OBJECTIVETo investigate the effects of salvianolic acid B (Sal B) on endothelin-1 (ET1)-induced contraction and cytoskeleton reorganization of rat hepatic stellate cells (HSCs).

METHODSHSCs were collected from Sprague-Dawley rats by in situ perfusion with pronase E and isolated by density-gradient centrifugation with Nycodenz. Cells were treated with ET-1, with or without Sal B or Y-27632 (a specific inhibitor of rho-associated protein kinases) pretreatment. HSC contraction was evaluated by collagen gel contraction assay. Cytoskeletal reorganization in response to ET-1 was evaluated by detecting changes in phosphorylation of myosin light chain 2 (MLC2) using glycerol-urea PAGE and the Odyssey Infrared Imaging System. Changes in actin stress fiber polymerization were detected by FITC-labeled phalloidin. Differences between the various cell treatment/pretreatment groups were statistically analyzed.

RESULTSCompared to the untreated control cells, the lattice area of ET-1-treated cells showed significant shrinkage (76.89% ± 3.84% vs. 37.10% ± 5.10%; P less than 0.01). Pretreatment with 105 M Sal B or 105 M Y-27632 significantly reduced ET-1-induced contraction (67.01% ± 4.14% and 77.28% ± 2.00%, respectively; bothP less than 0.01 vs. the ET-1-treated cells). The untreated control cells showed a basal MLC2 phosphorylation of (0.35 ± 0.05) mol PO4/mol MLC2. In contrast, ET-1 treatment elicited a rapid and sustained MLC2 phosphorylation, which was (0.87 ± 0.04) mol PO₄/mol MLC2 at 5 min post-treatment and with the maximal level of (0.96 ± 0.04) mol PO₄/mol MLC2 detected at 30 min post-treatment. The Sal B pretreatment led to a significant decrease in ET-1-induced MLC2 phosphorylation (by 63.1%) and an obvious disassembly of actin stress fibers.

CONCLUSIONSal B effectively inhibits ET-1-induced rat HSC contraction, through its suppressive effects on MLC2 phosphorylation and promotion of the disassembly of actin stress fibers.