| Research on male infertility,including oligoasthenozoospermia,is a hot topic in reproductive medicine worldwide.Huangjingzanyu capsule is a common treatment for oligoasthenozoospermia,exploring its pharmacodynamic mechanism increasingly important.We have found that Huangjing Zanyu capsule are effective antiapoptotic agents on previous studies.Huangjing Zanyu capsule may treat oligoasthenozoospermia by inhibiting apoptosis.In this study,a combination of animal and cell experiment experiments was used to investigate the mechanism of the effect of Huangjing Zanyu capsule on oligoasthenozoospermia,and the connotation of the harmony and equilibrium of Yin-Yang in traditional Chinese medicine was discussed from the perspective of modern biology.OBJECTIVES:(1)A rat model of cyclophosphamide-induced oligoasthenozoospermia was established and intervened with Huangjing Zangyu capsule to clarify the efficacy of the Chinese medicine.(2)The molecular biological mechanism of spermatogenesis of Huangjing Zanyu capsule was explored by centering on the Akt/mTOR autophagy pathway and p53 mediated apoptosis pathway,and observed the expression of proteins related to three key autophagy-apoptosis crosstalk nodes,including Akt/MDM2/p53,p53/AMPK/mTOR and Bcl-2/Beclin1.And we explored the significance of autophagy-apoptosis interaction in maintaining cell homeostasis.(3)The spermatogenic cell injury model induced by acrolein was established,and Huangjing Zanyu capsule was used to intervene to determine the modeling and dosing conditions.The effects of Huangjing Zanyu capsule on spermatogenic cell activity and autophagy related organelles were observed.(4)The function and homeostasis repair mechanism of Huangjing Zanyu capsule on spermatogenic cells were explored by centering on three key autophagy-apoptosis crosstalk nodes Akt/MDM2/p53,p53/AMPK/mTOR and Bcl-2/Beclin1,and the reverse verification was conducted to clarify the significance of autophagy-apoptosis interaction on maintaining cell homeostasis.METHODS:(1)Cyclophosphamide was used to construct oligoasthenozoospermia models in SD rats.Huangjing Zanyu capsule was used as intervention,and L-carnitine,a medicine commonly used in clinical treatment of male infertility,was used as positive control medicine for 28 days.After the experiment,the changes of testicular volume,testicular weight,sperm density,sperm activity,PR sperm rate,NP sperm rate,IM sperm rate,VCL,VSL,VAP,LIN,WOB,MAD,ALH,BCF and other indicators were detected.The pathological changes of testicular tissue were observed,and the submicroscopic structure of testicular tissue was observed by transmission electron microscope.The cell cycle of testicular cells was detected by flow cytometry.(2)Western blot combined with IHC was used to detect the expression levels of autophagyassociated proteins(Akt,p-Akt,mTOR,p-mTOR,Raptor,p-Raptor,ULK1,p-ULK1,Beclinl,LC3B and p62),apoptosis-associated proteins(p53,Bcl-2,Bax,Bcl-xL,Bad and Caspase-3)and autophagy-apoptosis crosstalk proteins(MDM2 and AMPK).The apoptosis of testicular cells were observed by TUNEL.(3)Acrolein was used to construct the GC-2 spd spermatogenic cell injury model and intervened with Huangjing Zangyu capsule.The cell viability was measured by CCK8 to select the optimal modeling and dosing concentration and time.(4)Western blot was used to detect key proteins of autophagy-apoptosis crosstalk nodes(Akt,p-Akt,MDM2,p53,AMPK,Raptor,Beclinl and Bcl-2),apoptosis-marker proteins(Bax,Bcl-XL and Bad)and autophagy-marker proteins(LC3B and p62).At the same time,Akt phosphorylation was inhibited with Akt inhibitor Ⅷ,and the expression changes of autophagy-apoptosis crosstalk nodes were observed when p-Akt was inhibited.GFP-LC3B was used to trace the autophagy flow.(5)All final data were statistically analyzed using SPSS20.0.RESULTS:(1)Cyclophosphamide could reduce testicular weight and several sperm parameters in rats.Huangjing Zangyu capsule can effectively improve sperm density,sperm activity,PR sperm rate,VCL,VSL,VAP,MAD,reduce IM sperm rate and BCF,and therefore improve the sperm quantity,sperm quality and the movement parameters of oligoasthenozoospermia rats.In morphology,cyclophosphamide could cause disappearance of spermatocytes and spermatids,and cause Sertoli cells to shrink and form cavities.Huangjing Zanyu capsule can repair the damaged Sertoli cells,restore the normal structure of seminiferous tubule,establish a suitable environment for spermatogenesis,and restore the ordered arrangements of spermatogenic cells at all levels.In ultrastructure,cyclophosphamide can cause abnormal increase of cytoplasmic autophagosomes and lysosomes,decreased the number of mitochondria and made them abnormally assembled,decreased secretion of Golgi apparatus,and hindered acrosome fusion.Huangjingzangyu capsule can effectively improve the quality of mitochondria and secretion efficiency of Golgi apparatus,restore the normal process of meiosis,repair the mitochondrial sheath and the structure of longitudinal column of sperm flagellum,and improve the motor ability of the flagellum.In terms of cell cycle,cyclophosphamide could cause abnormal increase in S and G2 phases of testicular cells.Huangjing Zanyu capsule can increase the number of testicular cells in G1 phase,reduce the number of cells in G2 phase,resist the inhibition of G1 phase caused by cyclophosphamide,and restore the cell cycle.(2)Molecular biological experiment results of testicular cells showed that cyclophosphamide down-regulated the protein expression levels of Akt,p-Akt,p53 and Bax,and up-regulated p-Raptor,ULK1,ULK1/p-ULK1,Beclinl and LC3-Ⅱ.Huangjing Zanyu capsule significantly up-regulated Akt,p-Akt,p-mTOR,Raptor,p62,MDM2,and downregulated ULK1,ULK1/p-ULK1,Beclinl,p53,Bax,AMPK.The result of TUNEL staining showed increased apoptosis after cyclophosphamide modeling,while Huangjing Zanyu capsule could alleviate the apoptosis.(3)IC50 was calculated using CCK8 to analyze the cell inhibition rate under different modeling conditions.It was found that 43 μM acrolein 9 h was the best modeling condition.CCK8 was used to analyze the cell viability under different dosing conditions,and it was found that the optimal dosing conditions of Huangjing Zanyu capsule were 40 μM,80 μM,160 μM for 24 h.Cyclophosphamide can decrease spermatogenic cell viability,increase the number of autophagosomes,decrease the number of mitochondria,and increase the number of lysosomes.Huangjing Zanyu capsule can effectively improve cell viability,reduce the number of autophagosomes,increase the number of mitochondria.There was no significant change in the number of lysosomes before and after intervention.(4)In vitro molecular biological experiments of spermatogenic cells showed that cyclophosphamide could down-regulate p-Akt and AMPK,and up-regulate Beclin1 and LC3Ⅱ.After Huangjing Zanyu capsule intervention,Bcl-2 and P62 were up-regulated,and p-Akt,Bax and Bad were down-regulated.When p-Akt was inhibited by Akt inhibitor Ⅷ,the expression of Beclinl and Bcl-2 was up-regulated and the expression of AMPK was downregulated.The inhibition of p-Akt attenuated the down-regulation of p53 and the up-regulation of MDM2 by Huangjing Zanyu capsule,and enhanced the up-regulation of Bcl-2,Raptor and AMPK.At the same time,we found that Huangjing Zanyu capsule can effectively reduce the number of autolysosome.CONCLUSION:(1)Huangjing Zangyu capsule helps restore the original cellular homeostasis of testicular cells,including repairing the structure of spermatogenic tubules and sperm flagella,improving mitochondrial quality and Golgi secretion efficiency,reconstructing the cellular environment and cell cycle of spermatogenesis,and restoring the meiotic activity of spermatogenic cells,thus improving the quantity,quality and motility of sperm in rats with oligoasthenozoospermia.(2)Huangjing Zanyu capsule can inhibit excessive autophagy and apoptosis in testicular tissue caused by cyclophosphamide.It regulated Akt/mTOR autophagy and p53 apoptosis pathways by regulating key proteins of the autophagy-apoptosis crosstalk node,ensuring that cell homeostasis is not damaged.Among them,Akt/MDM2/p53 is the key crosstalk node of autophagy-apoptosis interaction at this period.(3)Huangjing Zanyu capsule can effectively improve the cell viability and mitochondrial number of spermatogenic cells which was damaged by acrolein,the main metabolic component of cyclophosphamide,and reduce the number of autophagosomes,thus assisting spermatogenic cells to quickly restore their normal homeostasis in vitro.(4)In the early stage of autophagy,Huangjing Zanyu capsule can regulate the autophagy pathway mediated by Akt/mTOR by regulating the key proteins of autophagy-apoptosis crosstalk node,and block the autophagy flow at the same time.Through the above effects,Huangjing Zanyu capsule can control the autophagy degree of spermatogenic cells induced by acrolein within a certain range.Huangjing Zangyu capsule inhibited acrolein-induced spermatogenic cell apoptosis by up-regulating Bcl-2 and down-regulating Bax and Bad.Beclinl/Bcl-2 is the key crosstalk node of autophagy-apoptosis interaction in the early stage of autophagy.(5)Huangjing Zanyu capsule can activate Beclin1/Bcl-2 and Akt/MDM2/p53 crosstalk nodes in the process of autophagy development,and meanwhile regulate the Akt/mTOR pathway and p53 pathway to avoid excessive autophagy and apoptosis.And it can restore and maintain the homeostasis of spermatogenic cell,and restore the normal spermatogenesis.These results might be the evidence about the harmony and equilibrium of Yin-Yang in the treatment of oligoasthenozoospermia in traditional Chinese medicine. |
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