基于 Keap1-Nrf2-ARE 通路探讨加味天雄散调节弱精症大鼠精子活力的作用机制

目的基于Kelch样相关蛋白1-核因子E2相关因子-抗氧化反应元件信号通路(Keap1-Nrf2-ARE)及下游Maf、γ-谷氨酰半胱氨酸合成酶(γ-GCS)、血红素加氧酶(HO-1)蛋白表达探讨加味天雄散调节弱精症大鼠精子活力的作用机制。方法清洁级SD雄性大鼠60只随机分为对照组、模型组、安慰剂组、治疗组,每组15只。采用腺嘌呤[100 mg/(100 g·d)]灌胃10天造模,对照组灌胃等量生理盐水。造模结束后治疗组予以加味天雄散中药灌服,安慰剂组给予等量生理盐水,连续灌胃30天。RT-PCR检测附睾组织Keap1、Nrf2 m RNA表达;Western Blot检测附睾组织Keap1、Nrf2、Maf、γ-GCS、HO-1蛋白表达;检测附睾组织超氧化物歧化酶(SOD)、丙二醛(MDA)水平。结果与对照组比较,模型组Keap1 m RNA及蛋白表达、MDA水平升高(P<0.01),Nrf2m RNA、Nrf2、Maf、γ-GCS、HO-1蛋白表达及SOD水平降低(P<0.01)。与模型组和安慰剂组比较,治疗组Keap1m RNA及蛋白表达、MDA水平降低(P<0.01),Nrf2 m RNA、Nrf2、Maf、γ-GCS、HO-1蛋白表达及SOD水平升高(P<0.01)。结论加味天雄散能够通过调节弱精子症大鼠精子Keap1-Nrf2-ARE通路及下游Maf、γ-GCS、HO-1蛋白表达,调控氧化应激反应,从而改善精子活力。

Antioxidant Mechanism of Modified Tianxiong Powder on Improving Sperm Motility in Oligospermia Rats Model Based on Keap1-Nrf2-ARE Signaling Pathway

Objective To investigate the antioxidant mechanism of modified Tianxiong powder on regulating sperm motility in oligospermia rats based on the pathway of the Keap1-Nrf2-ARE and the downstream proteins of Maf,γglutamylcysteine synthetase(γ-GCS)and heme oxygenase 1(HO-1).Methods Totally 60 male SD rats were randomly divided into 4 groups ie,sham group,model group,placebo and treatment group.The model rats were gavaged with adenine[100 mg/(100 g·d)]for 10 days,the control group was gavaged with the equal volume of normal saline.The treatment group was gavaged with modified Tianxiong powder,the placebo group was gavaged with the equal volume of normal saline.Keap1 mRNA and Nrf2 mRNA expression of epididymis tissue were tested by RT-PCR.Keap1,Nrf2,Maf,γ-GCS,HO-1 protein expression of epididymis tissue was tested by Western Blot.SOD and MDA in epididymis tissue were tested.Results Compared with control group,Keap1 mRNA and protein expression as well as MDA level increased(P<0.01),Nrf2 mRNA and Nrf2,Maf,γ-GCS,HO-1 protein expression as well as SOD level decreased(P<0.01)in model group.Compared with model group and placebo group,Keap1 mRNA and protein expression as well as MDA level decreased(P<0.01),Nrf2 mRNA and Nrf2,Maf,γ-GCS,HO-1 protein expression as well as SOD level increased(P<0.01)in treatment group(P<0.01).Conclusion Modified Tianxiong powder can regulate oxidative stress and increase sperm motility by regulating Keap1-Nrf2-ARE pathway and downstream proteins of Maf,γ-GCS and HO-1 protein in oligospermia rats.