疏肝补肾毓麟汤通过Keap1/Nrf2/GPX4通路抑制睾丸细胞铁死亡改善肝郁肾虚型少弱精子症小鼠的精子质量  

Shugan Bushen Yulin Decoction(疏肝补肾毓麟汤)Can Improve Sperm Quality of Liver Depression and Kidney Deficiency Type of Oligasthenospermia Mice by Regulating Keap1/Nrf2/GPX4 Pathway to Inhibit Ferroptosis of Testicular Cells

作　　者：江晓翠 萧闵[1,2] 赵敏 刘祺 姜兴宇[3] 吕银娟 陈思易 曹继刚 JIANG Xiaocui;XIAO Min;ZHAO Min;LIU Qi;JIANG Xingyu;LYU Yinjuan;CHEN Siyi;CAO Jigang(Laboratory Animal Center,Hubei University of Chinese Medicine,Wuhan 430065,Hubei,China;Hubei Shizhen Laboratory,Wuhan 430065,Hubei,China;College of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,Hubei,China;Hubei Hospital of Traditional Chinese Medicine,Wuhan 430061,Hubei,China)

机构地区：[1]湖北中医药大学实验动物中心,湖北武汉430065 [2]湖北时珍实验室,湖北武汉430065 [3]湖北中医药大学基础医学院,湖北武汉430065 [4]湖北省中医院,湖北武汉430061

出　　处：《中华中医药学刊》2024年第5期46-51,I0017-I0019,共9页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金青年科学基金项目(82104704);湖北省中医药管理局中医药重点项目(ZY2023Z204);湖北省中医药管理局中医药指导性项目(ZY2023F134);武汉中青年名中医项目(武卫〔2019〕17号);湖北中医药大学校级科研平台项目(中医校〔2022〕80号);湖北中医药大学“青苗计划”项目(2022ZZXQ033)。

摘　　要：目的探讨疏肝补肾毓麟汤改善肝郁肾虚型少弱精子症小鼠生精功能和精子质量的作用及其可能机制。方法60只雄性KM小鼠,随机分为空白组、模型组、疏肝补肾毓麟汤高、中、低剂量组和司他丁-1(Ferrostatin-1,Fer-1)组(Fer-1是铁死亡抑制剂)。除空白组外,其余组以1.25 g·kg^(-1)腺嘌呤灌胃(Intragastrical administration,ig.)+慢性束缚刺激构建肝郁肾虚型少弱精子症小鼠模型。给予相应药物干预后:精密天平称取小鼠体质量、睾丸质量、附睾质量计算睾丸指数、附睾指数;苏木素-伊红(Hematoxylin-eosin staining,HE)染色观察睾丸组织病理变化并对睾丸生精功能进行评分;全自动精子分析仪检测精子密度和活动率;苯胺蓝染色法观察精子成熟度;布鲁斯蓝染色法观察睾丸组织铁沉积;免疫组化(Immunohistochemistry,IHC)检测睾丸组织谷胱甘肽过氧化物酶4(Glutathione peroxidase 4,GPX4)、溶质载体家族7成员11(Recombinant Solute Carrier Family 7 Member 11,SLC7A11)蛋白表达;生化法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)含量;蛋白免疫印迹法(Western Blot,WB)检测睾丸组织Kelch-样ECH相关蛋白1(Kelch-like ECH-associated protein-1,Keap1)、核因子E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)、GPX4、SLC7A11蛋白表达。结果与空白组比较,模型组小鼠睾丸指数、附睾指数、睾丸生精功能、精子密度及活动率、精子成熟度均显著下降(P<0.05,P<0.01);经疏肝补肾毓麟汤及Fer-1干预后,上述指标均有显著改善(P<0.05,P<0.01),且睾丸组织铁沉积显著降低,Keap1表达显著降低,Nrf2、SLC7A11、GPX4表达显著升高(P<0.05,P<0.01)。结论疏肝补肾毓麟汤能明显改善肝郁肾虚型少弱精子症,其机制可能与调控Keap1/Nrf2/GPX4信号通路抑制睾丸细胞铁死亡有关。Objective To investigate the effects and possible mechanism of Shugan Bushen Yulin Decoction(疏肝补肾毓麟汤)in improving spermatogenesis and sperm quality of liver depression and kidney deficiency type of oligoasthenospermia mice.Methods Sixty SPF male KM mice were randomly divided into blank group,model group,Shugan Bushan Yulin Decoction high-dose,medium-dose and low-dose groups and statin-1(Ferrostatin 1,Fer-1)group(Fer-1 is ferroptosis inhibitor)by ran⁃dom number method.Each group consisted of 10 animals.The other groups were given 1.25 g·kg^(-1) adenine intragastrium(ig)+chronic binding stimulation to construct oligasthenospermia mouse model with liver depression and kidney deficiency.Af⁃ter drug intervention,the body mass,testicle mass and epididymal mass of mice were measured with precision balance to calculate testicle coefficient and epididymal coefficient.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of testis and score the spermatogenic function of testis.Automatic sperm analyzer was used to detect the sperm density and motility.Sperm maturity was observed by aniline blue staining.Iron deposition in testicular tissue was observed by Bruce blue staining.The expressions of glutathione peroxidase 4(GPX4)and solute vector family 7 member 11(SLC7A11)in testicular tissue were determined by immunohistochemical(IHC).The activities of superoxide Dismutase(SOD),malondialdehyde(MDA)and cata⁃lase(CAT)in testicular tissue were detected by biochemical method.The expressions of Kelch-like ECH associated protein 1(Keap1),nuclear factor 2 associated factor 2(Nrf2),GPX4 and SLC7A11 in testicular tissue were detected by Western blotting(WB).Results Compared with those of the blank group,the testis coefficient,epididymis coefficient,testis spermatogenic func⁃tion,sperm density,motility rate and sperm maturity in the model group were significantly decreased(P<0.05,P<0.01).The above indexes were significantly improved after high and medium dose and Fer-1 intervention with Shugan

关 键 词：疏肝补肾毓麟汤 少弱精子症 Keap1/Nrf2/GPX4 铁死亡 精子质量 

分 类 号：R289.5[医药卫生—方剂学]