Background: Varicocele (VC) is a relatively common and treatable cause of male infertility. Mailuoshutong pill (MLST), a traditional Chinese patent medicine, is widely used for treating varicose vein disease, but the underlying mechanism of MLST on varicocele-associated male infertility is unclear.Objective: To reveal the phytochemical characterisation and multitarget mechanism of MLST on varicocele-associated male infertility.Methods: The components in MLST were determined using UHPLC-MS/MS. Through network analysis, we constructed the “Drug-Components-Targets-Disease” network and predicted the potential biological functions and signaling pathways of MLST. Finally, the therapeutic effects and potential mechanisms of MLST were discovered by pharmacological experiments.Results: By network analysis, the “Drug-Components-Targets-Disease” network was constructed, 62 components such as apigenin, limonin, kaempferol, and obacunoic acid may be the main active components of MLST for varicocele-associated male infertility, 28 targets such as VEGFA, PIK3CA, AKT1, and MTOR are considered as hub targets, signaling pathways such as HIF-1, Estrogen, PI3K/Akt, and mTOR may be key pathways for MLST against varicocele-associated male infertility. Through pharmacological experiments, we found that MLST ameliorated VC-induced testicular atrophy. Further histomorphology showed that MLST reduced VC-induced damage to testicular spermatogonia and seminiferous tubule, while MLST reduced ROS and MDA levels and increased antioxidant enzymes (GSH, GSH-Px, SOD, and CAT) levels. TUNEL staining and immunofluorescence showed that MLST reduced VC-induced apoptosis in testicular tissue, decreased BAX, and increased BCL2. Western blot results showed that MLST decreased the phosphorylation of PI3K, AKT, and mTOR proteins, and decreased the expression of HIF1α.Conclusion: The phytochemical characterisation and multitarget mechanism of MLST on varicocele-associated male infertility were discovered using network analysis and pharmacological experiments. We verified that MLST can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, reduce the expression of HIF1α, and further attenuate VC-induced oxidative stress and apoptosis in the testis. These findings provide evidence for the therapeutic role of MLST in varicocele-associated male infertility.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common urinary system disease that is prone to recurrence. It typically leads to varying degrees of pelvic pain and discomfort, as well as symptoms related to the urinary system in affected patients. QianLieJinDan tablets (QLJD), a traditional Chinese medicine, have shown promising therapeutic effects on CP/CPPS in clinical practice, but the underlying mechanisms of QLJD in treating CP/CPPS have not been determined. To reveal the phytochemical characterization and multitarget mechanism of QLJD on CP/CPPS. The concentrations of the components of QLJD were determined using UHPLC-Q Exactive Orbitrap-MS. Utilizing network pharmacology approaches, the potential components, targets, and pathways involved in the treatment of CP/CPPS caused by QLJD were screened. Molecular docking calculations were employed to assess the affinity between the components of the QLJD and potential targets, revealing the optimal molecular conformation and binding site. Finally, the therapeutic efficacy and potential underlying mechanisms of QLJD were investigated through pharmacological experiments. In this study, a total of 35 components targeting 29 CP-related genes were identified, among which quercetin, baicalin, icariin, luteolin, and gallic acid were the major constituents. Enrichment analysis revealed that the potential targets were involved mainly in the regulation of cytokines, cell proliferation and apoptosis, and the oxidative stress response and were primarily associated with the cytokine‒cytokine receptor interaction pathway, the IL-17 signaling pathway, the Th17 cell differentiation pathway, and the JAK-STAT signaling pathway. In vivo experiments demonstrated that QLJD effectively attenuated the infiltration of CD3⁺ T cells and the expression of ROS in a CP/CPPS model rat prostate tissue. Furthermore, through the inhibition of IL-6 and STAT3 expression, QLJD reduced the differentiation of Th17 cells, thereby ameliorating pathological injury and prostatic index in prostate tissue. The potential of QLJD as an anti-CP/CPPS agent lies in its ability to interfere with the expression of IL-6 and STAT3, inhibit Th17 cell differentiation, reduce inflammatory cell infiltration in rat prostate tissue, and alleviate oxidative stress damage through its multi-component, multi-target, and multi-pathway effects.