ABSTRACT
AIM: Atherosclerosis is a kind of chronic inflammatory disease. A crucial pathology change of atherosclerosis is the migration of activated VSMCs to the intima where they interact with leukocytes by expressing adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, monocyte chemoattractant protein-1 (MCP-1) expressed by VSMCs plays an important role in recruiting monocytes and macrophages. Leech (Whitmania pigra Whitman) is a traditional Chinese medicine to treat cardiovascular diseases including atherosclerosis, however previous research has rarely reported the molecular mechanism for its curative effect. Thus, our study focuses on the effects of leech extracts on the expression of inflammatory factors, adhesion molecules and MCP-1 in rat VSMCs. METHODS: In our present study, wound-healing assay and Boyden chamber model were applied to evaluate the anti-migration effect of LEE (Leech Enzyme Extracts) on LPS induced VSMCs. The anti-adhesion effect was assessed using DiI-labeled THP-1 and RAW264.7. RESULTS: LEE suppressed LPS-induced VSMCs migration and decreased the chemotaxis and adhesive capacity of THP-1 and RAW264.7 to LPS-stimulated VSMCs. LEE also attenuated the upregulation of a variety of pro-atherosclerotic factors by inhibiting the phosphorylation of p38 MAPK. LEE was also observed to prevent NF-κB p65 nuclear localization using immune-fluorescent staining. CONCLUSIONS: In conclusion, LEE suppresses LPS-induced upregulation of inflammatory factors, adhesion molecules and MCP-1 in rat VSMCs mainly via inhibiting the p38 MAPK/NF-κB pathways, thus partly uncovered LEE's molecular mechanisms for its therapeutic effect on atherosclerosis.
Subject(s)
Cell Adhesion/drug effects , Cell Movement/drug effects , Fibrinolytic Agents/pharmacology , Macrophages/drug effects , Monocytes/drug effects , Muscle, Smooth, Vascular/drug effects , Animals , Blotting, Western , Cells, Cultured , Fluorescent Antibody Technique , Humans , Leeches/enzymology , Lipopolysaccharides , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Monocytes/metabolism , Monocytes/pathology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Phosphorylation , RAW 264.7 Cells , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal TransductionABSTRACT
A fibrinolytic enzyme was purified from the dry body of Whitmania pigra Whitman. The fibrinolytic enzyme was purified to homogeneity with a yield of 0.003% and a purification of 630.7 fold. The molecular weight of the enzyme was estimated to be 26.7 kDa by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was tested by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and it showed that the enzyme was a novel fibrinolytic enzyme. The optimal pH and temperature of the enzyme were 8.5 and 55°C, respectively. Enzyme activity was enhanced by Na+, Mg2+, and K+. On the contrary, the proteolytic activity was significantly inhibited by Mn2+, Fe2+, Fe3+, ethylenediaminetetraacetic acid (EDTA), and ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Fibrinolytic and fibrinogenolytic assays showed that the enzyme preferentially hydrolyzed fibrinogen Aα-chains, followed by Bß- and γ-chains. The α-, ß-, and γ-γ-chains of fibrin were also degraded by the enzyme.
