| Male infertility is a common reproductive health issue,affecting about 15%of couples globally.oligoasthenozoospermia(OAT)is one of the main causes of male infertility,characterized by reduced sperm count,decreased motility,and abnormal morphology.In recent years,male infertility has become an increasingly urgent problem,imposing a heavy burden on many families and societies.However,the current methods for treating male infertility are still limited,especially for OAT.Traditional Chinese medicine(TCM)has a long history and rich theoretical system in the treatment of male infertility,and its effectiveness has been increasingly demonstrated in clinical studies.Huangjing Zanyu Capsule(HJZY)was developed based on the core pathogenesis of OAT proposed by Academician Qi Wang,which involves kidney deficiency,dampness-heat,blood stasis,and toxin.The treatment principles of HJZY are "tonifying the kidney and nourishing essence,clearing heat and promoting diuresis,and activating blood circulation and resolving stasis." It is the first new TCM drug approved by China to treat male infertility.Previous studies have found that HJZY can improve sperm mitochondrial membrane potential and function,which are closely related to male infertility.Based on this,we hypothesize that improving mitochondrial function may be a key mechanism of HJZY in treating OAT.In this study,we used animal experiments and network pharmacology to explore the molecular mechanisms of HJZY in treating OAT,focusing on mitochondrial autophagy and mitochondrial biogenesis.We also linked the concept of "qi" in TCM with mitochondria from the perspective of modern biology,explaining the treatment principles of HJZY in treating OAT by tonifying kidney qi and removing qi stagnation,which provides systematic theoretical support for TCM in treating male infertility from the perspective of "qi"Objectives:(1)To clarify the pharmacological effects of HJZY in treating OAT.(2)To explore the regulatory mechanism of HJZY on the interaction between mitochondrial autophagy and biogenesis.(3)To identify the pharmacological substance basis and potential mechanism of Huangjing Zanyu Capsule in treating OATMethods:(1)A rat model of OAT was established,and HJZY were used for intervention,with the positive drug levocarnitine as the control.The effects of HJZY on sperm density,sperm motility,reproductive hormone levels,testicular histology,germinal epithelial cells,and mitochondrial ultrastructure in rats with OAT were evaluated by semen analysis,HE staining,and transmission electron microscopy observation,among other methods.(2)Based on the concept of "qi" in traditional Chinese medicine and the relevance of mitochondria in modern biology,the PINK 1/Parkin pathway of mitochondrial autophagy and the PGC-1α/NRF1 pathway of mitochondrial biogenesis were investigated as entry points.Key signaling pathway proteins including PINK1,Parkin,TBK1,LC3,Sirtl,PGC-1α,NRF1,and their crosstalk factor AMPK were detected by molecular biology methods such as PCR,Western blot,and IHC.(3)Network pharmacology was used to construct a "drug-ingredient-target-pathwaydisease" network topology diagram from the perspectives of PPI,GO,and KEGG enrichment analyses.The core components of HJZY and key therapeutic targets were predicted using molecular docking methods.Results:(1)HJZY effectively counteracted the reproductive toxicity caused by cyclophosphamide,and increased sperm density and motility.The specific effects included regulation of reproductive hormone levels,improvement of mitochondrial morphology in reproductive cells,and restoration of the originally atrophied supporting cells to a fuller state.(2)HJZY could activate the PINK1/Parkin and PGC-1α/NRF1 pathways by promoting AMPK phosphorylation,and upregulated the expression levels of key proteins such as PINK1,Parkin,PGC-1α,and NRF1,achieving bidirectional regulation of mitochondrial autophagy and biogenesis.(3)The components of HJZY that entered the bloodstream were involved in multiple targets and signaling pathways related to the treatment of OAT according to PPI,GO,and KEGG enrichment analyses.Among them,isoquercetin may be the core component of HJZY for the treatment of OAT.It can interact with multiple key targets,especially AR,ESR1,and PARP1,which play critical roles in regulating reproductive hormones,promoting reproductive function,and improving mitochondrial function.Conclusion and Significance:(1)HJZY effectively improve sperm density and motility in rats with OAT.Its mechanism of action involves regulation of reproductive hormone levels,restoration of the intratubular environment of spermatogenesis,and promotion of spermatocyte maturation.These results indicate that HJZY have a repairing effect on the entire reproductive system,rather than being limited to a specific site or region.This study provides experimental support for the clinical application of HJZY in the treatment of OAT.(2)HJZY regulate the PINK1/Parkin and PGC-1α/NRF1 pathways through the key crosstalk factor AMPK,rebalancing the mechanisms and achieving dual regulation of mitochondrial quality and quantity to avoid disruption of mitochondrial homeostasis due to dysfunction of one pathway.This study explains the mechanism of action of HJZY in treating OAT from a modern biological perspective.It not only promotes the integration of traditional Chinese medicine theory and modern medicine,providing new perspectives and theoretical foundations for the study of traditional Chinese medicine in the treatment of male infertility,but also provides new ideas for solving the problem of male infertility.(3)Through network pharmacology and molecular docking analysis,it was found that the effective active ingredients of HJZY are closely related to male reproductive-related biological pathways,including regulation of reproductive hormones,improvement of mitochondrial function,promotion of sperm generation,development,and maturation.These findings are consistent with the results of experiments one and two,revealing that HJZY have the advantage of overall regulation and multiple targets in the treatment of OAT.This lays a foundation for further exploring the potential of traditional Chinese medicine in the treatment of male infertility and also contributes to the modernization of traditional Chinese medicine. |
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