基于Nrf2⁃NQO1/γ⁃GCS信号通路探讨续断种子方对少弱精子症模型大鼠附睾氧化损伤的保护机制

An exploration on the protective mechanism of XuduanZhongzi prescription against epididymis oxidative damage in oligoasthenospermiamodel rats based on Nrf2⁃NQO1/γ⁃GCS signaling pathway

目的:探讨续断种子方对少弱精子症模型大鼠附睾氧化损伤的保护机制。方法:将40只SD大鼠随机平均分为空白组、模型组、续断种子方组(10 g/kg)、左卡尼汀组(0.1 g/kg)。除空白组外,均诱导为少弱精子症。其中空白组、模型组给予生理盐水灌胃,续断种子方组给予续断种子方溶液灌胃,左卡尼汀组给予左卡尼汀灌胃,8周后HE染色观察附睾组织结构;通过精子质量检测大鼠附睾精子浓度和活动率;采用RT-qPCR、免疫组化检测附睾组织Nrf2、NQO1、γ-GCS mRNA和蛋白表达水平。结果:(1)HE染色:空白组附睾管排列紧密规则,组织结构完整,上皮细胞排列规整,管腔精子数目多,分布均匀。模型组附睾管出现结构萎缩,排列疏松,间质变大,细胞碎片增多,精子细胞明显减少。与模型组相比,续断种子方组、左卡尼汀组附睾管腔病变具有显著改善,腔内正常精子量增多,分布均匀。(2)各组精液质量比较结果:精子密度和精子活动率:相较于空白组,其余组精子密度、活动率均明显降低(P<0.05),模型组精子密度、活动率明显降低(P<0.05);与模型组比较,续断种子方组和左卡尼汀组精子密度和活动率均明显升高(P<0.05)。(3)RT-qPCR:与空白组相比,模型组大鼠附睾Nrf2、NQO1、γ-GCS mRNA和蛋白水平明显降低(P<0.05),与模型相比,续断种子方组、左卡尼汀组Nrf2、NQO1、γ-GCS mRNA和蛋白水平明显升高(P<0.05)。结论:续断种子方能够减轻少弱精子症大鼠氧化应激损伤,提高少弱精子症大鼠精子质量。其作用机制可能是促进少弱精子症大鼠附睾组织Nrf2-NQO1/γ-GCS信号通路活化,上调Nrf2、NQO1和γ-GCS蛋白表达相关。

Objective:To investigate the protective mechanism of XuduanZhongzi prescription against epididymal oxidative damage in oligoasthenospermiamodel rats.Methods:Forty SD rats were randomly divided into blank group,model group,XuduanZhongzi prescription group(10 g/kg)and L-carnitine group(0.1 g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the XuduanZhongzi prescription group was given XuduanZhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:(1)HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in XuduanZhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.(2)Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate inXuduanZhongzi prescription group and L-carnitine group were significantly increased(P<0.05).(3)RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:XuduanZhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.