二仙汤对少弱精子症小鼠Keap1/Nrf2/HO-1通路的影响

Effect of Erxian Decoction on Keap1/Nrf2/HO-1 pathway in oligoasthenospermia mice

目的:探讨二仙汤对少弱精子症小鼠生精功能和精子质量的影响及其作用机制。方法:48只成年雄性SPF级KM小鼠,随机数字法分为空白组,模型组,二仙汤高、中、低剂量组和维生素E组,每组8只。除空白组外,其余各组以1.25g·kg^(–1)腺嘌呤灌胃构建少弱精子症小鼠模型。造模完成后,二仙汤高、中、低剂量组分别灌胃给予11.7,5.85,2.93 g·kg^(–1)二仙汤,维生素E组灌胃给予0.013 g·kg^(–1)维生素E,连续灌胃21 d后取材。测定睾丸、附睾指数;苏木素-伊红染色观察睾丸组织病理变化并对睾丸生精功能评分;全自动精子分析仪检测精子密度和活动率;生化试剂盒检测睾丸组织超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、丙二醛(malondialdehyde,MDA);蛋白免疫印迹法检测睾丸组织Kelch-样ECH相关蛋白1(Kelch-like ECH related protein 1,Keap1)、核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)、血红素加氧酶1(heme oxygenase-1,HO-1)蛋白表达;实时荧光定量聚合酶链式反应检测睾丸组织Keap1、Nrf2、HO-1 m RNA表达。结果:与空白组比较,模型组小鼠附睾指数、睾丸指数,睾丸生精功能,精子密度及活动率,睾丸组织SOD、CAT活性,Nrf2、HO-1蛋白及其m RNA表达水平均降低,睾丸MDA含量、Keap1蛋白及其m RNA表达水平均升高(P<0.01);与模型组比较,二仙汤高、中剂量组小鼠上述指标均有显著逆转(P<0.05,P<0.01)。结论:二仙汤可能通过调控Keap1/Nrf2/HO-1信号通路减轻睾丸组织氧化应激损伤,提高生精功能,改善少弱精子症。

OBJECTIVE To explore the effects of Erxian Decoction(ED)on spermatogenesis and sperm quality and eluci⁃date its mechanism in oligoasthenospermia mice.METHODS Forty-eight adult male Kunming mice of specific pathogen free(SPF)grade were randomized into six groups of blank,model,ED high/medium/low-dose and vitamin E(n=8 each).Except for blank group,oligoasthenospermia murine model was constructed through an intragastric injection of 1.25 g·kg^(–1) adenine.After modeling,ED high/medium/low-dose group received 11.7,5.85 and 2.93 g·kg^(–1),while vitamin E group had 0.013 g·kg^(–1) vitamin E.Testicular and epididymal parameters were measured.Hematoxylin-eosin stain was employed for observing the patho⁃logical changes of testicular tissue and scoring testicular spermatogenic function.Automatic sperm analyzer was utilized for detect⁃ing sperm density/motility.The levels of superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)were detected by biochemical kits.The expressions of Kelch-like ECH related protein 1(Keap1),nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)were detected by Western blot.And real-time fluorescent quantitative polymerase chain reaction was utilized for detecting Keap1,Nrf2 and HO-1 mRNA expressions.RESULTS Compared with blank group,testis index,epididymal index,sperm density/activity,SOD/CAT/MDA,Nrf2,HO-1 protein/mRNA expression were lower while MDA,Keap1 protein/mRNA expression spiked in model group(P<0.01).As compared with model group,the above param⁃eters reversed markedly in high/medium-dose ED group(P<0.05,P<0.01).CONCLUSION ED may alleviate oxidative stress injury of testicular tissue by regulating Keap1/Nrf2/HO-1 signaling pathway and improving spermatogenesis function and oligoasthenospermia.