摘要
目的探究五子衍宗方对衰老大鼠睾丸支持细胞紧密连接损伤的保护作用及其分子机制。方法将30只16月龄SD雄性大鼠随机分为衰老模型组、五子衍宗方低剂量组(1 g/kg)和五子衍宗方高剂量组(4 g/kg),另取2月龄SD雄性大鼠作为青年对照组,每组10只。给予含药饲料或普通维持饲料喂养4个月后,麻醉处死大鼠,取睾丸组织,HE染色观察其形态,统计各组大鼠生精上皮厚度和生精小管直径,电镜观察大鼠睾丸支持细胞紧密连接超微结构,Western blot检测大鼠睾丸紧密连接相关蛋白ZO⁃1和Occludin(闭锁蛋白)蛋白表达,双标免疫荧光法观察大鼠睾丸支持细胞ZO⁃1、Occludin、Claudin⁃11(细胞紧密连接蛋白)、p⁃p38MAPK(磷酸化p38丝裂原活化蛋白酶)、MMP⁃9(基质金属蛋白酶9)、Occludin表达和共定位。结果HE结果显示,五子衍宗方可改善衰老大鼠睾丸组织形态结构,升高衰老大鼠生精上皮厚度和生精小管直径。电镜结果显示,五子衍宗方可改善衰老大鼠紧密连接的超微结构。Western blot结果显示,五子衍宗方可上调衰老大鼠ZO⁃1、Occludin、Claudin⁃11蛋白表达。免疫荧光结果显示,五子衍宗方可上调衰老大鼠睾丸支持细胞ZO⁃1、Occludin、Claudin⁃11表达,下调p⁃p38MAPK、MMP⁃9表达,减少MMP⁃9和Occludin共定位。结论五子衍宗方可通过抑制p38MAPK/MMP⁃9通路来改善衰老大鼠睾丸支持细胞紧密连接损伤。
AIM To investigate the mechanism and the protective effect of Wuziyanzong Recipe on injury of Sertoli cell tight junctions of aging rats.METHODS Thirty 16⁃month⁃old SPF SD male rats were randomly divided into aging model group,groups for interventions of low⁃dose(1 g/kg)and high⁃dose(4 g/kg)Wuziyanzong Recipe,and 2⁃month⁃old male rats were recruited in the young adult control group,with 10 rats in each group.The rats given 4⁃month normal diet or medicated diet respectively had their testis tissues removed rapidly after urethane anesthesia.Subsequently the testicular tissues were subjected to the determinations of morphological changes by HE staining;the thickness and diameter of seminiferous tubules;the ultrastructure of Sertoli cells tight junctions was observed by transmission electron microscope(TEM);the expressions of ZO⁃1 and Occludin proteins by were detected Western blot;the expression and localization of ZO⁃1,Occludin,Claudin⁃11 and p⁃p38MAPK proteins was observed by double⁃label immunofluorescence;the expression and co⁃localization of MMP⁃9 and Occludin was observed by double⁃label immunofluorescence.RESULTS Wuziyanzong Recipe improved the aging⁃related degeneration of testicular tissue morphology based on HE staining demonstration;increased the thickness of seminiferous epithelium and the diameter of seminiferous tubules;improved the ultrastructure of testicular Sertoli cells tight junction in aging rats based on TEM results;increased the expressions of tight junction proteins ZO⁃1 and Occludin based on Western blot results;increased the proteins expression of Occludin,Claudin⁃11 and ZO⁃1 and decreased the proteins expression of p⁃p38MAPK proteins in Sertoli cells;decreased the expression of MMP⁃9,and the co⁃localization of MMP⁃9 with Occludin based on double⁃label immunofluorescence revelation.CONCLUSION Wuziyanzong Recipe attenuates injury of Sertoli cell tight junctions of via inhibition of p38MAPK/MMP⁃9 pathway in aging rats.
作者
马琼艳
张长城
杨圆
张艳
吴杰
袁丁
赵海霞
MA Qiong-yan;ZHANG Chang-cheng;YANG Yuan;ZHANG Yan;WU Jie;YUAN Ding;ZHAO Hai-xia(School of Medical Sciences,China Three Gorges University,Yichang 443002,China)
出处
《中成药》
CAS
CSCD
北大核心
2021年第8期2017-2024,共8页
Chinese Traditional Patent Medicine
基金
国家自然科学基金资助项目(81573931,81774316,81873077)。
