百合育子方对少弱精子症大鼠睾丸锌稳态、CFTR和Keap1/Nrf2/HO-1信号通路影响

Effect of Baihe Yuzi Formula on Zinc Homeostasis,CFTR and Keap1/Nrf2/HO-1 Signaling Pathway in Oligoasthenospermia Rats

目的观察百合育子方(BYF)对少弱精子症(OAS)大鼠精液参数和睾丸组织结构的影响及机制。方法35只雄性SD大鼠随机选取7只作为正常组,其余28只大鼠予雷公藤多苷进行OAS造模后随机分为模型组、BYF低、高剂量组和左卡尼汀组各7只。BYF低、高剂量组分别予6.3、12.6 g/(kg·d)BYF灌胃,左卡尼汀组予左卡尼汀100 mg/(kg·d)灌胃,正常组和模型组给予等量生理盐水灌胃。检测精液参数;HE染色观察睾丸组织形态;检测血液和睾丸组织锌含量;锌离子探针检测睾丸游离锌离子含量;采用ELISA检测大鼠血清睾酮水平及睾丸组织碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、乙醇脱氢酶(ADH)、超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)含量;Western Blot检测金属反应元件结合转录因子1(MTF1)、锌转运蛋白(ZNT)4、ZnT8、锌铁转运蛋白(Zip)8、Zip12、核因子E2相关因子2(Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Keap1)、血红素氧合酶1(HO-1)、组蛋白脱乙酰基酶2(HDAC2)、囊性纤维化跨膜转运调节蛋白(CFTR)、水通道蛋白(AQP)3和AQP9蛋白表达。结果与正常组比较,模型组睾丸结构紊乱,可见少量精子细胞,游离锌水平降低;模型组精子计数和活力、血清和睾丸组织锌含量、血清睾酮水平、睾丸组织ADH、ALP、LDH、SOD、GSH-Px含量、ZnT4、ZnT8、Zip8、Zip12、MTF1、Keap1、Nrf2、HO-1、HDAC2、CFTR、AQP9蛋白表达降低,睾丸组织MDA含量、AQP3蛋白表达增加(P<0.05)。与模型组比较,干预组睾丸组织有不同程度的恢复,精子细胞数量增多,游离锌离子水平升高;干预组精子计数和活力、睾丸组织锌含量、睾丸组织ALP、LDH、SOD含量、ZnT4、ZnT8、Zip8、Zip12、Nrf2、HO-1、HDAC2蛋白表达升高,睾丸组织MDA含量降低(P<0.05);BYF高剂量组血清锌含量、血清睾酮水平、睾丸组织ADH含量、MTF1、Keap1、CFTR、AQP9表达增加,睾丸组织AQP3表达降低(P<0.05);左卡尼汀组血清锌含量、睾丸组织ADH、GSH-Px含量、KEAP1表达增加,睾丸组织AQP3表达降低(P<0.05)。结论百合育子方可改善OAS大鼠的精液参数和睾丸组织结构,其作用机制可能与调节锌稳态、CFTR/AQP和Keap1/Nrf2/HO-1信号通路,改善氧化应激,提高血清睾酮有关。

Objective To observe the effects and mechanism of Baihe Yuzi Formula(BYF)on semen parameters and testicular tissue structure.Methods Of the 35 male SD rats,7 rats were randomly selected as the normal group,and the remaining 28 rats were randomly divided into model group,BYF low-dose group,BYF high-dose group and leucovorin group after OAS modeling with tretinoin,7 in each group.The BYF low and high dose groups were given 6.3 and 12.6 g·kg-1·d-1 BYF by gavage,the leucovorin group was given leucovorin 100 mg·kg-1·d-1 by gavage,and the normal and model groups were given an equal amount of saline by gavage.Semen parameters was detected;HE staining was used to observe testicular tissue morphology;blood and testicular tissue zinc content were detected;zinc ion probe was used to detect testicular free zinc ion content;serum testosterone levels and testicular tissue alkaline phosphatase(ALP),lactate dehydrogenase(LDH),alcohol dehydrogenase(ADH),superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)levels were measured by ELISA;Western Blot was used to detect metal regulatory transcription factor 1(MTF1),zinc transporter(ZNT)4,ZnT8,zinc-iron transporter(Zip)8,Zip12,Kelchlike ECH-associated protein 1(Keap1),NF-E2-related factor 2(Nrf2),heme oxygenase 1(HO-1),histone deacetylase 2(HDAC2),cystic fibrosis transmembrane conductance regulator(CFTR),aquaporin(AQP)3,and AQP9 protein expression.Results Compared with the normal group,the model group had disturbed testicular structure,a few spermatocytes were seen,and free zinc levels were reduced;sperm count and viability,serum and testicular tissue zinc content,serum testosterone level,testicular tissue ADH,ALP,LDH,SOD,GSH-Px content,ZnT4,ZnT8,Zip8,Zip12,MTF1,Keap1,Nrf2,HO-1,HDAC2,CFTR,and AQP9 protein expression decreased in the model group,testicular tissue MDA content,AQP3 protein expression increased(P<0.05).Compared with the model group,testicular tissues of the intervention group showed different degrees of recovery,increased sperm cell number,and elevated free zinc ion levels;sperm count and viability,testicular tissue zinc content,testicular tissue ALP,LDH,and SOD content,testicular tissue protein expression of ZnT4,ZnT8,Zip8,Zip12,NRF2,HO-1,and HDAC2 increased in the intervention group,and testicular tissue MDA content decreased(P<0.05).Serum zinc content,serum testosterone level,testicular tissue ADH content,MTF1,KEAP1,CFTR,and AQP9 expression increased,testicular tissue AQP3 expression decreased in the BYF high-dose group(P<0.05);while serum zinc content,testicular tissue ADH,GSH-Px content,and KEAP1 expression increased,testicular tissue AQP3 expression decreased in the leucovorin group(P<0.05).Conclusion BYF could effectively improve semen parameters and testicular tissue structure in OAS rats,and its mechanism may be related to regulating zinc homeostasis,CFTR/AQP and Keap1/Nrf2/HO-1 signalling pathways,improving oxidative stress,and increasing serum testosterone.