Androgen Receptor Phosphorylation Induced By Bisphenol A And Kidney-Deficiency Causes Testicular Cell Apoptosis And The Protective Effect Of Icariin

【Backgound】BPA（Bisphenol A,BPA）is one of the typical environmental Endocrine disruptors（EDCs）,and its toxicity can especially lead to male reproductive dysfunction.However,its pathogenesis and treatment are not clear at present.It was proved that BPA causes male infertility by lowering androgen levels and causing apoptosis.In the male reproductive organs,BPA causes the most serious to the testicles.BPA not only directly induces the phosphorylation of Androgen receptor（AR）,but also induces Akt phosphorylation,which is a phosphorylation kinase at Ser791/792 of AR,leading to cell apoptosis.Previous researchs have also shown that infertlility mice caused by BPA or Kidney-deficiency are correlated in behavioristics,T and E₂ levels,sperm quality and quantity,fertility rate and DEPs（Differentially expressed proteins）.Therefore,in order to further clarify the correlation between these two models and therapeutic effect of Chinese herbs,this study located on three aspects:testicular cell apoptosis caused by BPA and Kidney-deficiency,the roles of AR and Akt phosphorylation in testicular cell apoptosis and the protective effect of icariin（ICA）.These provide a reliable basis for the prevention and treatment of male infertility caused by EDCs.【Methods】1.Animal models of BPA exposure and Kidney-deficiency were established,as well as ICA treatment models.Control group:intraperitoneal injection of normal saline 0.2ml per day for 7 consecutive days;Low dose BPA exposed model:intraperitoneal injection of BPA solution 1mg/kg per day for 7 consecutive days;Medium dose BPA exposed model:intraperitoneal injection of BPA solution 10mg/kg per day for 7 consecutive days;High dose BPA exposed model:intraperitoneal injection of BPA solution 100mg/kg per day for7 consecutive days;Kidney-Yang deficiency model:intraperitoneal injection of Hydrocortisone at 25mg/kg per day for 10 consecutive days.Kidney-Yin deficiency model:intraperitoneal injection of Hydrocortisone at 50mg/kg per day for 7 consecutive days.After modeling,mice were intragastric administration of 50mg/kg,100mg/kg and200mg/kg for 14 consecutive days.2.Testosterone and estradiol in serum of mice were detected by ELISA in BPA exposed（1mg/kg,10mg/kg,100mg/kg）and Kidney-deficiency models,and hormone changes were observed after ICA treatment at different concentrations（50mg/kg,100mg/kg,200mg/kg）.3.HE staining was used to observe the morphological changes in testicular tissues of BPA exposed（1mg/kg,10mg/kg,100mg/kg）and Kidney-deficiency mice models.4.TUNEL was used to observe the apoptosis of testicular cells induced by BPA exposure at different concentrations（1mg/kg,10mg/kg,100mg/kg）and Kidney-deficiency mice models,as well as the inhibition of apoptosis after ICA treatment（50mg/kg,100mg/kg,200mg/kg）.5.Immunohistochemical staining was used to observe the localization and expression levels of AR and p-Akt in testicular tissues of each model group and treatment group.6.Immunofluorescence staining was used to observe the location and expression level of p-AR in testicular tissues of each model group and the treatment group.7.The protein expressions of AR,p-AR,Akt and p-Akt in each model group and treatment group were observed by Westrern Blot.【Results】1.The concentration of BPA was negatively correlated with T and positively correlated with E₂ in the serum of BPA exposed mice.There was no significant statistical difference between Kidney-Yang deficiency group and BPA group（p>0.05）.2.In BPA model groups,the testicular tissue showed different degree of damage,including abnormal distribution of spermatogenic tubules,decreased number of Leydig cells,discontinuous basement membrane of spermatogenic tubules and disordered arrangement of spermatogenic cells.The higher the concentration of BPA was,the more obvious the damage was.3.BPA at the dose of of 10mg/kg and 100mg/kg could significantly induce testicular cell apoptosis（p<0.05）,which mostly occurred in spermatogenic cells near the basement membrane of spermatogenic tubules.The two Kidney-deficiency mice models also showed positive signs of apoptosis in different degrees.Compared with the BPA model group at 100mg/kg,the ratio of apoptosis cells in Kidney-Yang deficiency model group showed no statistical difference（p>0.05）,while that in Kidney-Yin deficiency model group was the opposite（p<0.05）.4.The expression of AR protein in testicular tissue was significantly down-regulated in BPA model groups and Kidney-deficiency model groups（p<0.05）.However,p-Akt was up-regulated only in Leydig cells of BPA models and Kidney-Yang deficiency mice model,which was significantly higher than control group（p<0.05）.5.BPA can phosphorylate AR,and its expression intensity was positively correlated with BPA concentration.p-AR was expressed in testicular spermatogenic cells,and there was no statistical difference between the BPA and the Kidney-Yang deficiency mice models（p>0.05）.6.ICA can up-regulate AR expression,inhibit spermatogenic cell apoptosis mediated by p-AR and p-Akt,and improve testicular histology and sex hormone expression（p<0.05）in models.ICA at 200mg/kg had pro-apoptotic effect on spermatogenic cells.7.Western Blot showed that the expression of testicular AR was down-regulated and p-AR/p-Akt were up-regulated after BPA exposure in animal models（p<0.05）.BPA concentration was negatively correlated with AR expression and positively correlated with p-AR and p-Akt expression.The Kidney-deficiency mice model also showed down-regulation of AR and up-regulation of p-AR and p-Akt（p<0.05）.After ICA treatment,the expression of AR could be up-regulated and the expression of p-AR and p-Akt could be down-regulated,and the best concentration of ICA was 100mg/kg.There was no significant statistical difference in the expression of Akt among model groups and treatment groups（p>0.05）,and the expression of the above proteins was the same as the morphological results.【Conclusions】1.BPA can interfere with the levels of sex hormones in the serum of mice,reduce the concentration of testosterone and increase the concentration of estradiol.With the increase of BPA concentration,its effects increase significantly.In addition,BPA can damage the normal histological morphology of testicular tissue,affect the distribution of spermatogenic tubules,reduce the number of Leydig cells and spermatogenic cells,and cause irreversible damage to testicular tissue.2.BPA at concentrations of 10mg/kg and 100mg/kg can significantly induce spermatogenic cell apoptosis in testis,which mostly occurs near the basement membrane of spermatogenic tubules.Moreover,the apoptotic effect of BPA was similar to that of Kidney-Yang deficiency model,but different from that of Kidney-Yin deficiency model.BPA can significantly down-regulate the expression of AR,and its inducing mechanism of spermatogenic cell apoptosis is phosphorylation of AR and its kinase Akt.3.After intraperitoneal injection of BPA at 100mg/kg for 7d,the animal showed significant sex hormone disorders in serum,testicular tissue damage and cell apoptosis.4.The phenotype of Kidney-Yang deficiency model is significantly different from that of Kidney-Yin deficiency model.In comparison with BPA model,it was found that Kidney-Yang deficiency model was more associated with BPA model in inducing testicular cell apoptosis.This suggests that male infertility caused by BPA may belong to the category of Kidney-Yang deficiency,and the specific similarities and differences between the two need to be further studied.5.ICA can up-regulate AR level,down-regulate p-AR and p-Akt,inhibit the phosphorylation of AR and Akt induced by BPA and Kidney-Yang deficiency,improve the histological morphology of testicular tissue,reduce the ratio of apoptotic cells and increase T expression,which means ICA has a good protective effect on BPA exposed mice as well as infertility caused by Kidney-Yang deficiency.However,the high dose of ICA（200mg/kg）can aggravate the apoptosis of testicular spermatogenic cells,and the mechanism needs to be further studied.At present,it has been confirmed that the optimal concentration of ICA for treating BPA exposure is 100mg/kg.