ABSTRACT
Sauchinone, a biologically active lignan isolated from the roots of Saururus chinensis (LOUR.) BAILL. (Saururaceae), is reported to exert a variety of biological activities, such as hepatoprotective, anti-inflammatory actions and inhibitory effects on bone resorption. In this study, we investigated the effect of sauchinone in suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, leading to a reduction in COX-2-derived prostaglandin E(2) (PGE(2)) and iNOS-derived nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages. Present study also demonstrates the effects of sauchinone in inducing heme oxygenase-1 (HO-1) expression and an increase in heme oxygenase (HO) activity in RAW264.7 macrophages. The effects of sauchinone on LPS-induced PGE(2), NO, tumor necrosis factor-α (TNF-α) and interlukine-1ß (IL-1ß) production were partially reversed by the HO-1 inhibitor Tin protoporphyrin was also seen in this study. In addition, we found that treatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) reduced sauchinone-induced HO-1 expression. Sauchinone also increased ERK phosphorylation. These results suggest that sauchinone inhibits pro-inflammatory mediators through expression of anti-inflammatory HO-1 via ERK pathway.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Benzopyrans/pharmacology , Dioxoles/pharmacology , Inflammation Mediators/antagonists & inhibitors , Inflammation/drug therapy , Plant Preparations/pharmacology , Saururaceae , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents/metabolism , Benzopyrans/chemistry , Benzopyrans/immunology , Benzopyrans/metabolism , Cell Line , Cyclooxygenase 2/immunology , Cyclooxygenase 2/metabolism , Dioxoles/chemistry , Dioxoles/immunology , Dioxoles/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Flavonoids/metabolism , Flavonoids/pharmacology , Heme Oxygenase-1/immunology , Heme Oxygenase-1/metabolism , Inflammation/physiopathology , Inflammation Mediators/immunology , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Metalloporphyrins , Mice , Molecular Targeted Therapy , Nitric Oxide/biosynthesis , Nitric Oxide/immunology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/drug effects , Phosphorylation/drug effects , Phytotherapy , Plant Preparations/chemistry , Plant Preparations/isolation & purification , Plant Roots , Protoporphyrins , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunologyABSTRACT
In this report we show that succinic groups are far more reactive to amino compounds than the carboxylic groups derived from Asp and Glu on the protein when using coupling via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (even by an 8 fold factor). Accordingly, a new carrier-protein was designed where both natural amino and carboxylic moieties were transformed into succinic residues. To prepare this hypersuccinylated carrier, all exposed carboxylic acids were first transformed into amino groups by reaction with ethylendiamine after activation with EDCI. Secondly, all these residues together with the ones from Lys were succinylated to prepare a fully succinylated protein. This was even more relevant considering that the amount of Lysine was 2-4 fold lower than Asp and Glu in most of the proteins. These "hyper-succinylated" proteins (KLH or BSA) offer significant improvements in protein reactivity compared to the native proteins (by a factor of 8-10). The optimization of the reaction, in which the presence of dioxane was found to be influential, permitted further improvements in the modification of the protein. Finally, this new strategy was successfully used to develop antibodies against the commercial anti-tumor molecule, ET-637-NH2. Using native KLH no response was found, whereas 1/64,000 serum dilutions gave very high values in ELISA procedures when immunization was performed using the hyper-succinylated KLH.
