Suppression of LPS-Induced Inflammation by Chalcone Flavokawain A through Activation of Nrf2/ARE-Mediated Antioxidant Genes and Inhibition of ROS/NF<i>κ</i>B Signaling Pathways in Primary Splenocytes

Abstract

Oxidative stress is an important contributing factor for inflammation. <i>Piper methysticum</i>, also known as <i>Kava</i>-<i>kava</i>, is a shrub whose root extract has been consumed as a drink by the pacific islanders for a long time. Flavokawain A (FKA) is a novel chalcone derived from the <i>kava</i> plant that is known to have medicinal properties. This study was aimed at demonstrating the antioxidant molecular mechanisms mediated by FKA on lipopolysaccharide- (LPS-) induced inflammation in BALB/c mouse-derived primary splenocytes. <i>In vitro</i> data show that the nontoxic concentrations of FKA (2-30 <i>μ</i>M) significantly suppressed the proinflammatory cytokine (TNF-<i>α</i>, IL-1<i>β</i>, and IL-6) release but induced the secretion of interleukin-10 (IL-10), an anti-inflammatory cytokine. It was also shown that FKA pretreatment significantly downregulated the LPS-induced ROS production and blocked the activation of the NF<i>κ</i>B (p65) pathway leading to the significant suppression of iNOS, COX-2, TNF-<i>α</i>, and IL-1<i>β</i> protein expressions. Notably, FKA favored the nuclear translocation of Nrf2 leading to the downstream expression of antioxidant proteins HO-1, NQO-1, and <i>γ</i>-GCLC via the Nrf2/ARE signaling pathway signifying the FKA's potent antioxidant mechanism in these cells. Supporting the <i>in vitro</i> data, the <i>ex vivo</i> data obtained from primary splenocytes derived from the FKA-preadministered BALB/c mice (orally) show that FKA significantly suppressed the proinflammatory cytokine (TNF-<i>α</i>, IL-1<i>β</i>, and IL-6) secretion in control-, LPS-, or Concanavalin A- (Con A-) stimulated cells. A significant decrease in the ratios of pro- and anti-inflammatory cytokines (IL-6/IL-10; TNF-<i>α</i>/IL-10) showed that FKA possesses strong anti-inflammatory properties. Furthermore, BALB/c mice induced with experimental pancreatitis using cholecystokinin- (CCK-) 8 showed decreased serum lipase levels due to FKA pretreatment. We conclude that with its potent antioxidant and anti-inflammatory properties, chalcone flavokawain A could be a novel therapeutic agent in the treatment of inflammation-associated diseases.