Protective effects of nicotine-treated peritoneal macrophage–conditioned medium on testicular function and spermatogenesis in an experimental rat model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder frequently linked to male reproductive dysfunction through sustained inflammation, oxidative stress, and germ-cell apoptosis. In this study, we evaluated the potential protective effects of conditioned medium derived from nicotine-treated peritoneal macrophages (N-MCM) against testicular injury and spermatogenic impairment in a Complete Freund’s Adjuvant (CFA)-induced rat model of RA, and compared its efficacy with that of untreated macrophage-conditioned medium (U-MCM) and methylprednisolone. Thirty-five adult male Wistar rats were divided into five groups ( n = 7 per group): healthy controls, arthritic controls, RA + U-MCM, RA + N-MCM, and RA + methylprednisolone. Arthritis was induced by subplantar injection of CFA, and successful induction was confirmed by progressive, significant increases in clinical arthritis scores and hind-paw diameter relative to healthy controls (Table 1). Treatments were administered intraperitoneally on days 7 and 14 after induction. On day 60, animals underwent comprehensive reproductive assessments, including computer-assisted sperm analysis (CASA), sperm functional tests (viability, morphology, membrane integrity, and chromatin integrity/DNA denaturation assessed by acridine orange staining), testicular histopathology with morphometric analysis (Johnsen and Cosentino scores), oxidative stress biomarkers (TAC, SOD, CAT, GPx, MDA, NO), and qRT-PCR quantification of apoptosis-related genes (Bax, Bcl-2, caspase-3, caspase-9). Induction of RA led to marked deterioration of sperm quality, disruption of testicular architecture, depletion of antioxidant defenses, and activation of the mitochondrial apoptotic pathway ( p ≤ 0.05). Treatment with N-MCM substantially improved sperm parameters, redox balance, and histological integrity, and reduced apoptosis-related gene expression to levels that did not differ significantly from healthy controls. These protective effects were significantly superior to those observed with both U-MCM and methylprednisolone ( p ≤ 0.05), and most reproductive endpoints in the N-MCM group did not differ significantly from healthy controls. Collectively, these findings suggest that nicotine priming may alter the macrophage secretome in a manner that confers immunomodulatory, antioxidant, and anti-apoptotic protection against RA-associated male reproductive impairment. As the secretome composition was not characterized and testicular inflammatory mediators were not directly measured, these mechanisms remain hypothetical. Nicotine-conditioned macrophage secretome may nonetheless represent a promising cell-free therapeutic candidate for preserving fertility in chronic inflammatory diseases, warranting further mechanistic and translational investigation.