雷公藤甲素对小鼠血睾屏障的损伤作用及机制

The damaging effect and mechanism of triptolide on the blood-testis barrier in mice

摘要

目的: 探讨雷公藤甲素对小鼠血睾屏障的损伤效应及分子机制。方法: 选用20~25 g健康雄性C57BL/6小鼠9只,随机分为对照组、雷公藤甲素低剂量给药组(0.2 mg/kg)和高剂量给药组(0.5 mg/kg),每组3只。持续给药28 d后,观察不同剂量雷公藤甲素干预后小鼠体重、睾丸体积及组织形态学改变,比较三组小鼠睾体比、生殖细胞凋亡水平、血睾屏障相关蛋白表达量及血睾屏障渗漏程度。结果: 与对照组相比,雷公藤甲素给药组小鼠体重涨幅显著降低,睾丸重量、睾体比显著下降(P均<0.01);HE染色显示雷公藤甲素给药组小鼠曲细精管管腔生殖细胞丢失,管腔结构出现空泡化;附睾精子数量减少,高剂量给药物组小鼠附睾精子数量进一步降低,可见大量坏死生殖细胞;TUNEL结果显示雷公藤甲素给药组小鼠曲细精管内细胞凋亡显著增加(P均<0.01);蛋白质印迹实验显示雷公藤甲素给药组小鼠血睾屏障相关蛋白(上皮钙黏蛋白、闭合蛋白、β-连环蛋白、血管内皮钙黏蛋白、肌动蛋白相关蛋白3、紧密连接蛋白11)表达均显著下调(P均<0.01);生物素示踪染色显示雷公藤甲素给药组小鼠睾丸的血睾屏障随给药浓度升高,屏障渗漏损伤程度逐步加剧。结论: 雷公藤甲素给药可构建不同程度的小鼠血睾屏障损伤模型,其作用机制与诱导支持细胞凋亡、下调血睾屏障相关蛋白、激活氧化应激多条通路协同调控密切相关。

Abstract

Objective: To investigate the damaging effects of triptolide on the blood-testis barrier and its underlying molecular mechanisms. Methods: Nine healthy male C57BL/6 mice (20-25 g) were randomly divided into control group, low-dose triptolide group (0.2 mg/kg), and high-dose triptolide group (0.5 mg/kg), with 3 animals per group. After continuous administration for 28 days, body weight, testicular volume, and testicular histomorphology were observed in mice exposed to different doses of triptolide. Intergroup comparisons of testis-to-body weight ratio, germ cell apoptosis level, expression of blood-testis barrier-related proteins and barrier leakage were performed among the three groups. Results: Compared with the control group, mice in triptolide-treated groups exhibited markedly reduced body weight gain, testicular weight and testis-to-body weight ratio (all P<0.01). HE staining revealed massive loss of germ cells and prominent vacuolization in the seminiferous tubular lumen of triptolide-exposed mice. The sperm count in epididymal tissues was decreased upon triptolide administration, and this reduction was further aggravated in the high-dose group, accompanied by abundant necrotic germ cells. TUNEL assay revealed markedly elevated apoptosis in seminiferous tubules of triptolide-treated mice (all P<0.01). Western blotting indicated that the expression levels of blood-testis barrier-related proteins (E-cadherin, occludin, β-catenin, VE-cadherin, Arp3, and Claudin 11) were significantly downregulated in triptolide-treated groups (all P<0.01). Biotin tracer staining demonstrated varying degrees of blood-testis barrier injury in triptolide-treated mice, and the severity of barrier leakage increased in a dose-dependent manner. Conclusion: Triptolide treatment can be used to stably establish a mouse model of blood-testis barrier injury. Its mechanism is closely related to the synergistic regulation of multiple pathways, including Sertoli cell apoptosis, downregulated expression of blood-testis barrier-related proteins and oxidative stress response.

关键词

血睾屏障 / 雷公藤甲素 / 生物素 / 细胞凋亡

Key words

blood-testis barrier / triptolide / biotin / apoptosis

参考文献

[1] 黄宝祥, 朱培林, 聂韡, 等 . 雷公藤的栽培与应用[J]. 江西林业科技, 2012(5): 26-29.

[2] Liu Q . Triptolide and its expanding multiple pharmacological functions[J]. Int Immunopharmacol, 2011, 11(3): 377-383.

[3] Li XX, Du FY, Liu HX, et al. Investigation of the active components in Tripterygium wilfordii leading to its acute hepatotoxicty and nephrotoxicity[J]. J Ethnopharmacol, 2015, 162: 238-243.

[4] 白雪, 谭天阳 . 雷公藤生殖及肝肾毒理机制研究进展[J]. 中国药物警戒, 2024, 21(10): 1192-1199.

[5] Cheng CY, Wong EW, Lie PP, et al. Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins: an unexpected turn of events[J]. Spermatogenesis, 2011, 1(2): 105-115.

[6] Jiang XH, Bukhari I, Zheng W, et al. Blood-testis barrier and spermatogenesis: lessons from genetically-modified mice[J]. Asian J Androl, 2014, 16(4): 572-580.

[7] Cheng CY, Mruk DD . The blood-testis barrier and its implications for male contraception[J]. Pharmacol Rev, 2012, 64(1): 16-64.

[8] Mruk DD, Cheng CY . The mammalian blood-testis barrier: its biology and regulation[J]. Endocr Rev, 2015, 36(5): 564-591.

[9] Chen H, Lui WY, Mruk DD, et al. Monitoring the integrity of the blood-testis barrier (BTB): an in vivo assay[J]. Methods Mol Biol, 2018, 1748: 245-252.

[10] 刘奕, 柳毅, 陈雅文, 等 . 血睾屏障损伤的影响因素及其相关机制[J]. 中华生殖与避孕杂志, 2021, 41(10): 937-942.

[11] Li MW, Mruk DD, Lee WM, et al. Disruption of the blood-testis barrier integrity by bisphenol A in vitro: is this a suitable model for studying blood-testis barrier dynamics?[J]. Int J Biochem Cell Biol, 2009, 41(11): 2302-2314.

[12] Lu Y, Luo B, Li J, et al. Perfluorooctanoic acid disrupts the blood-testis barrier and activates the TNFα/p38 MAPK signaling pathway in vivo and in vitro[J]. Arch Toxicol, 2016, 90(4): 971-983.

[13] Xiong J, Wang H, Guo G, et al. Male germ cell apoptosis and epigenetic histone modification induced by Tripterygium wilfordii Hook F[J]. PLoS One, 2011, 6(6): e20751.

[14] 王昊, 陈亮, 叶小云 . 雷公藤甲素对TM4细胞氧化应激及PI3K/AKT通路的影响[J]. 北京大学学报(医学版), 2018, 50(4): 607-612.

[15] Wang Y, Guo SH, Shang XJ, et al. Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response[J]. Acta Pharmacol Sin, 2018, 39(2): 311-327.

[16] 罗立, 卢树宏, 张嘉慧, 等 . 雷公藤甲素对小鼠睾丸支持细胞人白细胞分化抗原36蛋白表达及脂质代谢水平的影响[J]. 药物评价研究, 2022, 45(7): 1266-1273.

[17] 黄郑隽, 阙慧卿, 彭华毅, 等 . 雷公藤内酯醇对雄性大鼠的生殖毒性及其机制研究[J]. 中国中药杂志, 2015, 40(23): 4655-4659.

[18] Singla N, Challana S . Reproductive toxicity of triptolide in male house rat, Rattus rattus[J]. Sci World J, 2014, 2014: 879405.

[19] Hu J, Yu Q, Zhao F, et al. Protection of Quercetin against Triptolide-induced apoptosis by suppressing oxidative stress in rat Leydig cells[J]. Chem Biol Interact, 2015, 240: 38-46.

[20] 修晓宇, 刘永珍, 陆姮磊, 等 . 雷公藤甲素致小鼠睾丸精母细胞损伤的敏感靶细胞群研究[J]. 中国药学杂志, 2020, 55(12): 990-995.

[21] 任朝兴, 朱志铭, 顾春阳, 等 . 雷公藤甲素抑制PGC-1α诱导的小鼠睾丸和附睾损伤及其分子作用机制[J]. 生物加工过程, 2021, 19(6): 649-656.

[22] Pan Z, Gao Y, Liu S, et al. Wu-Zi-Yan-Zong-Wan protects mouse blood-testis barrier from Tripterygium wilfordii Hook. f. multiglycoside-induced disruption by regulating proinflammatory cytokines[J]. J Ethnopharmacol, 2021, 280: 114440.

[23] Zhang J, Bao X, Zhang M, et al. MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway[J]. Toxicol Appl Pharmacol, 2019, 370: 78-92.

[24] 张亚敏, 林文津, 徐小妹, 等 . 不同浓度雷公藤甲素肝损伤大鼠模型初步研究[J]. 中国医药导报, 2016, 13(19): 7-9.

基金资助

深圳市基础研究专项/自然科学基金资助项目(JCYJ20240813114409013)

深圳市龙华区医学会医学科研专项课题(2023LHMA08)