左归丸治疗男性不育症作用机制的网络药理学分析及实验验证OA

Objective To systematically analyze the active components,targets,and molecular mechanisms of Zuogui Pills in the treatment of male infertility using network pharmacology and in vitro experiments.Methods The active components and potential targets of Zuogui Pills were screened using the TCMSP and BATMAN-TCM databases,and male infertility-related disease targets were obtained from the GeneCards,OMIM,and DisGeNET databases.The intersection of drug and disease targets was identified,followed by construction of a protein-protein interaction(PPI)network and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.Single-cell sequencing data from the GEO database were used to validate the cellular localization of core targets.For the in vitro experiments,mouse spermatogonia(GC-1 spg)were divided into a control group,a model group,and a Zuogui Pills group.Flow cytometry was used to detect the apoptosis rate and cell cycle distribution.Western blot was used to detect the protein expression of phosphatidylinositol 3-kinase(PI3K),phosphorylated protein kinase B(p-AKT),and B-cell lymphoma 2(Bcl-2),and RT-qPCR was used to detect the mRNA expression of PI3K,p-AKT,and Bcl-2.Results Network pharmacology analysis identified 71 active components of Zuogui Pills,among which five components,including quercetin and stigmasterol,were identified as core components.A total of 260 drug-disease common targets were identified,and the PPI network indicated that AKT serine/threonine kinase 1(AKT1),tumor necrosis factor(TNF),and interleukin-6(IL-6)were key targets.KEGG enrichment analysis indicated that the phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway was significantly enriched.Single-cell sequencing confirmed high expression of AKT1 and Bcl-2 in testicular spermatogenic cells.In the in vitro experiments,compared with the control group,the model group showed a significantly increased apoptosis rate(P<0.001),a decreased proportion of cells in the G0/G1 phase,increased proportion of cells in the S phase,and decreased proportion of cells in the G2/M phase(P<0.01).Moreover,the protein and mRNA expression levels of PI3K,p-AKT,and Bcl-2 were significantly decreased(P<0.01).Compared with the model group,the Zuogui Pills group showed a decreased apoptosis rate,increased proportion of cells in the G0/G1 phase,decreased proportion of cells in the S phase,and increased proportion of cells in the G2/M phase(P<0.01),and the protein and mRNA expression levels of PI3K,p-AKT,and Bcl-2 were increased(P<0.01).Conclusion Zuogui Pills may act on targets such as AKT1 and Bcl-2 through core components such as quercetin,activate the PI3K/AKT signaling pathway,promote spermatogonial proliferation,and inhibit apoptosis,thereby improving spermatogenic dysfunction.