ORIGINAL ARTICLE Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB, Caspase-1/11, ASC, NOX, and NOS Isoforms Abdurrahman Dolunay, 1 Sefika Pinar Senol, 1 Meryem Temiz-Resitoglu, 1 Demet Sinem Guden, 1 Ayse Nihal Sari, 1 Seyhan Sahan-Firat, 1 and Bahar Tunctan 1,2 Abstract—The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), an intracellular signaling molecule that senses many environmental- and pathogen/host-derived factors, has been implicated in the pathogenesis of several diseases associated with inflammation. It has been suggested that NLRP3 inflammasome inhibitors may have a therapeutic potential in the treatment of NLRP3- related inflammatory diseases. The aim of this study was to determine whether inhibition of NLRP3 inflammasome prevents inflammatory hyperalgesia induced by lipopolysaccharide (LPS) in mice as well as changes in expression/activity of nuclear factor κB (NF-κB), caspase-1/11, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and endothelial/neuronal/inducible nitric oxide synthase (eNOS/nNOS/iNOS) that may regulate NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1 inflammasome formation and activity by using a selective NLRP3 inflammasome inhibitor, MCC950. Male mice received saline (10 ml/kg; i.p.), LPS (10 mg/kg; i.p.), and/or MCC950 (3 mg/kg; i.p.). Reaction time to thermal stimuli within 1 min was evaluated after 6 h. The mice were killed and the brains, hearts, and lungs were collected for measurement of NF-κB, caspase-1, caspase-11, NLRP3, ASC, NOX subunits (gp91 phox ; NOX2), and p47 phox ; NOXO2), nitrotyrosine, eNOS, nNOS, iNOS, and β-actin protein expression, NOS activity, and interleukin (IL)-1β levels. LPS-induced hyperalgesia was associated with a decrease in eNOS, nNOS, and iNOS protein expression and activity as well as an increase in expression of NF-κB p65, caspase-1 p20, caspase-11 p20, NLRP3, ASC, gp91 phox , p47 phox , and nitrotyrosine proteins in addition to elevated IL-1β levels. The LPS-induced changes were prevented by MCC950. The results suggest that inhibition of NLRP3/ASC/pro-caspase-1 inflammasome formation and activity prevents inflamma- tory hyperalgesia induced by LPS in mice as well as changes in NF-κB, caspase-11, NOX2, NOXO2, and eNOS/nNOS/iNOS expression/activity. KEY WORDS: lipopolysaccharide; inflammatory hyperalgesia; NLRP3 inflammasome; NF-κB; NOX; NOS. INTRODUCTION Inflammation, the innate immune response, produces a condition known as hyperalgesia, which is characterized by enhanced pain sensation and reduced pain threshold. Inflammatory pain is a common symptom of a variety of inflammatory diseases and can be triggered by different stimuli, such as infection, trauma, antigen, and radiation [1–3]. Because the progression of inflammatory pain in- fluences daily life and may affect the prognosis of patients, it is very important to prevent the development of inflam- matory pain by treating it at an early stage. However, the mechanisms underlying inflammatory pain are not completely understood. Among the mechanisms involved 1 Department of Pharmacology, Faculty of Pharmacy, Mersin University, Yenisehir Campus, Mersin, 33169, Turkey 2 To whom correspondence should be addressed at Department of Phar- macology, Faculty of Pharmacy, Mersin University, Yenisehir Campus, Mersin, 33169, Turkey. E-mail: btunctan@gmail.com 0360-3997/16/0000-0001/0 # 2016 Springer Science+Business Media New York Inflammation ( # 2016) DOI: 10.1007/s10753-016-0483-3