ABSTRACT
Lichens have been known to possess multiple biological activities, including anti-proliferative and anti-inflammatory activities. Vascular cell adhesion molecule-1 (VCAM-1) may play a role in the development of atherosclerosis. Hence, VCAM-1 is a possible therapeutic target in the treatment of the inflammatory disease. However, the effect of lobaric acid on VCAM-1 has not yet been investigated and characterized. For this study, we examined the effect of lobaric acid on the inhibition of VCAM-1 in tumor necrosis factor-alpha (TNF-alpha)-stimulated mouse vascular smooth muscle cells. Western blot and ELISA showed that the increased expression of VCAM-1 by TNF-alpha was significantly suppressed by the pre-treatment of lobaric acid (0.1-10 mug/ml) for 2 h. Lobaric acid abrogated TNF-alpha-induced NF-kappaB activity through preventing the degradation of IkappaB and phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 mitogen activated protein (MAP) kinase. Lobaric acid also inhibited the expression of TNF-alpha receptor 1 (TNF-R1). Overall, our results suggest that lobaric acid inhibited VCAM-1 expression through the inhibition of p38, ERK, JNK and NF-kappaB signaling pathways, and downregulation of TNF-R1 expression. Therefore, it is implicated that lobaric acid may suppress inflammation by altering the physiology of the atherosclerotic lesion.
Subject(s)
Animals , Mice , Atherosclerosis , Blotting, Western , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Extracellular Signal-Regulated MAP Kinases , Inflammation , Lichens , Muscle, Smooth, Vascular , NF-kappa B , Phosphorylation , Phosphotransferases , Physiology , Tumor Necrosis Factor-alpha , Vascular Cell Adhesion Molecule-1ABSTRACT
An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR cloning vectors with high ligation efficiencies and low blue or false-positive colonies were obtained.