gen Comp Endocrinol,2011,174(1):30-35.DOI:10.1016/j.ygcen.2011.08.001.
[9] BISSET K M,DHOPESHWARKAR A S,LIAO C Y,et al.The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain:Inhibition by phthalate esters in vitro[J].Neurochem Int,2011,59(5):706-713.DOI:10.1016/j.neuint.2011.06.019.
[10] 韩晓杰,段婷婷,庞雨,等.Western blot免疫印迹法检测磷酸化蛋白表达条件优化研究[J].现代生物医学进展,2017,17(21):4047-4050,4037.DOI:10.13241/j.cnki.pmb.2017.21.011.HAN X J,DUAN T T,PANG Y,et al.Optimization research on the Westem blot experimental conditions for detecting phosphorylated protein[J].Prog Mod Biomed,2017,17(21):4047-4050,4037.DOI:10.13241/j.cnki.pmb.2017.21.011.
[11] KUMAR N,SRIVASTAVA S,ROY P.Impact of low molecular weight phthalates in inducing reproductive malfunctions in male mice:Special emphasis on Sertoli cell functions[J].Gen Comp Endocrinol,2015,215:36-50.DOI:10.1016/j.ygcen.2014.09.012.
[12] 李玉秋,马明月.邻苯二甲酸酯对胚胎发育毒作用及其机制的研究进展[J].环境与职业医学,2016,33(6):606-609.DOI:10.13213/j.cnki.jeom.2016.16111.LI Y Q,MA M Y.Advances on effects of phthalate acid esters on development of embryos and related mechanism[J].J Environ Occup Med,2016,33(6):606-609.DOI:10.13213/j.cnki.jeom.2016.16111.
[13] JUREWICZ J,RADWAN M,SOBALA W,et al.Human urinary phthalate metabolites level and main semen parameters,sperm chromatin structure,sperm aneuploidy and reproductive hormones[J].Reprod Toxicol,2013,42:232-241.DOI:10.1016/j.reprotox.2013.10.001.
[14] CHEN Q,YANG H,ZHOU N Y,et al.Phthalate exposure,even below US EPA reference doses,was associated with semen quality and reproductive hormones:Prospective MARHCS study in general population[J].Environ Int,2017,104:58-68.DOI:10.1016/j.envint.2017.04.005.
[15] 李俊涛,曲晓伟,张蜀武,等.益肾生精胶囊对邻苯二甲酸二丁酯致生殖功能损伤大鼠精子质量及生殖激素水平的影响[J].中华男科学杂志,2016,22(12):1110-1115.DOI:10.13263/j.cnki.nja.2016.12.010.LI J T,QU X W,ZHANG S W,et al.Effects of Yishen-shengjing capsules on semen quality and gonadal hormone levels in rats with dibutyl phthalate-induced reproductive function injury[J].Nat J Androl,2016,22(12):1110-1115.DOI:10.13263/j.cnki.nja.2016.12.010.
[16] LI H G,DING X F,GUAN H T,et al.Inhibition of human sperm function and mouse fertilization in vitro by an antibody against cation channel of sperm 1:the contraceptive potential of its transmembrane domains and pore region[J].FertilSteril,2009,92(3):1141-1146.DOI:10.1016/j.fertnstert.2008.07.1751.
[17] DI BLASIO A M,VIGNALI M,GENTILINI D.The endocannabinoid pathway and the female reproductive organs[J].J Mol Endocrinol,2013,50(1):R1-R9.DOI:10.1530/jme-12-0182.
[18] ALMOG T,NAOR Z.Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions[J].Mol Cell Endocrinol,2008,282(1/2):39-44.DOI:10.1016/j.mce.2007.11.011.
[19] 钱晓菁,常青,许增禄,等.磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达[J].解剖学报,2008,39(2):214-218.DOI:10.3321/j.issn:0529-1356.2008.02.015.QIAN X J,CHANG Q,XU Z L,et al.The expression of phospho-MAPKs in the adult rat testis[J].Acta Anat Sin,2008,39(2):214-218.DOI:10.3321/j.issn:0529- 1356.2008.02.015.
[20] SUN Y H,ZHANG D J,MAO M,et al.Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line[J].Exp Ther Med,2017,14(2):1809-1817.DOI:10.3892/etm.2017.4704.
[21] LEE H L,LIN C S,KAO S H,et al.Gallic acid induces G1 phase arrest and apoptosis of triple-negative breast cancer cell MDA-MB-231 via p38 mitogen-activated protein kinase/p21/p27 axis[J].Anticancer Drugs,2017,28(10):1150-1156.DOI:10.1097/CAD.0000000000000565.
[22] 赵金昌,靳曙光,徐一波,等.ERK-MAPK通路在DBP致大鼠睾丸缝隙连接损伤中作用[J].中国公共卫生,2017,33(2):206-210.DOI:10.11847/zgggws2017-33-02-09.ZHAO J C,JIN S G,XU Y B,et al.Role of ERK-MAPK signaling pathway in dibutyl phthalate induced gap junction injury in testis of rat[J].Chin J Public Health,2017,33(2):206-210.DOI:10.11847/zgggws2017-33-02-09.
[23] LóPEZ-CARDONA A P,PéREZ-CEREZALES S,FERNáNDEZ-GONZáLEZ R,et al.CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways[J].FASEB J,2017,31(8):3372-3382.DOI:10.1096/fj.201601382RR.
[24] HOWLETT A C,BREIVOGEL C S,CHILDERS S R,et al.Cannabinoid physiology and pharmacology:30 years of progress[J].Neuropharmacology,2004,47(Suppl 1):345-358.DOI:10.1016/j.neuropharm.2004.07.030.
[25] MOLINA-HOLGADO E,VELA J M,ARéVALO-MARTíN A,et al.Cannabinoids promote oligodendrocyte progenitor survival:involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Aktsignaling[J].J Neurosci,2002,22(22):9742-9753.