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Abstract The Wannan black pig is a superior local breed in Anhui province, renowned for its exceptional meat quality and remarkable adaptability to various environmental conditions. Semen, being a crucial indicator of male sexual maturity and fertility, significantly influences the performance of breeding boars. The molecular basis for comprehending the fecundity of boars in practical production lies in understanding the disparities in sperm proteins among boars of varying ages. In this investigation, spermatozoa from three one-year-old and three seven-year-old Wannan black pigs were individually chosen. Employing a tandem mass tag (TMT)-based quantitative proteomics approach, a total of 4050 proteins were identified, out of which 130 proteins exhibited significant differences between the two groups. GO enrichment analysis revealed that these proteins primarily participated in energy metabolism, spermatogenesis, fertilization, and reproduction. KEGG enrichment analysis demonstrated that the differential proteins predominantly resided within the ribosome pathway. A protein-protein interaction (PPI) network was constructed to identify core proteins such as Small ribosomal subunit protein uS7 (RPS5). Ultimately, parallel reaction monitoring (PRM) was conducted on the selected differential proteins to validate result accuracy. The findings of this study establish a foundation for elucidating the molecular mechanism underlying variations in spermatozoa protein levels among Wannan Black Pig with different age.

Given the potent anti-inflammatory properties of the 1,2,3-triazole structure and the wide use of 2H-1,4-benzoxazin-3(4H)-one in developing treatments for neurodegenerative diseases, a series of 2H-1,4-benzoxazin-3(4H)-one derivatives were synthesized by introducing a 1,2,3-triazole moiety. Screening for anti-inflammatory activity in microglial cells revealed that compounds e2, e16, and e20 exhibited the most promising effects without significant cytotoxicity. These compounds effectively reduced LPS-induced NO production and significantly decreased the transcription levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, they downregulated the transcription and protein levels of the inflammation-related enzymes iNOS and COX-2 in response to LPS stimulation. To further investigate the anti-inflammatory mechanisms of these derivatives in microglia, the intracellular ROS levels and the activation of the Nrf2-HO-1 signaling pathway were analyzed. The results indicated that the 2H-1,4-benzoxazin-3(4H)-one derivatives significantly activated the Nrf2-HO-1 pathway, reduced LPS-induced ROS production, and alleviated microglial inflammation. Molecular docking studies suggested that compounds e2, e16, and e20 could interact with Nrf2-related binding sites, preventing its degradation by Keap1. Additionally, acute toxicity tests in mice demonstrated that compound e16 exhibited favorable safety.