The Effect And Mechanism Of Wuzi Yanzong Pill On Sertoli Cells And Blood Testis Barrier

1 Background and ObjectiveHeterosexual couples suffering from infertility issues increased year by year;male infertility accounts for roughly 40%of all infertility cases.Roughly 50%male infertility is caused by known etiologies such as varicocele,hypogonadism,testicμlar hypoplasia or cryptorchidism.The elevated temperature of the scrotum has been frequently implicated in the main pathogenesis of male infertilities,including occupation,ifestyle,posture,clothing,cryptorchidism,varicocele and chronic fever.The etiology and pathogenesis of these roughly 50%male infertility disorders are still not fully understood and these cases are referred to as idiopathic infertility.Wuzi Yanzong Pill(WYP)is widely applied to the treatment of Male infertility and it can significantly improve male reproductive ability.The main research direction of WYP was confined only to sperm function in adult male infertility and the changes of germ cells in the seminiferous tubule.Sertoli cells(SC)are indispensable for germ cell maturation and differentiation.and the blood testis barrier(BTB),which is created between adjacent SCs,plays a key role in the production of spermatozoa.Nevertheless,previous studies seldom explored the effects and it’s mechanism of WYP on the SCs based on observation of changes of SC and the BTB.Our study is to investigate the effects and its potential mechanism of WYP on the SC in juvenile rats and adult rats by local heating.So as to provide new themodern basis and new ideas for treatment of male infertility.2 Research methods:(1)SCs derived from Testes of male 20-day-old and 60-day-old SD rats were isolated and cultured in the complete medium(DMEM/F12 culture medium,10%fetal bovine serum(FBS),1%penicillin streptomycin).(2)Testicle Sertoli cells of male 20-day-old SD rats were randomly allotted into four experimental groups:normal group,low-dose WYP group(terminal concentration:5mg/ml),medium-dose WYP group(terminal concentration:10mg/ml),and high-dose WYP group(terminal concentration:15mg/ml).The SCs were cultured in the complete medium with drugs for 0h,4h,8h,16h,24h,and 32h.Testicle sertoli cells of male 60-day-old SD rats were randomly allotted into five experimental groups:normal group,model group,low-dose WYP group(terminal concentration:5mg/ml),medium-dose WYP group(terminal concentration:10mg/ml),and high-dose WYP group(terminal concentration:15mg/ml).And the SCs of model group,low-dose WYP group,medium-dose WYP group and high-dose WYP group were immersed in a thermostatically controlled water bath at 43℃ for 15 min.(3)MTT assay was performed to evaluate the effect of various concentrations of WYP on testicle sertoli cells of male 20-day-old and 60-day-old SD rats different hours.(4)Using immunohistochemistry to study the expression change of the proliferating cell antigen Ki67 on testicle sertoli cells of male 20-day-old and 60-day-old SD rats and the expression of AR proteins on testicle sertoli cells of male 60-day-old SD rats.(5)Western blot was used to detect the expression of Akt and p-Akt proteins on testicle sertoli cells of male 20-day-old SD rats.And Western blot was used to detect the expression of Akt,p-Akt,AR,CK-18,ZO-1,occludin proteins on testicle sertoli cells of male 60-day-old SD rats.3 Ruslts(1)MTT assay results:The testicle sertoli cells of male 20-day-old SD rats in WYP groups were significantly higher than those in the normal group(P<0.05),and the number of testicle sertoli cells was significantly increased with increasing dose of WYP.With the prolongation of WYP drug treating time,the growth of SCs reached the peak at 24h.The testicle sertoli cells of male 60-day-old SD rats in WYP groups were significantly higher than those in the normal group(P<0.05).And the number of testicle sertoli cells was significantly increased with increasing dose of WYP.(2)Immunohistochemistry results:The number of Ki67-positive cells in testicle sertoli cells of male 20-day-old SD rats in WYP groups were obviously higher than the normal group(P<0.05).And the number of Ki67-positive cells was significantly increased with increasing dose of WYP.The number of Ki67-positive cells in testicle sertoli cells of male 60-day-old SD rats in the model group was obviously lower than those in the normal group(P<0.05)and the number of Ki67-positive cells of WYP groups were obviously higher than the model group(P<0.05).The number of AR-positive cells in testicle sertoli cells of male 60-day-old SD rats in the model group was obviously lower the normal group(P<0.05)and the number of AR-positive cells in the WYP groups were obviously higher than those in the model group(P<0.05).(3)Western blot results:the expression of p-Akt on testicle sertoli cells of male 20-day-old SD rats in WYP groups were significantly higher than it in the normal group(P<0.05)and the expression of p-Akt was significantly increased with increasing dose of WYP.The expression of p-Akt and AR on testicle sertoli cells of male 60-day-old SD rats in the model group was obviously lower the normal group(P<0.05).And the expression of p-Akt and AR on testicle sertoli cells in the medium-dose WYP group and the high-dose WYP group were obviously higher than it in the model group(P<0.05).The expression of CK-18 on testicle sertoli cells of male 60-day-old SD rats in the model group was obviously higher the normal group(P<0.05).And the expression of CK-18 on testicle sertoli cells in WYP groups were obviously lower than it in the model group(P<0.05).The expression of ZO-1 and occludin on testicle sertoli cells of male 60-day-old SD rats in the model group was obviously lower the normal group(P<0.05).And the expression of ZO-1 and occludin on testicle sertoli cells in WYP groups were obviously higher than it in the model group(P<0.05)4 Conclusions1.WYP could stimulate proliferation of testicle sertoli cells of male immature rats through activating the PI3K/Akt signaling pathway induced Phosphorylation of AKT.WYP could stimulate proliferation of testicle sertoli cells of male immature rats and the optimum reaction time to stimulate proliferation of testicle sertoli cells of male immature rats was 24h.2.WYP can restore the activity of testicle sertoli cells of male mature rat’s proliferation after heat treatment.And WYP could stimulate proliferation of testicle sertoli cells of male mature rats.3.WYP can stimulate proliferation of testicle sertoli cells of male immature rats through inducing the up regulation of AR expression,and activating the PI3K/Akt signaling pathway.WYP can effectively prevent from SCs apoptosis and prevent the differentiation and maturation of SCs from dedifferentiating.And WYP can restore the function of BTB after heat treatment by raising the expression of ZO-1 and occluding.Furthermore,WYP Can preventing from spermatogenic dysfunction.