丹参酚酸B盐对大鼠肝星状细胞收缩以及对细胞骨架的影响

Effects of saivianolc acid B on endothelin-l-induced contraction and cytoskeleton organization of hepatic stcgate cells in rats

目的探讨丹参酚酸B盐（SalB）对内皮素-1（ET-1）诱导的大鼠肝星状细胞（HSC）收缩以及对细胞骨架的影响。方法采用肝脏原位灌流酶消化法、Nycodenz密度梯度离心法分离大鼠HSC。培养细胞分为对照组、ET-1组、SalB组和Y-27632组。ET-1组细胞予以10^-8mol／LET-1刺激，SalB组或Y-27632组细胞在ET-1刺激前分别予以10^-5mol／LSalB或10^-5mol／LY-27632预处理30min。用胶原凝胶收缩法观察HSC收缩情况。用甘油胶电泳和Odyssey荧光成像系统检测肌球蛋白轻链2（MLC2）的磷酸化水平。用FITC-鬼笔环肽特异性染肌动蛋白丝，观察SalB对HSC中肌动蛋白骨架的影响。两组间比较用t检验，多组间比较用方差分析，Q检验。结果对照组凝胶面积为76．89％±3．84％，ET-1组凝胶与对照组相比明显收缩，面积为37．10％±5．10％（q=25．51，P〈0．01）。10^-5mol／LSalB或10^-5mol／LY-27632预处理能显著抑制HSC收缩，凝胶面积分别为67．01％土4．14％和77．28％±2．00%，与ET-1组相比，口值分别为16．97、25．76，P值均〈0．01，差异均有统计学意义。在基础状态下MLC2磷酸化水平为（0．35±0．05）molPO4/molMLC2；ET-1刺激后，MLC2磷酸化水平迅速匕升，在ET-1刺激5min时为（0．87±0．04）molPO4／molMLC2，30min时达到高峰【（0．96±0．04）molPO4／molMLC2】。SalB能显著抑制ET-1诱导的MLC2磷酸化，可使ET-1刺激30man时MLC2磷酸化水平下降63．1％（q=26．67，P〈0．01），并且使肌动蛋白丝解聚，细胞骨架松弛。结论SalB能有效抑制ET-1诱导的大鼠HSC收缩。这种效应是基于SalB抑制ET-1诱导的MLC2磷酸化以及抑制HSC中肌动蛋白纤维的聚合。

Objective To investigate the effects of salvianolic acid B （Sal B） on endothelin-1 （ET- 1）-induced contraction and cytoskeleton reorganization of rat hepatic stellate cells （HSCs）. Methods HSCs were eollected from Sprague-Dawley rats by in situ perfusion with pronase E and isolated by density-gradicnt centrifugation with Nycodcnz. 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 Inflated Imaging System. Changes in actin stress fiber polymerization were detected by FITC-labeled phalloidin. Differences between the various cell trcatmcnt/prctrcatment groups were statistically analyzed. Results Compared to the untreated control cells, the lattice area of ET-l-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-l-induced contraction （67.01% ± 4.14% and 77.28% ±2.00%, respectively; bothP 〈 0.01 vs. the ET-l-treall cells）. The untreated control cells showed a basal MLC2 phosphorylation of （0.35 ± 0.05） mol PO4/mol MLC2. In contrast, ET-1 ireatment 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 pretrealment led to a significant decrease in ET-l-induced MLC2 phosphorylation Coy 63.1%） and an obvious disassembly of actin stress fibers. Conclusion Sal B effectively inhibits ET-l-induced rat HSC contraction, through its suppressive effects on MLC2 phosphorylation and promotion of the disassembly of actin slress fibers.