A new method to label fucoidan sulfate was established with tyramine and fluorescein isothiocyanate isomer I (FITC). Fluorescence spectrophotometry and high performance liquid chromatography verified the successful labelling of fucoidan by FITC. The results of the single-pass intestinal perfusion indicated that the jejunum and ileum are the main absorption sites, and there was carrier saturation. In addition, fucoidan sulfate at 1 mg/ml had no inhibitory effect on Caco-2 cell proliferation. Studies on the transmembrane transport mechanism showed that fucoidan can be absorbed because the apparent permeability coefficient of the drugs (P ) A → B was 3.78 + 0.03 ×10 and that of B → A was 1.42 + 0.19 ×10 . The peak absorption of fucoidan occurred at 120 min after administration; moreover, the higher the concentration used, the worse the absorption was, suggesting the saturation of transport carriers. The absorption was temperature dependent: the absorption at 37°C was much better than that at 4°C. Further, the absorption of fucoidan sulfate might rely on clathrin endocytosis as chlorpromazine (10 μg/ml) significantly inhibited it.

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.

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, Mg and K. On the contrary, the proteolytic activity was significantly inhibited by Mn, Fe, Fe, ethylenediaminetetraacetic acid (EDTA), and ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Fibrinolytic and fibrinogenolytic assays showed that the enzyme preferentially hydrolyzed fibrinogen Aα-chains, followed by Bβ- and γ-chains. The α-, β-, and γ-γ-chains of fibrin were also degraded by the enzyme.

SynopsisThe maximum at 220 nm in the circular dichroism spectrum of native collagen solution changed to a negative value after heat denaturation or collagenase hydrolysis. The enzyme induced rate of C1) change a t 220 nm was shown to be first order in collagen concentration. The specific rate constant k is actually a combined rate constant kfast and k,l,,v in which the ratio k,/k, is 4.1. The initial rates were linear with respect t o enzyme concentration, and the I<, was found to be 3.5 X 10-7 Ill. The rate of ultraviolet hyperchromicity at 220 nm on collagen hydrolysis was determined. The kf3't was the same as that obtained by C1).Both methods may be readily wed to assay for collagenase activity.The k,/k, ratio was 4.6.

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