脉络舒通丸对精索静脉曲张模型大鼠线粒体介导内源性细胞凋亡的影响

Effect of Mailuo Shutong Pills on mitochondria-mediated endogenous cell apoptosis in rats with varicocele

目的 探讨脉络舒通丸对精索静脉曲张模型大鼠线粒体介导的内源性细胞凋亡的影响。方法 36只雄性SD大鼠随机分为空白组、假手术组、模型组和脉络舒通丸低、中、高剂量(0.162、0.324、0.648 g/kg)组,精索静脉曲张模型制备成功7 d后,连续ig给药4周。苏木素-伊红(HE)染色法检测各组大鼠睾丸及附睾组织病理变化;透射电镜观察各组大鼠生精细胞线粒体超微结构;免疫荧光检测各组大鼠睾丸组织中线粒体动力相关蛋白1(dynamin-related protein 1,Drp1)、B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2 associated X protein,Bax)和细胞色素C氧化酶IV(cytochrome C oxidaseⅣ,COXⅣ)的表达;免疫组化检测各组大鼠睾丸组织中半胱氨酸天冬氨酸蛋白酶-9(cystein-asparate protease-9,Caspase-9)和Caspase-3的表达。结果 假手术组与空白组大鼠睾丸及附睾组织病理染色结果相似,睾丸及附睾组织结构完整;与假手术组比较,模型组大鼠睾丸及附睾组织出现明显的病理性损伤;药物治疗后大鼠睾丸及附睾的病理性损伤得到不同程度的改善。假手术组与空白组大鼠睾丸线粒体形态结构相似,均可见大量线粒体广泛分布;与假手术组比较,模型组睾丸组织线粒体结构遭到严重破坏;药物治疗后线粒体仍可见不同程度的损伤,但较模型组明显改善。免疫荧光结果显示,Drp1与Bax在假手术组与空白组中荧光表达微弱,模型组较假手术组二者的荧光表达水平显著升高;Drp1与Bax在各治疗组的荧光表达趋势相似,经药物治疗后二者的荧光强度较模型组均降低。免疫组化结果显示,Caspase-9与Caspase-3在细胞间质及生精小管管壁周围分别呈褐色及棕色表达;假手术组与空白组二者均零星分布;与假手术组比较,模型组Caspase-9、Caspase-3表达增加(P<0.01);与模型组比较,各治疗组中Caspase-9、Caspase-3的表达水平明显降低(P<0.05、0.01)。结论 精索静脉曲张模型大鼠睾丸及附睾组织结构损伤明显,线粒体结构遭到明显破坏,睾丸中Drp1、Bax、Caspase-9及Caspase-3表达显著升高。脉络舒通丸可能是通过对睾丸中线粒体结构的保护作用,抑制生精细胞中Caspase级联反应,从而调控线粒体介导的内源性细胞凋亡。

Objective To investigate the effect of Mailuo Shutong Pills(脉络舒通丸) on mitochondria-mediated endogenous cell apoptosis in rats with varicocele.Methods A total of 36 male SD rats were randomly divided into blank group,sham group,model group,Mailuo Shutong Pills low-,medium-and high-dose(0.162,0.324,0.648 g/kg) groups.After 7 d of successful preparation of the varicocele model,drugs were continuous ig for four weeks.Hematoxylin-eosin(HE) staining was used to detect the histopathological changes of testicle and epididymis tissue in each group.Transmission electron microscopy was used to observe the changes in mitochondrial ultra microstructure of spermatogenic cells in each group.Immunofluorescence was used to detect dynamin-related protein 1(Drp1),B-cell lymphoma-2 associated X protein(Bax) and cytochrome C oxidase IV(COX IV) expressions in mitochondrial testicular tissue of rats in each group;Immunohistochemistry was used to detect the expressions of cystein-asparate protease-9(Caspase-9) and Caspase-3 in testicular tissue of rats in each group.Results The pathological staining results of testis and epididymis of rats in sham group and blank group were similar,and the structure of the testis and epididymis was intact.Compared with sham group,the testis and epididymis in model group showed obvious pathological damage.After drug treatment,the pathological damage of testis and epididymis in each group was improved to varying degrees.The morphological structure of testicular mitochondria was similar between sham group and blank group,and a large number of mitochondria were widely distributed.Compared with sham group,the mitochondrial structure of testicular tissue in model group was severely damaged.After drug treatment,the mitochondria still showed varying degrees of damage,but it was significantly improved compared with model group.Immunofluorescence results showed that the fluorescence expression levels of Drp1 and Bax in sham group and blank group were weak,and the fluorescence expression levels of Drp1 and Bax in model group were significantly higher than those in sham group.The fluorescence expressions of Drp1 and Bax in each treatment group had a similar trend,and the fluorescence intensity of Drp1 and Bax after drug treatment were lower than that of the model group.The results of immunohistochemistry showed that Caspase-9 and Caspase-3 were expressed in brownness and brown in the mesenchyme and around the wall of seminiferous tubules,respectively.Both in the sham group and blank group were scattered.Compared with sham group,the expressions of Caspase-9 and Caspase-3 in model group were increased(P < 0.01).Compared with model group,the levels of Caspase-9 and Caspase-3 in each treatment group were significantly decreased(P < 0.05,0.01).Conclusion In rats with varicocele model,the tissue structure of testis and epididymis was significantly damaged,mitochondrial structure was significantly damaged,and the expressions of Drp1,Bax,Caspase-9 and Caspase-3 in the testis were significantly increased.Mailuo Shutong Pills may inhibit the Caspase cascade in spermatogenic cells through its protective effect on mitochondrial structure in testis,thereby regulating mitochondria-mediated endogenous cell apoptosis.