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In silico analysis and experimental validation of molecular mechanisms of salvianolic acid A-inhibited LPS-stimulated inflammation, in RAW264.7 macrophages.

ABSTRACT: OBJECTIVES:The aim of this study was to explore mechanisms by which salvianolic acid A (SAA) revealed its anti-inflammatory activity, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. MATERIALS AND METHODS:Nitric oxide (NO) concentration was determined by the Griess reaction and cell viability was assessed by MTT assay. Interleukin-6, TNF? and interleukin-1? were determined by ELISA. The RAW264.7 cells were transfected with siRNA against p38 or HO-1. Expressions of COX-2, inducible NO synthase (iNOS), NF-?B, HO-1, p-p38 and phosphorylation of I?B kinase ?/? were detected by western blotting. Potential targets of SAA were analysed by homology modelling, target prediction, protein-protein interaction prediction and docking studies. RESULTS:Salvianolic acid A suppressed LPS-triggered production of NO, TNF? and Interleukin-6. It also reduced protein expression of inducible NO synthase and COX-2, and reduced translocation of NF-?B to nuclei. Moreover, SAA promoted expression of phosphorylated p38, and downstream HO-1. Zn (II) protoporphyrin IX, a specific inhibitor of HO-1, or siRNA against HO-1 could effectively increase transfer of NF-?B. SAA was predicted to target amyloid-beta protein-like protein and arachidonate 5-lipoxygenase, that could regulate p38 and HO-1. CONCLUSIONS:In silico analysis and experimental validation together demonstrated that SAA exhibited its anti-inflammatory effect via the p38-HO-1 pathway in LPS-stimulated RAW264.7 cells, reduced transfer of NF-?B to the nuclei and thus reduced production of inflammatory mediators.

SUBMITTER: Huang J

PROVIDER: S-EPMC6496881 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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In silico analysis and experimental validation of molecular mechanisms of salvianolic acid A-inhibited LPS-stimulated inflammation, in RAW264.7 macrophages.

Huang J J Qin Y Y Liu B B Li G Y GY Ouyang L L Wang J H JH

Cell proliferation 20130824 5

<h4>Objectives</h4>The aim of this study was to explore mechanisms by which salvianolic acid A (SAA) revealed its anti-inflammatory activity, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.<h4>Materials and methods</h4>Nitric oxide (NO) concentration was determined by the Griess reaction and cell viability was assessed by MTT assay. Interleukin-6, TNFα and interleukin-1β were determined by ELISA. The RAW264.7 cells were transfected with siRNA against p38 or HO-1. Expressions of COX-2, ind ...[more]

PMID: 24033467

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Project description:Recent studies confirm that chronic low-grade inflammation is closely associated with metabolic syndromes, and anti-inflammatory therapy is a potential approach for treating cardiovascular diseases and type 2 diabetes. Accumulating evidence suggests that GPR120 activation is a feasible solution to ameliorating chronic inflammation and improving glucose metabolism. In this study we investigated whether ginsenoside Rb2 (Rb2), which exhibited regulatory activities in glucose and lipid metabolism, affected GPR120 expression in lipopolysaccharide (LPS)-activated mouse macrophage RAW264.7 cells, and examined the contribution of GPR120 activation to reducing the LPS-induced inflammatory response. LPS (100 ng/mL) activated the macrophages, resulting in dramatic increases in TNF-α, IL-6, IL-1β and NO production. Treatment with a ω-3 fatty acid α-linolenic acid (ALA, 50 μmol/L) produced moderate reduction in LPS-stimulated inflammatory cytokines and NO production (TNF-α and IL-6 were decreased by 46% and 42%, respectively). Pre-incubation with Rb2 (1 or 10 μmol/L) for 12 h before ALA treatment dramatically amplified the inhibitory effects of ALA (TNF-α and IL-6 were decreased by 74% and 86%, respectively). Compared to the treatment with ALA alone, pre-incubation with Rb2 resulted in a more prominent reduction in LPS-stimulated expression of iNOS and COX-2 and LPS-stimulated IKK/NF-κB phosphorylation and MAPK pathway activation. Rb2 (0.1-100 μmol/L) dose- and time-dependently increased both mRNA and protein expression of GPR120 in RAW264.7 cells, but treatment with Rb2 alone did not exert anti-inflammatory effect in LPS-activated RAW264.7 cells. In RAW264.7 cells transfected with GPR120 shRNA, the ameliorating effects of Rb2 on LPS-induced inflammation were abolished. In conclusion, Rb2 exerts anti-inflammatory effect in LPS-stimulated mouse macrophage RAW264.7 cells in vitro by increasing GPR120 expression and subsequently enhancing ω-3 fatty acid-induced GPR120 activation.

Dataset Information

In silico analysis and experimental validation of molecular mechanisms of salvianolic acid A-inhibited LPS-stimulated inflammation, in RAW264.7 macrophages.

Publications

In silico analysis and experimental validation of molecular mechanisms of salvianolic acid A-inhibited LPS-stimulated inflammation, in RAW264.7 macrophages.

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