The anti-inflammatory activities of ethanol extract from Dan-Lou prescription in vivo and in vitro

Background: Although, Dan-Lou prescription (DLP) is used for antagonizing check discomfort and heartache, the pharmacological mechanism has not been clearly illustrated. Our present study aimed to design inflammatory models induced by LPS in vivo and in vitro to investigate the anti-inflammation of DLP ethanol extract (EEDL) and the potential mechanisms. Methods: EEDL was prepared and then analyzed by high performance liquid chromatography (HPLC). Further, the anti-inflammatory effects of EEDL in vivo was evaluated by measuring inflammation-associated factors includingcytokines, chemokines and acute phase proteins in lipopolysaccharide (LPS)-induced mice serum and liver. The anti-inflammatory mechanism exploration of EEDL was performed in LPS-stimulated RAW 264.7 cells. Different effects of EEDL on nitric oxide (NO) and prostaglandin (PG)E-2 secretion were investigated by Griess reagent method and enzyme-linked immunosorbent assay (ELISA) respectively. Then the mRNA and protein expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured by real-time reverse-transcription polymerase chain reaction (RT-PCR), ELISA and Western blot. Other chemokines and acute phase proteins were determined by proteome profile array. Finally, the ELISA based transcription factor assay was applied to measure the DNA-binding activity of nuclear transcription factor (NF)-kappa B p65. Results: Eight compounds from EEDL have been identified as gallic acid, salvianic acid, puerarin, daidzin, paeoniflorin, salvianolic acid B, cryptotanshinone, and tanshinone IIA, with amounts of 0.26, 9.84, 10.41, 2.55, 9.44, 3.82, 0.24 and 0.3 mg/kg, respectively. In vivo, EEDL administration antagonized the up-regulation of more than 17 kinds of cytokines, chemokines and acute phase proteins in LPS-treated mice serum, and inhibited LPS-induced IL-6 mRNA and protein expression in mice liver tissue. In vitro, LPS-induced NO and PGE2 over-productions were decreased by EEDL treatment. The mRNA and protein expression of iNOS, COX-2 and IL-6 were similarly inhibited by EEDL treatment, which might be attributed to decrease the DNA-binding activity of NF-kappa B p65. Conclusion: EEDL was valid for anti-inflammation and the potential molecular mechanisms might be due to the inhibition of of LPS-induced iNOS/NO, COX-2/PGE2 and cytokines expression by antagonizing the activation of NF-kappa B p65.