J Huang1, Y Qin, B Liu, G Y Li, L Ouyang, J H Wang. 1. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China.
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.
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.
Authors: Michael J Keiser; Bryan L Roth; Blaine N Armbruster; Paul Ernsberger; John J Irwin; Brian K Shoichet Journal: Nat Biotechnol Date: 2007-02 Impact factor: 54.908