Pharmacological inhibition of TLR4/NF-κB with TLR4-IN-C34 attenuated microcystin-leucine arginine toxicity in bovine Sertoli cells

This study investigated the pharmacological inhibition of the toll-like receptor 4 (TLR4) genes as a measure to attenuate microcystin-LR (MC-LR) reproductive toxicity. Bovine Sertoli cells were pretreated with TLR4-IN-C34 (C34) for 1 hour. Thereafter the pretreated and non-pretreated Sertoli cells were cultured in medium containing 10% heat-activated fetal bovine serum + 80 mu g/L MC-LR for 24 hours to assess the ability of TLR4-IN-C34 to attenuate the toxic effects of MC-LR. The results showed that TLR4-IN-C34 inhibited MC-LR-induced mitochondria membrane damage, mitophagy and downregulation of blood-testis barrier constituent proteins via TLR4/nuclear factor-kappaB and mitochondria-mediated apoptosis signaling pathway blockage. The downregulation of the mitochondria electron transport chain, energy production and DNA replication related genes (mt-ND2, COX-1, COX-2, ACAT, mtTFA) and upregulation of inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor-alpha, IL-1 beta, interferon-gamma, IL-4, IL-10, IL-13 and transforming growth factor beta 1) were modulated by TLR4-IN-C34. Taken together, we conclude that TLR4-IN-C34 inhibits MC-LR-related disruption of mitochondria membrane, mitophagy and downregulation of blood-testis barrier proteins of the bovine Sertoli cell via cytochrome c release and TLR4 signaling blockage.