Effects of salvianolic acid B on endothelin-1-induced contraction and cytoskeleton organization of hepatic stellate cells in rats

摘要

Objective To investigate the effects of salvianolic acid B (Sal B) on endothelin-1 (ET1)-induced contraction and cytoskeleton reorganization of rat hepatic stellate cells (HSCs).Methods HSCs 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.Results Compared 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 ＜ 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 ＜ 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.Conclusion Sal 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.

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