| Objective:This study adopts D-galactose(D-gal)replication mice senescence model,to investigate the protective effect of ASP on testis of D-gal induced senescence mice,in order to explain whether ASP can regulate the senescence of testis cells by inhibiting the activation of p53-p21-pRb signaling pathway,and to provide experimental and theoretical basis for exploring a new method for delaying the senescence of male reproductive organs.It provides theoretical and experimental basis for elucidating new methods of angelica to delay the senescence of male reproductive organs.Methods:1.Establishment of aging mice model and group administration of D-gal: Forty male C57BL/6J mice were randomly divided into the normal,ASP normal,aging model and ASP aging model groups,with 10 mice in each group.The aging model group mice were subcutaneously injected with D-gal(120mg/kg,qd×42);the normal group mice were subcutaneouslyinjected with saline of the same dose at the same time;the ASP normal group mice were subcutaneously injected with the same amount of saline,qd×15,and following intraperitoneally injected with ASP(140mg/kg,qd×27);the ASP aging model group were the same as the aging model group,from the 16 th day of establishment of the aging model group on,the mice were subcutaneously injected with the same dose and the same time of ASP.During the modeling period,the biological changes of aging of mice in each group were observed,and the relevant indicators were detected at the second day after the model replication.2.Peripheral blood was collected from the inner canthus vein and the content of serum testosterone was measured.3.The testes were sampled and testicular index determined.4.The histopathological morphology of testis was observed by paraffin section and HE staining.5.Frozen sections of testicular tissue were prepared,the aging of testicular cells was detected by SA-?-Gal stain.6.The tissue homogenate of testes was prepared,the activity of superoxide dismutase(SOD)was evaluated by enzymatic method,the total antioxidant capacity(T-AOC)was detected by ABTS,the content of malondialdehyde(MDA)was measured by thiobarbituric acid method.7.The expression of aging-related proteins P53 and P21 was detected by Western blotting.Results:1.The mice were continuously injected with D-gal subcutaneously for42 days,and their hair color was gradually dull,easy to fall off,poor skin elasticity,listlessness,lethargy,lethargy,eating and drinking were significantly reduced,and their body mass increased slowly,showing obvious signs of natural aging.However,no obvious signs were found in the control group and the ASP aging model group.2.Serum testosterone results showed that the serum testosterone level of mice in the aging model group was significantly decreased,compared with the aging model group,the serum testosterone level of ASP aging model group was not significantly decreased.3.There was no significant difference in testicular wet weight and testicular organ index between the groups.4.Histopathological observations showed that the aging model mice testicular tissue structural damage is apparent,born of the seminiferous tubule cells layer decreases,sperm cells appeared degeneration,leydig cells decreased significantly,compared with aging model group,the ASP aging model group testicular histology structure damage significantly reduce,sperm cells degeneration and leydig cells reduce are not obvious.5.Aging staining results showed that the density of positive cells with SA-?-gal staining was significantly increased in the aging model group.Compared with the aging model group,the density of positive cells withSA-?-gal staining was decreased in the ASP aging model group.6.Oxidative and antioxidant results showed that SOD activity and T-AOC in homogenate of testicular tissue of mice in aging model group were significantly decreased,while MDA content was significantly increased.Compared with aging model group,SOD activity and T-AOC in ASP aging model group were increased,while MDA content was decreased.7.Western blotting results showed that the expressions of P53 and P21 protein in the aging model group were significantly increased,and the expressions of P53 and P21 protein in the ASP aging model group were significantly decreased compared with the aging model group.Conclusion:1.Aging mice constructed with D-gal can cause damage to testicular structure and function,showing biological manifestations of aging.2.ASP has antagonistic effect on D-gal,and has potencial protective effect on testes of mice.3.ASP antagonistic effect of senescence agent on testicular cells by enhancing antioxidant capacity and inhibiting activation of p53-p21-pRb signaling pathway. |
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