Leptin is upregulated in epididymitis and promotes apoptosis and IL-1β production in epididymal epithelial cells by activating the NLRP3 inflammasome

Abstract

Epididymitis, one of the most common urological disease, is a significant cause of male infertility. Leptin is capable of modulating both reproduction and immune response. We analyzed the serum and seminal plasma levels of leptin in infertile patients with or without chronic epididymitis. Experimental epididymitis models were generated by administrating 200 μg Lipopolysaccharide (LPS) to Sprague-Dawley rats. The expression of leptin in epididymis were detected using qPCR, Western blots 6–72 h after injection, and using immunohistochemistry 72 h after injection. Besides, rat epididymal epithelial cells were isolated as an in vitro model and were treated with leptin (5–40 ng/ml, 6–48 h), LPS (1ug/ml, 6 h), and NLRP3 inflammasome inhibitor MCC950 (10 μM, 2 h). Cell Counting Kits-8 assay and Annexin V/PE assay were used to evaluate cell viability and apoptosis. Quantitive PCR and ELISA assay were used to detected inflammatory cytokines interleukin-1beta (IL-1β) production. Western Blots were used to detect molecular related to cell apoptosis, IL-1β maturation, and NLRP3 inflammasome. We found that patients with chronic epididymitis presented a significantly higher level of seminal plasma leptin and correlated declined sperm progressive motility. Leptin and leptin receptor expression in epididymis was significantly upregulated 24 h after LPS administration both in mRNA and protein level, and highly expressed in the epididymis epithelium 72 h after LPS administration. In epididymal epithelial cells, leptin reduced cell viability and promoted apoptosis in a concentration-dependent manner via cleavage of caspase-9, caspase-3, and PARP. Leptin enhanced the LPS-induced production of IL-1β, which was associated with increased IL-1β maturation and caspase-1 activation. Furthermore, NLRP3 inhibitor MCC950 attenuated the effects of leptin or co-treatment with LPS on NLRP3, ASC expression, IL-1β maturation, and caspase-1 activation, which indicated that leptin promotes IL-1β production via activating the NLRP3 inflammasome. These data suggested that leptin may act as a potential evaluation and treatment target for epididymitis and male subfertility.