Male factors are estimated to account for about 50% of infertility cases, and testicular inflammation is considered one of the main issues. Reducing inflammation in the testis can restore testis/sperm function. Based on this, we considered whether a safe, plant-based compound could be applied to reduce inflammation and related infertility. We focused on guayusa (Ilex guayusa Loes;), a tea leaf native to South America, which has anti-inflammatory properties. This study investigated its effects on the secretion of pro-inflammatory cytokines on a testis-derived Leydig cell line.
Mouse Leydig tumour cells (I-10) were treated with guayusa extracts, followed 1 h later by nigericin to induce inflammatory response. After 3 h, supernatants were collected, and levels of inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured. Lactate dehydrogenase (LDH), a cell death marker, was measured. Treating with nigericin increased IL-1β, IL-6 and LDH secretion, all of which were reduced by guayusa treatment. Since nigericin is known as an activator of the NLRP3 inflammasome, the inhibitors of NLRP3 inflammasome including glibenclamide (ATP-sensitive potassium channel), MCC950 (NLRP3 inhibitor) and AC-YVAD-cmk (caspase-1 inhibitor), were treated simultaneously with guayusa. While no significant changes were observed with MCC950 and AC-YVAD-cmk, treatment with glibenclamide tended to decrease IL-1β secretion and significantly reduced IL-6 secretion. To investigate the cytokine-suppressive mechanism of guayusa, we measured the protein expression of Gasdermin D (GSDMD), which controlling cell death by creating holes in the cell membrane. Treatment with guayusa significantly reduced GSDMD protein compared to treatment with nigericin alone. Consistently, treatment with necrosulfonamide (NSA), a GSDMD and MLKL phosphorylation inhibitor, resulted in decreased secretion of IL-1β and IL-6.
These findings suggest that the inhibitory effect of guayusa for IL-1β and IL-6 reduction induced by nigericin may be meditated through potassium ion channels and GSDMD rather than via the NLRP3 inflammasome system.