雷公藤内酯醇对雄性大鼠生殖毒性的机制研究(Ⅱ)
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摘要
目的:考察雷公藤内酯醇对大鼠睾丸细胞相关凋亡基因mRNA表达的影响,研究雷公藤内酯醇生殖毒性的分子机制。方法:以低(0.025 mg·kg~(-1))、中(0.05 mg·kg~(-1))、高(0.1 mg·kg~(-1))剂量的雷公藤内酯醇对健康雄性Wistar大鼠连续灌胃染毒30 d,每天一次,于末次染毒24 h后处死大鼠,取睾丸组织进行病理学检查,并通过实时荧光定量PCR测定Bcl-2、Bax、Fas、FasL、CREM和Caspase-3基因mRNA的表达情况。结果:与阴性对照组相比,雷公藤内酯醇染毒组睾丸组织生精细胞明显减少,精索内几乎无精子;高剂量组Bcl-2、CREM mRNA表达降低;而Bax mRNA表达水平在中、高剂量组时呈显著地高表达;Fas和FasL mRNA表达水平在高剂量组显著上升;Caspase-3 mRNA表达水平呈现依赖剂量的高表达,中、高剂量时呈现显著性差异。结论:提示在本实验染毒剂量范围内,特别是高剂量的雷公藤内酯醇能够使生殖细胞相关凋亡基因Bcl-2、Bax、Fas、FasL、CREM和Caspase-3不同程度的表达异常,这很可能是雷公藤内酯醇诱导大鼠生殖细胞凋亡的重要原因,为进一步深入阐述雷公藤内酯醇雄性生殖毒性的分子机制提供了依据。
OBJECTIVE To investigate the molecular mechanism of Triptolide's reproductive toxicity by investigating the effects of triptolide on the expression of apoptosis gene mRNA in rats. METHODS Male Wistar rats were treated with low(0.025 mg·kg~(-1)), medium(0.05 mg·kg~(-1)) and high(0.1 mg·kg~(-1)) doses of triptolide by gavage for 30 days, The rats were excuted 24 h after the last exposure. Testis tissues were collected for pathological examination. The expression of Bcl-2, Bax, Fas, FasL, CREM and caspase-3 mRNA was determined by real-time PCR. RESULTS Compared with the negative control group, the spermatogenic cells in the testes of the triptolide-exposed group were significantly reduced, with almost no sperm in the spermatic cord. The expression of Bcl-2 and CREM mRNA was decreased in the high-dose group; the expression level of Bax mRNA was significantly higher in the medium-and high-dose groups; the expression level of Fas and FasL mRNA was significantly increased in the high-dose group; the expression level of caspase-3 mRNA was high in a dose-dependent manner, with significant differences in the mid-and high-dose groups. CONCLUSION The results indicated that in the range of exposure dose, especially at a high dose, triptolide could cause the abnormal expression of Bcl-2, Bax, Fas, FasL, CREM and Caspase-3 in different degrees Maybe this was the important cause of the apoptosis induced by triptolide in rat germ cells, and provided the basis for the further explanation of the molecular mechanism of triptolide male reproductive toxicity.
OBJECTIVE To investigate the molecular mechanism of Triptolide's reproductive toxicity by investigating the effects of triptolide on the expression of apoptosis gene mRNA in rats. METHODS Male Wistar rats were treated with low(0.025 mg·kg~(-1)), medium(0.05 mg·kg~(-1)) and high(0.1 mg·kg~(-1)) doses of triptolide by gavage for 30 days, The rats were excuted 24 h after the last exposure. Testis tissues were collected for pathological examination. The expression of Bcl-2, Bax, Fas, FasL, CREM and caspase-3 mRNA was determined by real-time PCR. RESULTS Compared with the negative control group, the spermatogenic cells in the testes of the triptolide-exposed group were significantly reduced, with almost no sperm in the spermatic cord. The expression of Bcl-2 and CREM mRNA was decreased in the high-dose group; the expression level of Bax mRNA was significantly higher in the medium-and high-dose groups; the expression level of Fas and FasL mRNA was significantly increased in the high-dose group; the expression level of caspase-3 mRNA was high in a dose-dependent manner, with significant differences in the mid-and high-dose groups. CONCLUSION The results indicated that in the range of exposure dose, especially at a high dose, triptolide could cause the abnormal expression of Bcl-2, Bax, Fas, FasL, CREM and Caspase-3 in different degrees Maybe this was the important cause of the apoptosis induced by triptolide in rat germ cells, and provided the basis for the further explanation of the molecular mechanism of triptolide male reproductive toxicity.
引文
[1] Mei ZL, Yang XL, Xu HB. Pharmacological Study of Triptolide[J]. Chin Hosp Pharm J(中国医院药学杂志), 2003, 23(9): 557-558.
[2] Shamon LA, Pezzuto JM, Graves JM, et al. Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii[J]. Cancer Lett, 1997, 112(1): 113-117.
[3] Yuan YL, Zhou XP. Reproductive toxicity research progress of Tripterygium wilfordii Hook.f[J]. Chin J Tradit Chin Med Pharm(中华中医药杂志), 2013, 28(10): 2997-3000.
[4] Xiong W, Chen J. Study advances in clinical and animal experiment on reproductive toxicity of Tripterygium wilfordii[J]. J Mod Clin Med (现代临床医学), 2014, 40(6): 403-405.
[5] Cao L, Lin J. Research progress of triptolide and its clinical application[J]. Chin J Biochem Pharm (中国生化药物杂志), 2011, 32(6): 502-504.
[6] Liu XM, Liu ZH, Zhang J, et al. Study progress on clinical application and side effects of Tripterygium wilfordii[J]. J Pharm Pract(药学实践杂志), 2015, 33(2): 110-113.
[7] Ni B, Jiang ZZ, Huang X, et al. Male reproductive toxicity and toxicokinetics of triptolide in rats[J]. Arzneimittelforsch, 2008, 58(12): 673-680.
[8] Wang XZ, Xu SW, Zhang CH. Effects of nitrobenzene on apoptosis and the mRNA levels of Bcl-2 and Bax in mouse reproductive cells[J]. Acta Sci Circumst (环境科学学报), 2009, 29(1): 186-190.
[9] Zhang M, Qiu XX, Zhu CL, et al. The Effect of Bushenyichongkangshuai Decoction on Ovarian Granule Cell Apoptosis Factors Bcl-2,Bax,Caspase-3 of DOR Rat Model[J]. J Zhejiang Chin Med Univ (浙江中医药大学学报), 2014, 38(1): 68-72.
[10] Luo DM, She RP, Hu YX, et al. Damage of BisphenolA on Reproductive System and Effects the Expression of Bax and Bcl-2 Genes Induced in Male Rat[J]. Sci Tech Eng (科学技术与工程), 2008, 8(7): 1673-1677.
[11] Shen ZX, Liu CX. The Effects of Exercise on the Myocardial Bcl-2 and Bax mRNA Expression in the Aged Mice[J]. J Sport Sci (体育与科学), 2007, 28(2): 66-68.
[12] Nagata S, Golstein P. The Fas death factor[J]. Sci, 1995, 267(5203): 1449-1456.
[13] Lee J, Richburg JH, Younkin SC, et al. The Fas system is a key regulator of germ cell apoptosis in the testis[J]. Endocrinol, 1997, 138(5): 2081-2088.
[14] Ogi S, Tanji N, Yokoyama M, et al. Involvement of Fas in the apotosis of mouse germ cells induced by experimental cryptorchidism[J]. Urol Res, 1998, 26(1): 17-21.
[15] Suda T, Takahashi T, Golstein P, et al. Molecular cloning and expression o f the fas ligand, a novel member of the tumor necrosis factor family[J]. Cell, 1993, 75(6): 1169-1178.
[16] French LE, Hahne M, Viard I, et al. Fas and Fas ligand in embryos and adult mice: Ligand expression in several immuneprivileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover[J]. J Cell Biol, 1996, 133(2): 335-343.
[17] Woolveridge I, de Boer-Brouwer M, Taylor MF, et al. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes[J]. Biol Reprod, 1999, 60(2): 461-470.
[18] Jin Q, Peng YF. FAS/FASL ligand-mediated apoptosis of spermatogenic cell in male reproductive system[J], J Reprod Med, 2012, 21(5): 503-506.
[19] Wang J, Jia SH, Chen YX, et al. Study on the relationship between CREM gene polymorphism and idiopathic spermatogenesis dysfunction[J], Chin J Androl(中国男科学杂志), 2010, 24(12): 14-17.
[20] Martianov I, Choukrallah MA, Krebs A, et al. Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells[J]. BMC Genomics, 2010, 11: 530.
[21] Song XD, Liu H, Li QY, et al. The Relationship Between Caspase-3 and Spermatogenic Cell Apoptosis[J]. Mod Prevent Med(现代预防医学), 2007, 34(2): 223-224.
[22] Abe H, Shibata MA, Otsuki Y. Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells[J]. Mol Hum Reprod, 2006, 12(9): 535-541.
[2] Shamon LA, Pezzuto JM, Graves JM, et al. Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii[J]. Cancer Lett, 1997, 112(1): 113-117.
[3] Yuan YL, Zhou XP. Reproductive toxicity research progress of Tripterygium wilfordii Hook.f[J]. Chin J Tradit Chin Med Pharm(中华中医药杂志), 2013, 28(10): 2997-3000.
[4] Xiong W, Chen J. Study advances in clinical and animal experiment on reproductive toxicity of Tripterygium wilfordii[J]. J Mod Clin Med (现代临床医学), 2014, 40(6): 403-405.
[5] Cao L, Lin J. Research progress of triptolide and its clinical application[J]. Chin J Biochem Pharm (中国生化药物杂志), 2011, 32(6): 502-504.
[6] Liu XM, Liu ZH, Zhang J, et al. Study progress on clinical application and side effects of Tripterygium wilfordii[J]. J Pharm Pract(药学实践杂志), 2015, 33(2): 110-113.
[7] Ni B, Jiang ZZ, Huang X, et al. Male reproductive toxicity and toxicokinetics of triptolide in rats[J]. Arzneimittelforsch, 2008, 58(12): 673-680.
[8] Wang XZ, Xu SW, Zhang CH. Effects of nitrobenzene on apoptosis and the mRNA levels of Bcl-2 and Bax in mouse reproductive cells[J]. Acta Sci Circumst (环境科学学报), 2009, 29(1): 186-190.
[9] Zhang M, Qiu XX, Zhu CL, et al. The Effect of Bushenyichongkangshuai Decoction on Ovarian Granule Cell Apoptosis Factors Bcl-2,Bax,Caspase-3 of DOR Rat Model[J]. J Zhejiang Chin Med Univ (浙江中医药大学学报), 2014, 38(1): 68-72.
[10] Luo DM, She RP, Hu YX, et al. Damage of BisphenolA on Reproductive System and Effects the Expression of Bax and Bcl-2 Genes Induced in Male Rat[J]. Sci Tech Eng (科学技术与工程), 2008, 8(7): 1673-1677.
[11] Shen ZX, Liu CX. The Effects of Exercise on the Myocardial Bcl-2 and Bax mRNA Expression in the Aged Mice[J]. J Sport Sci (体育与科学), 2007, 28(2): 66-68.
[12] Nagata S, Golstein P. The Fas death factor[J]. Sci, 1995, 267(5203): 1449-1456.
[13] Lee J, Richburg JH, Younkin SC, et al. The Fas system is a key regulator of germ cell apoptosis in the testis[J]. Endocrinol, 1997, 138(5): 2081-2088.
[14] Ogi S, Tanji N, Yokoyama M, et al. Involvement of Fas in the apotosis of mouse germ cells induced by experimental cryptorchidism[J]. Urol Res, 1998, 26(1): 17-21.
[15] Suda T, Takahashi T, Golstein P, et al. Molecular cloning and expression o f the fas ligand, a novel member of the tumor necrosis factor family[J]. Cell, 1993, 75(6): 1169-1178.
[16] French LE, Hahne M, Viard I, et al. Fas and Fas ligand in embryos and adult mice: Ligand expression in several immuneprivileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover[J]. J Cell Biol, 1996, 133(2): 335-343.
[17] Woolveridge I, de Boer-Brouwer M, Taylor MF, et al. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes[J]. Biol Reprod, 1999, 60(2): 461-470.
[18] Jin Q, Peng YF. FAS/FASL ligand-mediated apoptosis of spermatogenic cell in male reproductive system[J], J Reprod Med, 2012, 21(5): 503-506.
[19] Wang J, Jia SH, Chen YX, et al. Study on the relationship between CREM gene polymorphism and idiopathic spermatogenesis dysfunction[J], Chin J Androl(中国男科学杂志), 2010, 24(12): 14-17.
[20] Martianov I, Choukrallah MA, Krebs A, et al. Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells[J]. BMC Genomics, 2010, 11: 530.
[21] Song XD, Liu H, Li QY, et al. The Relationship Between Caspase-3 and Spermatogenic Cell Apoptosis[J]. Mod Prevent Med(现代预防医学), 2007, 34(2): 223-224.
[22] Abe H, Shibata MA, Otsuki Y. Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells[J]. Mol Hum Reprod, 2006, 12(9): 535-541.