Anti-inflammatory effect of Acalypha australis L. via suppression of NF-κB signaling in LPS-stimulated RAW 264.7 macrophages and LPS-induced septic mice

Abstract

We evaluated the anti-inflammatory activity of methanol extracts of Chinese medicinal plants from Beijing and determined which extract was the most effective. We found the methanol extract of Acalypha australis L. (AAL) to be the most effective. AAL has been used for clearing heat, toxic material, and hemostasia in Chinese medicine. Although these uses are closely related to inflammation, the anti-inflammatory effect of AAL has not yet been described and its underlying mechanism remains unclear. Therefore, we aimed to identify anti-inflammatory effect of AAL and its underlying mechanism in vitro and in vivo. In RAW 264.7 macrophages, cytotoxicity was evaluated by MTT assay and nitric oxide (NO) was measured with Griess reagent. To confirm the production of pro-inflammatory cytokines and its mRNA expression, enzyme immunoassay (EIA) and quantitative real-time PCR (qRT-PCR) were performed. Further, protein expression was analyzed by western blotting. Septic shock was induced by intraperitoneal injection of LPS (25 mg/kg) in mice. One hour before LPS injection, AAL (25 and 50 mg/kg) was administered orally. In LPS-stimulated macrophages, AAL inhibited NO production at concentrations without cytotoxicity. Additionally, AAL reduced not only inducible nitric oxide synthase (iNOS) expression but the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by attenuating nuclear factor-kappa B (NF-κB)-related proteins (NF-κB p65, phosphorylation of inhibitor κB-α; p-IκB-α, phosphorylation of inhibitor κB kinase-α/β; p-Ikk-α/β). Moreover, AAL enhanced the survival rate of mice through the inhibition of iNOS expression and IL-6 and interleukin-1β (IL-1β) production in LPS-induced septic mice. Furthermore, AAL also reduced the expression of NF-κB-related proteins. These finding suggest that AAL is related to the modulation of inflammatory reactions by blocking NF-κB activation in LPS-stimulated RAW 264.7 macrophages and LPS-induced septic mice.

Keywords: Acalypha australis L.; Anti-inflammation; Lipopolysaccharide; NF-κB; RAW 264.7 macrophages; Septic shock.