Hirudo Enzyme Extract Inhibits The Expression Of ICAM-1、VCAM-1 And MCP-1 Induced By LPS In RAT Vascular Smooth Muscle Cells

Atherosclerosis is a disease of lipid metabolism disorder in the arterial wall, now recognized to be driven by inflammatory processes. vascular smooth muscle cells (VSMCs), as the vascular stromal cells, are involved in all stages of the disease progression. It is a crucial event that activated VSMCs can migrate from the media to the intima followed with proliferation in response to a variety of atherogenic stimuli. Besides endothelial cells are thought to be the major cell type responsible for interacting with mononuclear leucocyte, VSMCs are also involved in this process mediated by the expression of a variety of adhesion molecules including intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1). Moreover, in consideration of the fact that there is chemotaxis between VSMCs and monocytes modulated by Monocyte Chemotactic Protein-1 (MCP-1) within the sub-intima, VSMCs could play an important role in retaining monocytes and macrophages within the lesion formation. Our previous studies have investigated the anti-atherosclerosis effect of an extract from hirudo using ApoE-/- mice, and found the treatment of hirudo enzyme extract (HEE) could obviously attenuate the area of atherosclerosis lesion by blocking NF-κB translocation. Our present study is aimed at uncovering the molecular mechanisms by which HEE inhibits LPS-induced overexpression of pro-inflammatory mediators, adhesion molecules and MCP-1 in VSMCs.Rat thoracic aortic smooth muscle cells were obtained in the method of digestion with type Ⅱ collagenase. Wound-Healing Assay and modified Boyden Chamber Model accompanied with DiI were applied to evaluate the anti-migration and anti-adhesion effect of HEE on LPS-induced VSMCs in vitro. The effect of HEE on LPS-induced nuclear translocation of NF-κB p65 in VSMCs was explored by immune-fluorescent staining and the levels of varieties of cytokines, including inflammatory factor, chemokine, adhesion molecules and signal molecules of MAPK, were determined with RT-PCR or Western Blotting in the process of the whole experiment.The potential performance of HEE was equal to simvastatin (10μM), positive control, on inhibiting VSMCs migration in the concentration of 200μg/mL.200μg/mL HEE could also decrease the chemotaxis of THP-1 and RAW264.7 to LPS-stimulated VSMCs, and the inhibition rate of those two experiments were 60% and 80% respectively. The outstanding anti-migration effect of simvastatin was simply between 200μg/mL and 400μg/mL of HEE’s. Treating with HEE could prevented NF-κB p65 nuclear localization induced by LPS in VSMCs at concentrations from 100 to 400μg/mL. RT-PCR and Western Blotting showed that HEE not only attenuated LPS-induced overexpression of inflammatory factor, chemokine and adhesion molecules significantly by inhibiting the phosphorylation of p38 MAPK but also suppressed an LPS-induced increase in the mRNA expression of them in a concentration-dependent manner in VSMCs.HEE suppresses LPS-induced overexpression of adhesion molecules and chemokine in rat VSMCs in vitro mainly via inhibiting the p38-MAPK/NF-κB pathways. Our results provide a beneficial experimental basis for using HEE as a therapeutic agent to treat atherosclerosis.