Lipopolysaccharide and palmitic acid synergistically induced MCP-1 production via MAPK-meditated TLR4 signaling pathway in RAW264.7 cells

Abstract

Background: Obesity increases the risk of developing diabetes mellitus. Clinical studies suggest that risk factors like palmitic acid (PA) and lipopolysaccharide (LPS) exist simultaneously in diabetes with obesity. Combination of PA and LPS even at low concentration can induce strong inflammatory reaction. Monocyte chemoattractant protein-1 (MCP-1) is an important inflammatory chemokine related to insulin resistance and type II diabetes. Our previous study using PCR array revealed that LPS and PA synergistically induce MCP-1 mRNA expression in macrophage cells RAW264.7, while the protein expression of MCP-1 in this case was not investigated. Moreover, the underling mechanism in the synergistic effect of MCP-1 expression or production induced by treatment of LPS and PA combination remains unclear.

Methods: Protein secretion of MCP-1 was measured by the enzyme-linked immunosorbent assay (ELISA) and mRNA levels of MCP-1 and Toll-like receptor 4 (TLR4) were measured by real-time PCR. Statistical analysis was conducted using SPSS software.

Results: LPS could increase MCP-1 transcription as well as secretion in RAW264.7, and PA amplified this effect obviously. Meanwhile, combination of LPS with PA increased TLR4 mRNA expression while LPS alone or PA alone could not, TLR4 knockdown inhibited MCP-1 transcription/secretion induced by LPS plus PA. Moreover, not NF-κB inhibitor but inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK were found to block MCP-1 generation stimulated by LPS plus PA.

Conclusion: LPS and PA synergistically induced MCP-1 secretion in RAW264.7 macrophage cells, in which MCP-1 transcription mediated by MAPK/TLR4 signaling pathways was involved. Combined treatment of PA and LPS in RAW264.7 cells mimics the situation of diabetes with obesity that has higher level of PA and LPS, MAPK/TLR4/ MCP-1 might be potential therapeutic targets for diabetes with obesity.

Keywords: Lipopolysaccharide; MAPK; MCP-1; Palmitic acid; TLR4.

Fig. 1

The markedly synergistic effect of PA on LPS-stimulated MCP-1 secretion from RAW264.7. RAW264.7 cells were treated with LPS 1 ng/ml, in the absence or presence of PA 100 μM for 24 h. After treatment, MCP-1 secretion in the culture medium were measured by ELISA, and MCP-1 was showed as pg/mL (a) or fold of control (b). Cells were collected and subject to MCP-1 (c) and CCR2 (d) mRNA analysis by real-time PCR, or cell survival was assayed by CCK8 kit (e). ^*** P < 0.01; ^* 0.01 < P < 0.05; ⁺ P > 0.05. The data from three independent experiments were represented as mean ± SD

Fig. 2

Time course of MCP-1 secretion and mRNA expression by RAW264.7 cells treated with LPS, PA, or LPS plus PA. RAW264.7 cells were treated with 1 ng/ml of LPS, 100 μM of PA, or LPS plus PA for different times as indicated. At each time point, culture medium was collected and RNA isolated from cells. MCP-1 in culture medium (a) and MCP-1 mRNA in cells (b) were quantified using ELISA and real-time PCR, respectively. The data from three independent experiments are represented as mean ± SD, and the MCP-1 secretion was showed by the fold of control. Two-way repeated measures ANOVA were applied to determine the statistical significance. c The effect of LPS or the combination of LPS and PA on MCP-1 mRNA transcription. RAW264.7 cells were treated with 1 ng/ml LPS or 1 ng/ml LPS plus 100 μM PA for 12 h, followed by addition of 10 μg/ml actinomycin D The cells were harvested after the addition of actinomycin D for 2 h, and MCP-1 mRNA was quantified using real-time PCR. Student's t test were used. ^*** P < 0.01. The data were represented as mean ± SD from three independent experiments

Fig. 3

RAW264.7 cells were transfected with 200 pmol TLR4 siRNA or negative control siRNA for 12 h, then cells were treated with 1 ng/ml LPS, 100 μM PA, or LPS plus PA for 24 h, finally MCP-1 secretion was quantified using ELISA. TLR4 knockdown by siRNA was confirmed using real-time PCR (a). MCP-1 secretion was showed by the fold of control (b). ^*** P < 0.01; ^* 0.01 < P < 0.05; ⁺ P > 0.05. The data were represented as mean ± SD from three independent experiments

Fig. 4

The effect of pharmacological inhibitors of MAPK and NF-κB signaling pathways on MCP-1 secretion stimulated by LPS alone or the combination of LPS and PA. A-B: RAW264.7 cells were treated with 1 ng/ml LPS alone (a) or 1 ng/ml LPS + 100 μM PA (b) in the absence or presence of 10 μM SB-203580 (SB), an inhibitor for the p38 MAPK pathway, 2.5 μM SP-600125 (SP), an inhibitor for the JNK pathway, or 2.5 μM PD 98059 (PD), an inhibitor for the ERK pathway, for 24 h. C-D: RAW264.7 cells were also treated with 1 ng/ml LPS alone (c) and the combination of 1 ng/ml LPS + 100 μM PA (d) in the absence or presence of 0.5 μM Bay-117,085 (Bay), an inhibitor for the NF-κB pathway, for 24 h. After treatment, MCP-1 in culture medium was quantified using ELISA. The RAW264.7 were pretreated with all the inhibitors for 30 min, following LPS or LPS plus PA treatment for 24 h. MCP-1 secretion was showed by the fold of control, and the data from three experiments were presented. ^*** P < 0.01; ^* 0.01 < P < 0.05; ⁺ P > 0.05

Fig. 5

MAPK inhibitor diminished TLR4 upregulation induced by LPS + PA. a. RAW264.7 cells were treated with LPS 1 ng/ml, PA 100 μM, or LPS 1 ng/ml plus PA 100 μM for 24 h, then TLR4 mRNA in cells was quantified using real-time PCR. The data from three independent experiments are represented as mean ± SD. b. RAW264.7 cells were pretreated with 10 μM SB, 2.5 μM PD, or 2.5 μM SP for 30 min, followed by LPS plus PA treatment for 24 h, then TLR4 expression was quantified by real-time PCR. The data from three experiments were presented. ^*** P < 0.01; ⁺ P > 0.05