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.

Figure 1

Cytotoxic effects of MOK extract on RAW 264.7 cells. Different concentrations of MOK (0.5-10 mg/ml) were used for cell treatment for 24 h, with or without LPS (1 µg/ml). WST-1 assay was used to determine cell viability. Values are shown as means ± standard error of the mean. LPS, lipopolysaccharide.

Figure 2

Effects of MOK extract on NO production and iNOS expression in RAW 264.7 cells stimulated by LPS. (A) After treatment with the indicated MOK extract concentrations for 30 min, cells were stimulated for 24 h with LPS (1 µg/ml). Griess reaction was used to measure NO concentrations in the culture media. Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 1st bar, untreated control cells (a) or 2nd bar, cells treated with LPS only (b). (B) After treatment with MOK extract for 30 min, cells were stimulated with LPS for an additional 5 h. Total RNA was isolated, and then RT-PCR was used to measure the mRNA levels of iNOS with GAPDH expression as an internal control. (C) After treatment with MOK extract for 30 min, cells were stimulated with LPS for an additional 24 h. The cell lysates were extracted and western blotting was used to analyze iNOS protein levels with β-actin expression as an internal control. Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. NO, nitric oxide; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Effects of MOK extract on the production of PGE₂ and expression of COX-2 in RAW 264.7 cells stimulated by LPS. (A) After treatment with various concentrations of MOK extract for 30 min, cells were then stimulated with LPS. At 16 h, culture media were collected and measured by an enzyme immunoassay for PGE₂ concentration. Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 1st bar, untreated control cells (a) or 2nd bar, cells treated with LPS only (b). (B) After treatment with MOK extract for 30 min, cells were stimulated with LPS for an additional 5 h. RT-PCR was used to measure the mRNA levels of COX-2 with GAPDH expression as an internal control. (C) After treatment with MOK extract for 30 min, cells were stimulated with LPS for an additional 24 h. Western blotting was used to analyze the protein level of COX-2 in total cell lysates with β-actin as an internal control. Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. PGE₂, prostaglandin E₂; COX, cyclooxygenase; LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Effects of MOK extract on production of pro-inflammatory cytokines induced by LPS in RAW 264.7 cells. Cells were incubated with MOK extract for 6 h with or without LPS. (A) RT-PCR was used to measure the mRNA level of each cytokine (TNF-α, IL-1β and IL-6) with GAPDH as an internal control (ratios are in relation to GAPDH). (B) Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05 and ^**P<0.01 vs. 1st bar, untreated control cells (a) or 2nd bar, cells treated with LPS only (b). Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; TNF, tumor necrosis factor; IL, interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Effects of MOK extract on phosphorylation of MAPKs induced by LPS in RAW 264.7 cells. After treatment with the indicated MOK extract concentrations for 30 min, cells were stimulated for 5 min with LPS (for ERK) or for 15 min with LPS (for JNK and p38 MAPK). (A) The cellular proteins were used to detect the phosphorylated or total forms of the MAPK molecules ERK1/2, JNK and p38 MAPK with β-actin as the housekeeping control protein. (B) Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 1st bar, untreated control cells (a) or 2nd bar, cells treated with LPS only (b). Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Figure 6

Effects of MOK extract on the expression of NF-κB induced by LPS in RAW 264.7 cells. Graded concentrations of MOK extract were used to pretreat cells for 30 min, followed by stimulation for 30 min with LPS. RAW 264.7 cells were harvested, and then the nuclei and cytosol were isolated. (A and B) Western blot analysis was used to detect the expression of NF-κB p65 in the nuclei with TATA-binding protein (TBP) as an internal control. Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05 and ^**P<0.01 vs. 1st bar, untreated control cells (A) or 2nd bar, cells treated with LPS only (b). Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. (C) The translocation of NF-κB to the nucleus from the cytoplasm was observed by fluorescence microscopy after staining with anti-NF-κB p65 antibody (green, Alexa Fluor 488) and counterstaining with DAPI (blue). Original magnification, ×200. NF-κB, nuclear factor-κB; LPS, lipopolysaccharide.

Figure 7

Effects of MOK extract on ROS production and expression of antioxidant genes and proteins (MnSOD, HO-1, SOD2 and CAT) in RAW 264.7 cells stimulated with LPS. (A) After treatment with graded concentrations of MOK extract for 30 min, cells were stimulated for 24 h with or without LPS. Then, the cells were homogenized, and an in vitro ROS/RNS assay kit used to measure ROS levels in RAW 264.7 cells stimulated with LPS. Data represent means ± standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 1st bar, untreated control cells (A) or 2nd bar, cells treated with LPS only (b). (B) After treatment with MOK extract for 30 min, the cells were stimulated for an additional 5 h with LPS. RT-PCR was used to measure the mRNA levels of MnSOD and HO-1 with GAPDH expression as an internal control. (C) After treatment with MOK extract for 30 min, the cells were stimulated for an additional 24 h with LPS. Western blotting was used to analyze the protein levels of SOD2 and CAT in total cell lysates with β-actin as an internal control. Mean densitometric values of the three independent experiments were analyzed and are expressed as bar charts. ROS, reactive oxygen species; RNS, reactive nitrogen species; LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; SOD, superoxide dismutase; HO-1, heme oxygenase-1; CAT, catalase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.