Anti-inflammatory and antioxidant effects of MOK, a polyherbal extract, on lipopolysaccharide-stimulated RAW 264.7 macrophages

Abstract

MOK, a pharmacopuncture medicine consisting of 10 herbs, has a long history as treatment for various inflammatory conditions. To investigate the mechanisms of action of MOK, its anti-inflammatory and antioxidative effects were assessed in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). RAW 264.7 cells were treated with different concentrations of MOK extract for 30 min prior to stimulation with or without LPS for the indicated times. Nitric oxide (NO) production was measured using Griess reagent, while the mRNA levels of inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and the antioxidant enzymes Mn superoxide dismutase and heme oxygenase-1, were determined using reverse transcription-polymerase chain reaction analysis. Western blotting was used to determine the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, superoxide dismutase (SOD)2, catalase (CAT) and heme oxygenase-1 (HO-1), and the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK and p38. Western blotting and immunocytochemistry were used to observe the nuclear expression of nuclear factor (NF)-κB p65. Additionally, reactive oxygen species (ROS) and prostaglandin (PG)E2 production were determined using the ROS assay and an enzyme immunoassay. With MOK treatment, there was a notable decrease in NO and PGE2 production induced by LPS in RAW 264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expression. Furthermore, with MOK treatment, there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 MAPK, by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells. In addition, MOK treatment led to an increase in the antioxidant enzymes SOD, CAT and HO-1 in LPS-stimulated cells, with a concomitant decrease in ROS generation. These results indicate that the inflammatory responses in activated macrophages are inhibited by MOK through downregulation of the transcription levels of inflammatory mediators and inhibition of the MAPK/NF-κB pathway. Moreover, MOK protects against oxidative damage by upregulating the expression of antioxidant enzymes and generating ROS scavengers.