基于Nrf2/HO-1通路的加味大黄虫庶虫颗粒对精索静脉曲张模型大鼠附睾生精细胞凋亡的影响

Antagonistic effect of modified Dahuang Zhechong Granule against the apoptosis of epididymal spermatogenic cells in varicocele rats based on Nrf2/HO-1 pathways

目的:探讨加味大黄蟅虫颗粒对精索静脉曲张模型大鼠附睾组织的保护作用及与核因子NF-E2相关性因子(Nrf2)、血红素加氧酶-1(HO-1)蛋白表达的关系。方法:60只SPF级SD大鼠,按随机数字表法分成假手术组、模型组、迈之灵组及加味大黄蟅虫颗粒低、中、高剂量组。采用Turner法构建精索静脉曲张大鼠模型。造模完成后,加味大黄蟅虫颗粒低、中、高剂量组分别给予浓度为0.6、1.2、2.4 g/ml的大黄蟅虫丸生药水冲剂灌胃,假手术组和模型组予以等量的生理盐水灌胃,迈之灵组则予54 mg/kg迈之灵片,以上各组灌胃体积均为1 ml/100 g,1次/d,持续8周。给药8周后全部处死以上各组大鼠,取出左侧附睾检测精子质量及观察局部显微超微结构的变化;Annexin V-FITC法检测左侧附睾生精细胞凋亡情况;免疫组织化学法检测左侧附睾组织中Nrf2、HO-1的表达。结果:模型组中左侧附睾组织内发生明显病理损伤,各级生精细胞和精子数量重度减少,细胞结构部分破坏,溶酶体、线粒体、内质网、细胞核、细胞膜等部分亚细胞结构消失,而加味大黄蟅虫颗粒各剂量组及迈之灵组中左侧附睾组织的病理损伤具有一定程度的改善。与模型组相比,加味大黄蟅虫颗粒中、高剂量组及迈之灵组大鼠左侧附睾的精子浓度及活率显著增高(P<0.05)。与迈之灵组比较,加味大黄蟅虫颗粒高剂量组精子浓度及活率显著升高(P<0.05)。模型组中左侧附睾生精细胞凋亡率显著高于假手术组(P<0.05),加味大黄蟅虫颗粒中、高剂量组及迈之灵组中生精细胞凋亡率显著低于模型组(P<0.05)。免疫组织化学法显示,模型组大鼠左侧附睾组织中Nrf2/HO-1棕黄色阳性表达呈散在排列,且细胞辉度低;而加味大黄蟅虫颗粒高剂量组、迈之灵组中棕黄色阳性表达的细胞辉度明显提升。结论:加味大黄蟅虫颗粒可以有效修复精索静脉曲张大鼠附睾组织的病理损伤,提高Nrf2/HO-1的蛋白表达,进而拮抗生精细胞的凋亡,为精子的发育成熟提供有利条件。

Objective:To investigate the effects of modified Dahuang Zhechong Granule(DZG)on the epididymal tissue of varicocele(VC)rats and the expressions of the nuclear factor erythroid 2(NF-E2)-related factor(Nrf2)and heme oxygenase-1(HO-1)protein.Methods:Sixty SD rats were randomly divided into six groups of an equal number:sham operation,VC model control,aescuven forte(AF)and low-,medium-and high-dose DZG.The VC model was established by ligation of the left renal vein with the Turner’s method,followed by intragastrical administration of normal saline to the rats in the sham operation and VC model control groups,AF Tablets at 54 mg/kg to those in the AF group,and modified DZG at 0.6,1.2 and 2.4 g/ml to those in the low-,medium-and high-dose DZG groups respectively,all once daily for 8 weeks.Then,all the animals were sacrificed and their left epididymides harvested for examination of semen quality,observation of local ultrastructural changes,measurement of the apoptosis of spermatogenic cells by Annexin V-FITC,and determination of the expressions of Nrf2 and HO-1 in the epididymal tissue by immunohistochemistry.Results:Evident pathological damage was observed in the left epididymal tissue of the VC model controls,with significantly reduced numbers of spermatogenic cells and sperm at all levels,partially destroyed cellular structure,and disappearance of some subcellular structures such as the lysosome,mitochondrion,endoplasmic reticulum,nucleus and cell membrane,which were all improved to some extent in the DZG and AF group.Sperm concentration and motility in the left epididymis were significantly higher in the medium-and high-dose DZG and AF groups than in the VC model controls(P<0.05),even more significantly in the high-dose DZG than in the AF group(P<0.05).The apoptosis rate of spermatogenic cells was markedly higher in the VC model control than in the sham operation group(P<0.05),but lower in the medium-and high-dose DZG and AF groups than in the VC model controls(P<0.05).Immunohistochemistry showed positive expressions of Nrf2 and HO-1 proteins,brown,scattered and with a low luminance of the cells,in the left epididymis tissue of the VC model control rats,but with a significantly higher cell luminance in the high-dose DZG and AF groups.Conclusion:Modified Dahuang Zhechong Granule can effectively repair pathological damage to the epididymis of varicocele rats,increase the expressions of Nrf2 and HO-1 proteins,antagonize the apoptosis of spermatogenic cells and provide a favorable condition for sperm maturation.