| Objective To explore the protective effect and mechanism of Wuzi yanzong prescription(WZ)on testicular reproductive dysfunction through regulating the proliferation and differentiation of spermatogonial stem cells(SSCs)in natural aging rats.This study will provide a new idea for preventing decline in testicular reproductive dysfunction with aging.Methods The 18-month old male SD rats were randomly divided into aging model group,WZ low,middle and high dose group,with 10 rats in each group.Ten 2-month old rats were used as young control group.WZ low,middle and high dose groups were given by gastric gavage of Wuzi yanzong prescription 0.4 g/kg,0.8 g/kg and 1.6 g/kg,respectively.Young control group and aging model group were given normal saline and withdrawal 2 days a week,lasting for 4 months.(1)The rats were sacrificed after the final treatment of WZ and then weighed body mass,and the testis were obtained to calculate testicular weight and testicular index;(2)The testicular tissue morphology was observed using HE staining;(3)The microstructure of Sertoli cells was observed by electron microscopy;(4)The levels of testosterone(T)in testis of rats were detected by ELISA;(5)The activity of SOD,GSH and the levels of MDA in testis were detected by biochemical assay;(6)The numbers of Sertoli cells were detected by immunofluorescence;(7)The relative m RNA and protein expression levels of GDNF,PLZF,BMP4 and SCF in testicular tissue were detected by Real-time quantitative PCR and Western Blot;(8)The numbers of spermatogonial stem cells and the expression levels of PCNA were detected by immunofluorescence assay;(9)The expression and localization of p-JAK2/JAK2,p-STAT3/STAT3 signaling pathway proteins were detected by Western Blot and immunofluorescence;(10)The c-kit expression and localization of spermatogonial stem cell differentiation marker was used by immunofluorescence;(11)The expression and localization of spermatagonial stem cell differentiation related signaling pathway proteins PI3 K,Akt and p-Akt were detected by Western Blot and immunofluorescence.Results(1)Compared with young control group,the testicular weight,testicular index and T levels in aging model group were significantly decreased.However,WZ low,middle and high dose groups significantly increased testicular weight,testicular index and T levels.(2)Compared with young control group,testicular tissues from aging model group showed seminiferous tubules arraged loosening,sermatogenic cells disordering and partly shedding,and germ cells numbers reducing.Conversely,treatment with WZ obviously improved the structure of testis and restored spermatogenic cells numbers.(3)The results of biochemical tests showed that the activity of SOD and GSH in aging model group significantly decreased,the content of MDA significantly increased compared with young control group.Conversely,the activity of SOD and GSH increased significantly,and MDA content decreased after WZ treatment.(4)The results of electron microscopy showed that testicular Sertoli cells in young control group were intact with abundant endoplasmic reticulum and mitochondria.With advancing age,endoplasmic reticulum was swollen,and mitochondria appeared to be chaotic and vacuolated.The WZ significantly improved the ultrastructure of the Sertoli cells.(5)Immunofluorescence results showed no significant difference in the number of Sertoli cells in each group.(6)The results of RT-q PCR and Western Blot showed that the expressions of GDNF,PLZF,BMP4 and SCF in testis of aging model group were significantly lower than that of young control group.The expression levels of GDNF,PLZF,BMP4 and SCF were significantly increased after the treatment with WZ.(7)Immunofluorescence results showed that compared with young control group,the number of spermatogenic stem cells and PCNA expression decreased in aging model group.After WZ treatment,the numbers of spermatogenic stem cells were increased and PCNA expression was up-regulated.(8)Western Blot and immunofluorescence results showed that the spermatogonial stem cell proliferation associated signaling pathway proteins p-JAK2,p-STAT3 were reduced,and after WZ treatment,the expression of p-JAK2,p-STAT3 were significantly increased.(9)Immunofluorescence results showed that the expression of c-kit,a differentiation-related protein of spermatogonial stem cells,was significantly reduced in aging model group,and the expression of c-kit was significantly increased after WZ treatment.(10)Western Blot and immunorescence results showed that WZ significantly increased the expression of PI3 K and p-Akt in spermatogonial stem cell differentiation-related signaling pathway proteins.Conclusion WZ effectively improve the decline of testicular function caused by aging,and its mechanism may be related to enhancing the secretion function of Sertoli cells,improving the niche of spermatogonial stem cells and promoting the proliferation and differentiation of spermatogonial stem cells in aging rats. |
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