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, 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, inducible NO synthase (iNOS), NF-κB, HO-1, p-p38 and phosphorylation of IκB kinase α/β 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, TNFα and Interleukin-6. It also reduced protein expression of inducible NO synthase and COX-2, and reduced translocation of NF-κB 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-κB. 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-κB to the nuclei and thus reduced production of inflammatory mediators.

Figure 1

Effects of salvianolic acid A ( SAA ) on lipopolysaccharide ( LPS )‐induced production of nitric oxide and viability of RAW264.7 cells. Cells were treated with LPS (1 μg/ml) alone or LPS plus different concentrations of SAA (125, 250 and 500 μm) for 24 h. Amounts of nitric oxide were quantified using Griess reagent (a) and cell viability was measured by MTT assay (b). Cell morphological changes were observed using phase contrast microscopy (c: medium; d: 500 μm SAA; e: 1 μg/ml LPS; f: 125 μm SAA plus 1 μg/ml LPS; g: 250 μm SAA plus 1 μg/ml LPS; h: 500 μm SAA plus 1 μg/ml LPS, c–h ×400 magnification). Data are presented as mean ± SD of three independent experiments, ^a P < 0.01 versus LPS group. Con, control; Bar, 10 μm.

Figure 2

Effect of salvianolic acid A ( SAA ) on lipopolysaccharide ( LPS )‐induced production of TNF α, IL‐1β and IL‐6. Cells were treated with 1 μg/ml LPS alone or LPS plus different concentrations of SAA (125, 250 and 500 μm) for 24 h, and levels of TNF α (a), IL‐1β (b) and IL‐6 (c) were measured by ELISA. Data are presented as mean ± SD of three independent experiments, ^a P < 0.01, ^b P < 0.01 versus LPS group. Con, control.

Figure 3

Effect of salvianolic acid A ( SAA ) on iNOS and COX‐2 expression in lipopolysaccharide ( LPS )‐treated cells. Cells were treated with SAA (125, 250 and 500 μm) in the presence of 1 μg/ml LPS for 24 h. Whole cell lysates were prepared and analysed for expressions of iNOS, COX‐2 and β‐actin by western blot analysis (a); relative density of bands normalized to β‐actin was assayed by Bio‐Rad Quantity One 4.62 (b). Data are presented as mean ± SD of three independent experiments. Con, control.

Figure 4

Inhibitory effects of salvianolic acid A ( SAA ) on the protein levels of p‐IKK α/β and NF‐κB (p65) in lipopolysaccharide ( LPS )‐treated cells. Cells were treated with SAA (125, 250 and 500 μm) in the presence of 1 μg/ml LPS for 24 h. Western blot analysis was used to determine levels NF‐κB (p65) in nuclei and p‐IKK α/β in cytoplasm (a); relative density of bands normalized to β‐actin was assayed by Bio‐Rad Quantity One 4.62 (b). Data are presented as mean ± SD of three independent experiments. Con, control.

Figure 5

Effects of mitogen‐activated protein kinase ( MAPK ) specific inhibitors on salvianolic acid A ( SAA ) suppression of nitric oxide production in lipopolysaccharide ( LPS )‐treated cells. Cells were treated with LPS (1 μg/ml) alone or LPS plus different concentrations of inhibitors (1, 5 and 10 μm) for p38 (SB203580, a), JNK (SP600125, b) and ERK (PD98059, c) for 1 h, then SAA (500 μm) was added for 24 h. Levels of nitric oxide were quantified using Griess reagent. Data are presented as mean ± SD of three independent experiments, ^a P < 0.01, ^c P < 0.01, ^d P < 0.01 versus LPS group, ^b P < 0.01 versus LPS + 500 μm SAA group. Con, control. Cells were treated with SAA (125, 250, 500 μm) in the presence of 1 μg/ml LPS for 24 h, and western blot analysis was carried out to determine levels of p38 and phosphorylated p38 proteins (d); relative density of bands normalized to β‐actin was assayed by Bio‐Rad Quantity One 4.62 (e).

Figure 6

Effects of salvianolic acid A ( SAA ) on production of haem oxygenase‐1 (HO‐1) in lipopolysaccharide ( LPS )‐treated cells. After cells were cultured in LPS (1 μg/ml) alone or LPS plus SB203580 (10 μm) or LPS plus different concentrations of ZnPP (40 μm) for 1 h, they were treated with SAA (500 μm) for 24 h. Cells were then transfected with p38, HO‐1 or control siRNA for 24 h, and cultured in LPS (1 μg/ml) for 1 h. They were then treated with SAA (500 μm) for 24 h. HO‐1 level was examined by western blot analysis in (a) and relative density of bands normalized to β‐actin, was assayed by Bio‐Rad Quantity One 4.62 in (b). Level of nitric oxide was tested using Griess reagent in (c). Western blot analysis was also used to examine levels of NF‐κB (p65) in nuclei and p‐IKK α/β in cytoplasm in (d); relative density of bands normalized to β‐actin was assayed by Bio‐Rad Quantity One 4.62 in (e). Data are presented as mean ± SD of three independent experiments, ^a P < 0.01 versus LPS group, ^b P < 0.01, ^c P < 0.01 versus LPS group + 500 μm SAA, Con, control.

Figure 7

Identification of two potential targets amyloid‐beta protein‐like protein and arachidonate 5‐lipoxygenase of SAA .

Figure 8

Identification of of salvianolic acid A‐inhibited lipopolysaccharide‐stimulated inflammatory pathways.