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 lipopoly-saccharide (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.

Dexamethasone induces aberrant macrophage immune function and apoptosis

“…While this implies that the microenvironment may change, which would support tumor growth due to prostaglandin production by COX-2 enzymatic activity, other mechanisms are possible. Decreased levels of COX-2 are relative to the abnormal immune function of macrophages (17,18). In the present study, it was revealed that KLF9, a direct target of GCs, could reduce LPS-induced COX-2 levels, indicating that KLF9 is a potent inhibitor of immune function in macrophages.…”

“…In addition, COX-2 has been revealed to modulate the fate of colon cancer cell lines by generating prostanoids (15)(16)(17). These observations indicate that COX-2 expression and prostanoid generation may play a fundamental role in the initiation and promotion of cancers arising from inflamed tissues, such as cholangiocarcinoma (18). However, it is unknown whether COX-2 can modulate apoptosis of immune cells, such as macrophages.…”

Anti-Inflammatory Activities of Kukoamine A From the Root Bark of Lycium chinense Miller

“…The levels of NO were measured using Griess reagent. 23,24 After cell treatment, 100 µL of the supernatant from the cell culture was mixed with 100 µL of Griess reagent and incubated in the dark for 10 minutes at room temperature. The absorbance of the mixture was measured at 550 nm using a microplate reader.…”

Antipseudomonal and Immunomodulatory Properties of Esc Peptides: Promising Features for Treatment of Chronic Infectious Diseases and Inflammation

“…The LPS is recognized as a pathogen-associated molecular pattern by specific receptors expressed on immune cells, i.e., Toll-like receptors (TLRs), which are then activated. In particular, the LPS interacts with monocytes and macrophages through TLR4 and plays a crucial role in the inflammation process by inducing the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and the expression of the enzyme cyclooxygenase-2 (COX-2) which controls the formation of several inflammatory mediators catalyzing conversion of arachidonic acid to different prostaglandins including prostaglandin E2 [ 4 , 5 , 6 , 7 , 8 ]. Notably, inflammation is the primary physiological response of the immune system to external and internal stimuli (e.g., infections or tissue injuries), a response that restores the integrity of damaged tissues; the regulation of inflammation is essential for the tissue homeostasis in damaged biological compartments [ 9 , 10 , 11 , 12 ].…”