AIM OF THE STUDY: Aging-induced testicular inflammation impairs male fertility. The purpose of this study was to investigate the effectiveness and mechanism of C. pilosula water extract (CPWE) in preventing testicular inflammation in D-galactose-induced aging mice. MATERIALS AND METHODS: The "The Plant List" database (www.theplantlist.org) provided verified plant taxonomy. D-galactose was intraperitoneally injected to induce an aging mice model, with high, medium, and low dosages of CPWE used as pharmacological interventions. The concentrations of superoxide dismutase (SOD), malondialdehyde (MDA), testosterone and in mouse serum or testicle samples after CPWE treatment were quantified using biochemical method. Hematoxylin and eosin (HE) staining was employed to assess the morphological features of testicular tissues, whereas immunohistochemical (IHC) analysis and enzyme-linked immunosorbent assay (ELISA) were conducted to evaluate the presence and levels of inflammatory cytokines interleukin-6 (IL-6) and interleukin-1β (IL-1β) within testicular samples of mice. Differentially expressed genes were identified using transcriptome sequencing
the genes and pathways regulated by CPWE, as well as immune cell infiltration, were examined using bioinformatics analysis. The expression of target gene and pathway-related protein was confirmed using real-time quantitative PCR and Western blotting. RESULTS: Treatment with CPWE alleviated the pathological alterations in the testicular tissues of aged mice, increased the concentrations of SOD and testosterone in the serum, and decreased the levels of MDA, IL-6 and IL-1β in the testes. The expression of C-C motif chemokine ligand 21a (Ccl21a) and C-C motif chemokine ligand 27b (Ccl27b) genes was downregulated after treatment with CPWE. The protein levels associated with the C-type lectin domain family 7, member A (CLEC7A)/inflammasome signaling pathway, including IL-1β, Caspase 8 (CASP8), and nuclear factor-kappa B (NF-κB), were found to be downregulated after treatment with CPWE. T cells, B cells, and macrophages showed a strong association with aging and the modulatory effects of CPWE. CONCLUSIONS: The results indicate that CPWE regulates the CLEC7A/inflammasome pathway, thereby inhibiting inflammasomes activation and reducing the expressions of proinflammatory cytokines such as IL-6 and IL-1β, as well as chemokines such as Ccl21a and Ccl27b, providing substantial protection against age-related testicular inflammatory injury.