Effect of pulegone on the NLPR3 inflammasome during inflammatory activation of THP-1 cells

Pulegone is a key active component of Schizonepeta essential oil and has been determined to have anti-inflammatory properties. However, the underlying molecular mechanisms with regard to the NLR family pyrin domain containing 3 (NLRP3) inflammasome, also known as the NALP3 inflammasome, have remained to be elucidated. NLRP3 represents a potential link between inflammation and immunity and may play possible key role in various pathologies. In the present study, the modulatory effects of pulegone on the NLRP3 inflammasome were investigated. THP-1 cells induced with phorbol myristate acetate were divided into various groups, including the Normal (control), lipopolysaccharide (LPS), LPS + ATP/nigericin, LPS + ATP/nigericin + 0.2% DMSO and pulegone (0.2, 0.1 and 0.05 mg/ml) groups. ELISA was used to detect the levels of interleukin (IL)-1β and IL-18 in the cell supernatants and the influence of potassium ions was assessed. PCR was used to determine the expression levels of NLRP3, caspase-1, IL-1β and IL-1α in the cell lysates. Furthermore, NLRP3 and apoptosis-associated speck-like protein (ASC) were detected via immunofluorescence assays and fluorescence microscopy was employed to determine the reactive oxygen species (ROS) levels in the THP-1 cells. The results indicated reduced levels of IL-18 and IL-1β in the supernatant of the cells of the pulegone groups when compared with those in the LPS + ATP/nigericin group. In addition, reduced mRNA production of inflammasome-associated genes was detected in the cell lysates after pulegone treatment. The immunofluorescence analyses indicated significantly reduced protein expression levels of NLRP3 and ASC in the pulegone groups, as well as co-localization of the NLRP3 and ASC proteins. The pulegone groups also exhibited significantly reduced ROS levels. Furthermore, a high concentration of potassium ions significantly reduced the secretion of IL-1β after induction/stimulation. In conclusion, the present study suggested that pulegone exerts its anti-inflammatory effects on LPS + ATP/nigericin-induced THP-1 cells via inhibition of NLRP3 expression, and its regulatory mechanism is associated with potassium channel and ROS pathways. It was hypothesized that pulegone first inhibits ROS signaling, to then inhibit NLRP3 expression as a downstream event. It appeared that NLRP3 may be situated further downstream and represented the link between inflammation and immunity.