| Background With the development of society,the age of male childbearing is increasing.How to improve age-related decline in male reproductive function has become one of the research hotspots in recent years.Wuzi Yanzong Wan,a classic traditional Chinese medicine formula,has the properties of kidney nourishing and essence strengthening,and is widely used to treat male infertility with a long history.Blood-testis barrier are injured with aging,resulting in testicular dysfunction,and Wuzi Yanzong Wan effective rejuvenates the aging-related decline of testicular function.However,whether the improvement effects of Wuzi Yanzong Wan on aging-related testicular ineffectiveness is dependent on the restoration of blood-testis barrier function remains unclear.Objective To study the protective effect and mechanism of Wuzi Yanzong Wan on blood-testis barrier injury in natural aging mice.Methods Thirty 2-month-old SPF C57BL/6J male mice were fed with normal diet until15 months old,and then randomly divided into 3 groups: aging model group,low-dose(2g/kg)and high-dose(8 g/kg)of Wuzi Yanzong Wan treated group,with 10 mice in each group.Another ten 1-month-old SPF C57BL/6J male mice were used as adult control group.The adult control group and aging model group were received a normal diet,while the Wuzi Yanzong Wan treated groups were given a diet containing Wuzi Yanzong Wan at two different doses.After 3 months,the mice in the adult control group were 4 months old,while the mice in the aging model group and Wuzi Yanzong Wan treated groups were 18 months old.Mice were weighed,and then sacrificed after anesthesia,and the testis,epididymis and seminal vesicle tissues were quickly removed.The following indicators were detected:(1)The weight of testis,epididymis and seminal vesicles were weighed and their indexes were calculated;(2)The epididymis tissue was taken,the sperm concentration was detected by blood cell count plate,and the sperm viability was detected by eosin Y staining.(3)Hematoxylin-eosin staining(HE)staining was used to observe the morphological changes of testicular,and Image J statistically analyzed the changes of seminferous tubules diameter and seminferous epithelium height in each group.(4)Testicular Sertoli cells were labeled with Sox9 by immunofluorescence,and the number of Sertoli cells in each group was counted(5)The ultrastructural changes of Sertoli cell tight junction,autophagosomes and mitochondria were observed by transmission electron microscopy.(6)The expression of tight junction related proteins(ZO-1,Occludin and Claudin11),ectoplasmic speialization-related proteins(N-Cadherin,E-Cadherin and β-Catenin),gap junction-related proteins(Cx43)and cytoskeletal proteins(Vimentin)were detected by Western blot;(7)The expression and localization of ZO-1 and β-Catenin in mice testis were detected by immunofluorescence.(8)The protein expression and localization of autophagy related proteins(LC3B,p62 and Atg5)in mice sertoli cells were detected by immunofluorescence method.(9)The expression levels of autophagy related proteins(Beclin1,Atg5,LC3Ⅱ,LC3Ⅰ and p62)in testicular of mice were detected by Western blot and the ratio of LC3Ⅱ to LC3Ⅰ was calculated.(10)The protein expression and localization of p-AKT and p-mTOR in testicular sertoli cells were detected by immunofluorescence.(11)The expression of p-PI3 K,PI3K,p-AKT,AKT,p-mTOR and mTOR in testis of mice were detected by Western blot.(12)The expression and localization of Raptor,an important component of mTORC1 in mice testicular sertoli cells,p-p70 S6 K,p-rps6 and Rictor,an important component of mTORC2,were detected by immunofluorescence.(13)The expression of Raptor,p-rps6 and Rictor in testis of mice were detected by Western blot.Results(1)Wuzi Yanzong Wan significantly increased testis weight,testis index,epididymal index,seminal vesicle weight and seminal vesicle index of aging mice,but had no significant effect on the epididymal weight.(2)Wuzi Yanzong Wan can significantly increase the sperm concentration and sperm viability of aging mice.(3)HE staining showed that Wuzi Yanzong Wan could significantly improve the atrophy of spermatogenic tubules,reduce the shedding of spermatogenic cells,and restore the order of cell arrangement in testis.The statistical results of Image J software showed that Wuzi Yanzong Wan had no significant effect on seminferous tubules diameter,but could significantly increase seminferous epithelium height in testicular tissue.(4)Immunofluorescence results showed that Wuzi Yanzong Wan significantly increased the number of Sertoli cells in the testis of aging mice.(5)Transmission electron microscopy results showed that Wuzi Yanzong Wan significantly restored the tight junction damage of Sertoli cells in the testis of aging mice,increased autophagosome,restored mitochondrial morphology,and reduced vacuoles in the tissue.(6)Western blot results showed that Wuzi Yanzong Wan significantly up-regulated the expression of tight junction-related proteins(ZO-1 and Claudin11),ectoplasmic speialization-related proteins(N-Cadherin,E-Cadherin and β-Catenin),and the gap junction-related protein Cx43,while it had no significant effect on the expression of tight junction-related protein Occludin and cytoskeletal protein Vimentin.(7)Immunofluorescence results showed that Wuzi Yanzong Wan significantly up-regulated the expression levels of ZO-1 and β-Catenin in the testis of aging mice,without affecting their expression location.(8)Immunofluorescence results showed that Wuzi Yanzong Wan significantly up-regulated the expression of LC3 B and Atg5 in the Sertoli cells of aging mice,while down-regulated the expression of p62.(9)Western blot results showed that Wuzi Yanzong Wan significantly up-regulated the expression of Beclin1 in the testicular tissue of aging mice,without affecting the expression of Atg5,p62,and the ratio of LC3Ⅱ/LC3Ⅰ in the testicular of aging mice.(10)Immunofluorescence results showed that Wuzi Yanzong Wan significantly down-regulated the expression of p-AKT and p-mTOR in the Sertoli cells of aging mice.(11)Western blot results showed that Wuzi Yanzong Wan significantly down-regulated the ratio of p-PI3K/PI3 K,and had no significant effect on the ratio of p-AKT/AKT and p-mTOR/mTOR in the testicular of aging mice.(12)Immunofluorescence results showed that Wuzi Yanzong Wan significantly down-regulated the expression of Raptor,p-p70 S6 K,and p-rps6 proteins,and up-regulated the expression of Rictor protein in the Sertoli cells of aging mice.(13)Western blot results showed that Wuzi Yanzong Wan had no significant changes in the expression of Raptor,Rictor,and p-rps6 proteins in the testicular of aging mice.Conclusion Wuzi Yanzong Wan improves the injury of blood-testis barrier and aging-related testicular ineffectiveness by restoring AKT/mTOR-mediated autophagy and mTORC1-mTROC2 balance in Sertoli cells during aging. |
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