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镉对大鼠血睾屏障的损伤及黄芪甲苷的保护作用
Cadmium damages the blood-testis barrier in rats and the protective effect of Astragaloside IV
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。
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，AAof 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.
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
Cadmium / Astragaloside IV / Zonula occludens-1 / Claudin-11 / P-p38 MAPK / Immunohistochemistry / Western blotting / Rat
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