镉对大鼠血睾屏障的损伤及黄芪甲苷的保护作用

doi:10.3969/j.issn.0529-1356.2014.05.021

摘要/Abstract

摘要：

目的观察黄芪甲苷和SB203580对镉致大鼠血睾屏障破坏及相关蛋白表达改变的保护效应，探讨黄芪甲苷的保护机制。方法 21只成年雄性SD大鼠随机分为7组：单纯镉组［0.1%氯化镉腹腔内注射，1mg/（kg·d）］，镉+黄芪甲苷组［镉剂量同上，同时注射黄芪甲苷，10mg/（kg·d）］，镉+SB203580组［镉剂量同上，同时注射SB203580，100μg/（kg·d）］，以上各组又分为连续处理5d和10d两个时间组，对照组腹腔内注射等量生理盐水。各实验和对照组动物均为3只。取睾丸做光学显微镜、电子显微镜观察以及免疫组织化学染色和Western blotting检测。结果 HE染色观察对照组支持细胞核染色较浅且不规则，镉组支持细胞内有空泡形成，镉+黄芪甲苷组与镉+SB203580组未见明显形态异常。免疫组织化学染色观察对照组闭锁小带-1蛋白（ZO-1）、紧密连接蛋白-11（claudin-11）阳性产物在生精上皮靠近基底部表达。镉组ZO-1、claudin-11阳性产物表达均显著降低（P＜0.05），镉+黄芪甲苷组与镉+SB203580组阳性产物表达低于对照组但明显高于镉组（P＜0.05）。超微结构观察对照组血睾屏障紧密连接形态完整，呈连续的电子密度较深致密线，镉组血睾屏障紧密连接及支持细胞均见不同程度破坏，镉+黄芪甲苷组与镉+SB203580组破坏程度较相应处理时间镉组为轻。Western blotting结果显示，镉组磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）水平明显增强（P＜0.05），镉+黄芪甲苷组与镉+SB203580组p-p38MAPK水平虽高于对照组，但较镉组明显减弱（P＜0.05）。结论镉致大鼠血睾屏障ZO-1、claudin-11表达降低，紧密连接超微结构损伤，黄芪甲苷具有保护作用，其保护机制与抑制p38MAPK磷酸化有关。

关键词: 镉, 黄芪甲苷, 闭锁小带-1蛋白, 紧密连接蛋白-11, P-p38MAPK, 免疫组织化学, 免疫印迹法, 大鼠

Abstract:

Objective To observe the effect of astragaloside IV (A) and SB203580 antagonize cadmium(Cd) toxicity on expression of associated protein and blood-testis barrier(BTB) in rats and to study the protective mechanism of A on it. Methods Totally 21 SD male rats were randomly divided into 7 groups, 3 rats per group：Cd ［intraperitoneally injected with 0.1%CdCl2，1mg/(kg·d)］，Cd+A ［at the above dose of CdCl2，at the same time with A，10mg/(kg·d)］，and Cd + SB203580 ［at the above dose of CdCl2，at the same time with SB203580，100μg/(kg·d)］， each of the above groups was further divided into continuous five and ten days treatment groups. The control group was intraperitoneally injected with equal dosage of normal saline. The testes were studied by light, electron microscopy, immunohistochemistry and Western blotting. Results In the control group，irregular and lightly stained nuclei of Sertoli cell(Sc) in seminiferous tubules were observed by HE staining. A continuous electron density line of tight junction（TJ）and normal ultrastructure of BTB were observed. After Cd treatment，the vesicular formation in the Sc was observed. The ultrastructural damage of Sc and TJ was observed. Compared with the corresponding time point of Cd group，these were weakened in morphology of testis and ultrastructure of TJ after Cd + A or Cd + SB203580 treatment. The positive products of zonula occludens-1（ZO-1） and claudin-11 were localized mainly in the base of the seminiferous tubule. After Cd treatment，the average absorbance (AA) of ZO-1 and Claudin-11 was decreased significantly compared with that of the control group（P＜0.05）. After Cd + A or Cd + SB203580 treatment，AAof ZO-1 and Claudin-11 were increased significantly compared with that of the Cd group（P＜0.05），though lower than that of the control group. The result of Western blotting showed that phosphorylation-p38MAPK in Cd group was increased significantly compared with that of the control group（P＜0.05）.After Cd + A or Cd + SB203580 treatment, it was decreased significantly compared with that of the Cd group（P＜0.05）. Conclusion Cd decreases ZO-1 and Claudin-11 expression and damages ultrastructure of TJ in BTB, as Ⅳ has protective effect on it, and is related to inhibiting activation of p38 MAPK pathway.

Key words: Cadmium, Astragaloside IV, Zonula occludens-1, Claudin-11, P-p38 MAPK, Immunohistochemistry, Western blotting, Rat

宁巍廖晓岗* 王毅姚志勇毛胜艳. 镉对大鼠血睾屏障的损伤及黄芪甲苷的保护作用[J]. 解剖学报, 2014, 45(5): 704-709.

NING Wei LIAO Xiao-gang* WANG Yi YAO Zhi-yong MAO Sheng-yan. Cadmium damages the blood-testis barrier in rats and the protective effect of Astragaloside IV[J]. AAS, 2014, 45(5): 704-709.

参考文献

［1］He P, Lu Y, Liang Y, et al. Exposure assessment of dietary cadmium: findings from shanghainese over 40 years, China［J］. BMC Public Health,2013,13:590.
［2］Siu ER, Mruk DD, Porto CS, et al.Cadmium-induced testicular injury［J］.Toxicol Appl Pharmacol,2009,238(3):240-249.
［3］Lee J, Lim KT. Expression of TNF-α and IL-6 in HMC-1 cells treated with bisphenol A is attenuated by plant-originating glycoprotein (75 kDa) by blocking p38 MAPK［J］.Naunyn Schmiedebergs Arch Pharmacol,2010,382（1）:51-61.
［4］Mao ShY, Liao XG, Jiang R, et al. Protective function of astragaloside on cadmium-treated Sertoli cell in rat ［J］. Laser Journal, 2011,32(4):84-86.(in Chinese)
毛胜艳, 廖晓岗, 姜蓉, 等. 黄芪甲苷对镉致大鼠支持细胞损伤的保护作用［J］.激光杂志,2011,32（4）:84-86.
［5］Shi ZhH, Liao XG, Zou C, et al. Protective effect of astragalus mongholicus against cadmium toxicity on the expression of vimentin & e-cadherin and the ultrastructure of Sertoli cell in rats ［J］. Chinese Journal of Histochemistry and Cytochemistry, 2012,21(3):284-289.(in Chinese)
石之虎, 廖晓岗, 邹聪, 等. 黄芪对镉致大鼠睾丸支持细胞超微结构及波形蛋白、E-钙黏蛋白表达改变的保护作用［J］.中国组织化学与细胞化学杂志,2012,21（3）:284-289.
［6］Chang YX, Sun YG, Li J, et al.The experimental study of astragalus membranaceus on meridian tropsim: the distribution study of astragaloside IV in rat tissues［J］.J Chromatogr B Analyt Technol Biomed Life Sci,2012,911:71-75.
［7］Li X, Wang X, Han C, et al. Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway［J］.Free Radic Biol Med,2013,60:168-176.
［8］Pérez CV, Theas MS, Jacobo PV, et al. Dual role of immune cells in the testis: protective or pathogenic for germcells［J］? Spermatogenesis,2013,3(1):e23870.
［9］Erkanl ?entürk G, Ersoy Canilliolu Y, Umay C, et al. Distribution of zonula occludens-1 and occludin and alterations of testicular morphology after in utero radiation and postnatal hyperthermia in rats［J］.Int J Exp Pathol，2012，93(6):438-449. 
［10］Lui WY, Lee WM, Cheng CY. Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11［J］. Endocrinology,2001,142(5):1865-1877. 
［11］Lydka M, Bilinska B, Cheng CY,et al. Tumor necrosis factor α-mediated restructuring of the Sertoli cell barrier in vitro involves matrix metalloprotease 9 (MMP9), membrane-bound intercellular adhesion molecule-1 (ICAM-1) and the actin cytoskeleton［J］.Spermatogenesis,2012,2(4):294-303.
［12］Xiao X, Cheng CY, Mruk DD. Intercellular adhesion molecule-2 is involved in apical ectoplasmic specialization dynamics during spermatogenesis in the rat［J］. J Endocrinol,2013,216(1):73-86.
［13］Wong CH, Mruk DD, Lui WY, et al. Regulation of blood-testis barrier dynamics: an in vivo study［J］. J Cell Sci.,2004,117（5）:783-798.
［14］Siu ER, Wong EW, Mruk DD, et al. An occludin-focal adhesion kinase protein complex at the blood-testis barrier: a study using the cadmium model［J］. Endocrinology,2009,150(7):3336-3344.
［15］Mazaud-Guittot S. Dissecting the phthalate-induced Sertoli cell injury: the fragile balance of proteases and their inhibitors［J］.Biol Reprod,2011,85(6):1091-1093.
［16］Qu YZ, Li M, Zhao YL, et al. Astragaloside IV attenuates cerebral ischemia-reperfusion-induced increase in permeability of the blood-brain barrier in rats［J］. Eur J Pharmacol,2009,606(1-3):137-141.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[5]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[6]	吕海燕沈西雅赵富生朱梅. 松茸多糖对 1-甲基-4-苯基-吡啶离子诱导 PC12 细胞损伤的影响 [J]. 解剖学报, 2024, 55(1): 49-54.
[7]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[8]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[9]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[10]	刘丽平朱梦妮徐慧 . 白细胞介素6对脂多糖诱导所致类子痫前期大鼠有核红细胞的影响 [J]. 解剖学报, 2023, 54(6): 722-729.
[11]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[12]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[13]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[14]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.
[15]	刘哲川李坤马帅男孟家琪王艳芹. 利拉鲁肽对百草枯诱导的小鼠帕金森病模型炎症及线粒体融合/分裂的影响 [J]. 解剖学报, 2023, 54(6): 676-681.