百合育子方对少弱精子症大鼠附睾组织CFTR、AQP、ZIP离子通道和氧化应激的影响

Effect of Baihe Yuzi Prescription on CFTR,AQP,and ZIP Ion Channels and Oxidative Stress in Epididymis of Oligoasthenozoospermia Rats

目的:观察百合育子方(BYP)对少弱精子症(OAS)大鼠附睾囊性纤维化跨膜转运调节蛋白(CFTR)、水通道蛋白(AQP)、锌转运蛋白(ZIP)表达和附睾氧化应激的影响,探讨其干预OAS的作用机制。方法:35只大鼠适应性饲养1周后,随机选取7只作为正常组,28只大鼠采用雷公藤多苷30 mg·kg^(-1)造模,4周后随机分成模型组、左卡尼汀组和BYP低、高剂量组,每组7只。正常组和模型组给予等量生理盐水,左卡尼汀组给予左卡尼汀100 mg·kg^(-1),BYP低、高剂量组分别给予BYP 6.3、12.6 g·kg^(-1)灌胃,每日1次,连续4周。实验结束后检测大鼠精子计数和活力,苏木素-伊红(HE)染色观察附睾组织病理结构,蛋白免疫印迹法(Western blot)检测CFTR、AQP9、AQP3、ZIP8、ZIP12等蛋白的表达,酶联免疫吸附测定法(ELISA)检测附睾组织α-糖苷酶(α-GC)、唾液酸(SA)、肉毒碱、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)的含量,用电感耦合等离子体质谱仪测定附睾组织锌含量,锌离子探针检测游离锌离子含量。结果:与正常组比较,模型组大鼠精子计数和活力降低(P<0.05),附睾组织结构紊乱;附睾组织α-GC、肉毒碱含量降低(P<0.05),MDA水平升高,SOD,GSH-Px和锌水平下调(P<0.05);附睾头部和尾部CFTR、ZIP12表达下调,AQP3表达上调,附睾尾部ZIP8表达上调(P<0.05);与模型组比较,BYP可明显改善精子计数和活力,恢复OAS大鼠附睾的组织结构,增加组织α-GC、肉毒碱含量(P<0.05);上调附睾头部和尾部CFTR、ZIP12表达,降低附睾尾部ZIP8和附睾头部AQP3的表达(P<0.05);提高附睾组织SOD、GSH-Px水平和总锌含量,下调MDA水平(P<0.05)。结论:百合育子方可能通过调节OAS大鼠附睾CFTR、AQP3、ZIP12离子通道表达和局部抗氧化机制来恢复其组织结构和功能,改善其精液质量。

Objective:To observe the effects of Baihe Yuzi prescription (BYP) on the cystic fibrosis transmembrane conductance regulator (CFTR),aquaporin (AQP),zinc/iron-regulated transporter-like protein(ZIP) and local oxidative stress in epididymis of oligoasthenozoospermia (OAS) rats,and to explore the mechanism of its intervention in OAS.Method:After 35 rats were acclimatized for 1 week,7 rats were randomly selected as the normal group,and the remaining 28 rats were given tripterygium glycosides (TG) 30 mg·kg^(-1).After 4 weeks of modeling,they were randomly divided into 4 groups:model group,BYP low-dose group (LBYP),BYP high-dose group (HBYP) and levocarnitine group,with 7 rats in each group.The rats in the normal group and model group were given normal saline at the same dosage.The levocarnitine group rats were given L-carnitine oral liquid (100 mg·kg^(-1)) by gavage.The LBYP group rats were given BYP 6.3 g·kg^(-1),and the HBYP group rats were given BYP 12.6 g·kg^(-1) by gavage once a day for consecutive 4 weeks.After the end of the intervention,sperm count and motility of all rats were detected,the histopathological structure of epididymis was observed by hematoxylin-eosin (HE) staining,and the expressions of CFTR,AQP9,AQP3,ZIP8,ZIP12 and other proteins were detected by Western blot.The contents of α-glycosidase (α-GC),sialic acid (SA),carnitine,superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and malondialdehyde(MDA) were detected by enzyme-linked immunosorbent assay (ELISA).Total zinc content was measured using an inductively coupled plasma mass spectrometer.Free zinc ion content was detected by zinc ion probes.Result:Compared with those in the normal group,the sperm count and motility of rats were decreased and the epididymal structure was disordered in the model group.The contents of α-GC and carnitine were decreased in epididymis (P<0.05).MDA levels were increased,while SOD,GSH-Px and zinc levels were decreased (P<0.05).The expressions of CFTR and ZIP12 in the head and cauda of the epididymis were down-regulated,and AQP3 expression was up-regulated.The expression of ZIP8 in the cauda epididymis was up-regulated (P<0.05).Compared with the model group,BYP can significantly improve the sperm count and motility,the epididymal structure of OAS rats and the levels of α-GC and carnitine (P<0.05).The expressions of CFTR and ZIP12 in the head and cauda of the epididymis were up-regulated,while the expressions of ZIP8 in the cauda epididymis and AQP3 in the head of the epididymis were decreased (P<0.05).The SOD and GSH-Px levels and total zinc content in epididymis were increased,and the MDA levels were decreased (P<0.05).Conclusion:BYP may improve the sperm quality and repair epididymal tissue structure and function of OAS rats,by regulating the expressions of CFTR,AQP3,and ZIP12 ion channels and local antioxidant mechanism.