endothelin-1 and salvianolic-acid-B

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).. 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.. 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 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.. 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.

Topics: Actins; Animals; Benzofurans; Cardiac Myosins; Cell Shape; Cells, Cultured; Cytoskeleton; Endothelin-1; Hepatic Stellate Cells; Male; Myosin Light Chains; Phosphorylation; Rats; Rats, Sprague-Dawley

The contraction of hepatic stellate cells (HSCs) has a critical role in the regulation of intrahepatic vascular resistance and portal hypertension. Previous studies have confirmed that salvianolic acid B (Sal B) is effective against liver fibrosis. In the present study, we evaluated the effect of Sal B on portal hypertension and on HSCs contractility. Liver cirrhosis was induced in rats by peritoneal injection of dimethylnitrosamine and the portal pressure was measured. HSCs contraction was evaluated by collagen gel contraction assay. Glycerol-urea gel electrophoresis was performed to determine the phosphorylation of myosin light chain 2 (MLC2). F-actin stress fiber polymerization was detected by fluorescein isothiocyanate-labeled phalloidin. Intracellular Ca(2+) and RhoA signaling activation were also measured. Sal B effectively reduced the portal pressure in DMN-induced cirrhotic rats. It decreased the contraction by endothelin-1 (ET-1)-activated HSCs by ∼66.5% and caused the disassembly of actin stress fibers and MLC2 dephosphorylation. Although Sal B reduced ET-1-induced intracellular Ca(2+) increase, blocking Ca(2+) increase completely by BAPTA-AM, a Ca(2+) chelator, barely affected the magnitude of contraction. Sal B decreased ET-1-induced RhoA and Rho-associated coiled coil-forming protein kinase (ROCK) II activation by 66.84% and by 76.79%, respectively, and inhibited Thr(696) phosphorylation of MYPT1 by 80.09%. In vivo, Sal B lowers the portal pressure in rats with DMN-induced cirrhosis. In vitro, Sal B attenuates ET-1-induced HSCs contraction by inhibiting the activation of RhoA and ROCK II and the downstream MYPT1 phosphorylation at Thr(696). We consider Sal B a potential candidate for the pharmacological treatment of portal hypertension.

Topics: Actins; Animals; Benzofurans; Cardiac Myosins; Cells, Cultured; Dimethylnitrosamine; Endothelin-1; Hepatic Stellate Cells; Histocytochemistry; Liver; Liver Cirrhosis, Experimental; Male; Myosin Light Chains; Phosphorylation; Portal Pressure; Protein Phosphatase 1; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

To observe the contraction effect of endothelin-1 (ET-1) on human hepatic stellate cells (HSCs) and the inhibition of salianic-acid B (SA-B) on ET-1, to explore the acting link and the possible mechanism.. HSC were isolated from human normal liver tissue by enzyme digestion and Nycondenz density gradient centrifugation. The contraction of ET-1 on passage HSCs and the intervention of SA-B with three doses (low-, middle-, and high-) on the contraction were observed by collagen gel contraction. ET-1 and SA-B were directly added to the serum-free medium of HSCs, then calcium ion concentration was detected by laser scanning confocal microscope.. Collagen gel contraction experiments showed that ET-1 could induce the contraction of HSC directly (P < 0.01). Three doses of SA-B significantly inhibited the contraction effects of ET-1 on HSCs (all P < 0.01). After adding the ET-1, HSCs morphology changed obviously with the number of cells decreased. However, SA-B inhibited the changes. Laser scanning confocal microscope experiments revealed that ET-1 stimulated the transiently rapid increase of intracellular calcium ion concentration, and the effects was obviously inhibited when SA-B was added.. SA-B could inhibit the contraction of HSCs induced by ET-1, and its mechanism might be related to the lowing of free calcium ion concentration in HSCs. This anti-contraction effect of SA-B is perhaps one of the mechanisms of its anti-fibrosis and anti-portal hypertension effects.

Topics: Benzofurans; Cells, Cultured; Endothelin-1; Hepatic Stellate Cells; Humans; Isometric Contraction

To investigate the effects of salvianolic acid B (SA-B) on portal hypertension induced by endothelin-1 in rats.. Twenty-eight Sprague-Dawley rats were randomly divided into four groups: ET-1 group, ET-1+SA-B group, ET-1+ET(A)R blocker (BQ-123) group and ET-1+ET(B)R blocker (BQ-788) group. The rats of ET-1+SA-B group underwent intragastrical administration of salvianolic acid B for five days before ET-1 injection, while in three other groups' drinking water was given. In BQ-123 group or BQ-788 group, an intravenous injection of BQ-123 or BQ-788 via femoral vein was administered 30 minutes prior to ET-1 injection. Then changes of portal pressure, cervical artery pressure and heart rate were monitored continuously.. After ET-1 injection, the portal pressure of all rats in the ET-1 group increased significantly, while slightly in groups that pretreated with SA-B, BQ-123 or BQ-788.. SA-B can attenuate the elevated portal pressure induced by ET-1 with effect similar to ETR blocker.

Topics: Animals; Antihypertensive Agents; Benzofurans; Drugs, Chinese Herbal; Endothelin Receptor Antagonists; Endothelin-1; Hypertension, Portal; Injections, Intravenous; Male; Oligopeptides; Peptides, Cyclic; Piperidines; Portal Pressure; Random Allocation; Rats; Rats, Sprague-Dawley