基于解毒养精法探讨仙方活命饮提升弱精子症小鼠精子质量的效应机制

Xianfang Huoming Decoction improves sperm quality in asthenospermia mice:An investigation based on the detoxification and sperm-nourishing method

目的:探讨基于解毒养精法探讨仙方活命饮提升弱精子症小鼠精子质量的效应机制。方法:选取30只SPF级BALB/c雄性小鼠(8周),根据随机数表法分为6组,A组:空白对照组,B组:模型组,C组:仙方活命饮低剂量组,D组:仙方活命饮中剂量组,E组:仙方活命饮高剂量组,F组:左卡尼汀+维生素E组。根据人与小鼠用药量换算方法计算给药量,B~F组小鼠每周腹腔注射1次甲氨蝶呤,每次0.5 mg/kg,共注射8周,造成热毒型弱精子症小鼠模型;A组注射等量生理盐水,共注射8周。第9周开始各组分别进行药物治疗,A、B组:PBS 0.1 ml/d,C~E组:7.13、14.2、28.52 g/(kg·d)仙方活命饮,F组:左卡尼汀+维生素E(30∶1)0.55 g/(kg·d),共4周。12周后各组小鼠经过相对应处理后,将双侧附睾剪碎制备精子悬浊液,观察精子总数与活动精子数。分离睾丸组织进行荧光染色、RT-PCR、Western印迹检测Nrf-2/HO-1信号通路相关mRNA与蛋白表达。结果:与模型组相比,仙方活命饮低、中、高剂量组能显著改善弱精子症模型小鼠精子活率[(22.89±14.96)%)vs(47.98±5.12)%、(48.53±6.03)%、(49.31±6.24)%,P<0.01)]与浓度[(22.36±16.02)×10^(6)/ml vs(39.04±4.50)×10^(6)/ml、(40.76±6.57)×10^(6)/ml、(41.04±8.39)×10^(6)/ml,P<0.01],降低睾丸组织ROS水平[(16.82±4.74)%)vs(12.08±3.26)%、(10.77±2.21)%、(9.56±2.08)%,P<0.01)],上调睾丸组织中Nrf-2、HO-1 mRN与蛋白表达水平(P<0.05或P<0.01)。结论:仙方活命饮可能通过上调Nrf-2/HO-1信号通路mRN与蛋白表达,抑制氧化应激的发生,为临床使用仙方活命饮治疗弱精子症提供了理论依据。

Objective:To investigate the mechanism of Xianfang Huoming Decoction(XHD)improving sperm motility in mice with asthenospermia(AS).Methods:Thirty normal BALB/c mice were randomly divided into six groups,blank control,AS model control,low-dose XHD,medium-dose XHD,high-dose XHD and levocarnitine+vitamin E(LC+VE).The AS model was established in the latter five groups by injection of methotrexate at 0.5 mg/kg once a week,and the mice in the blank control group were injected with the same volume of normal saline,all for 8 weeks.From the ninth week,the animals in the blank control and AS model control groups were treated with PBS at 0.1 ml/d,those in the low-,medium-and high-dose XHD groups with XHD at 7.13,14,2 and 28.52 g/kg/d respectively,and those in the LC+VE group with LC+VE(30:1)at 0.55 g/kg/d,all for 4 weeks.Then,the bilateral epididymides were harvested from all the mice for preparation of a sperm suspension and observation of the total numbers of sperm and motile sperm.The testis tissues were obtained for to determination of the expressions of Nrf-2-and HO-1-related mRNA and proteins by fluorescence staining,RT-PCR and Western blot.Results:Compared with the AS model controls,the mice treated with low-,medium-and high-dose XHD showed dramatically increased sperm concentration([22.36±16.02]vs[39.04±4.50],[40.76±6.57]and[41.04±8.39]×10^(6)/ml,P<0.01)and motility([22.89±14.96]%vs[47.98±4.74]%,[48.53±6.03]%and[49.31±6.24]%,P<0.01),decreased level of reactive oxygen species(ROS)([16.82±14.96]%vs[12.08±3.26]%,[10.77±2.21]%and[9.56±2.08]%,P<0.01),and up-regulated expressions of Nrf-2-and HO-1-related mRNA and proteins in the testis tissue(P<0.05 or P<0.01).Conclusion:Xianfang Huoming Decoction inhibits the development of oxidative stress by up-regulating the expressions of Nrf-2-and HO-1-related mRNA and proteins in the testis tissue,which has provided theoretical evidence for its clinical application in the treatment of asthenospermia.