CAJ | 학술논문

目的探讨丹参酚酸B盐(Sal B)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10-8 mol/L ET-1刺激,SalB组或Y-27632组细胞在ET-1刺激前分别予以10-5 mol/L Sal B或105 mol/L Y-27632预处理30 min.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Sal B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).104mol/L Sal B或10-5 mol/L Y-27632预处理能显著抑制HSC收缩,凝胶面积分别为67.01％±4.14％和77.28％±2.00％,与ET-1组相比,q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) mol PO4/mol MLC2; ET-1刺激后,MLC2磷酸化水平迅速上升,在ET-1刺激5min时为(0.87±0.04) molPO4/mol MLC2,30min时达到高峰[(0.96±0.04)mol POJmol MLC2].SalB能显著抑制ET-1诱导的MLC2磷酸化,可使ET-1刺激30min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 Sal B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.
목적 탐토단삼분산B염(Sal B)대내피소-1(ET-1)유도적대서간성상세포(HSC)수축이급대세포골가적영향.방법 채용간장원위관류매소화법、Nycodenz밀도제도리심법분리대서HSC.배양세포분위대조조、ET-1조、SalB조화Y-27632조.ET-1조세포여이10-8 mol/L ET-1자격,SalB조혹Y-27632조세포재ET-1자격전분별여이10-5 mol/L Sal B혹105 mol/L Y-27632예처리30 min.용효원응효수축법관찰HSC수축정황.용감유효전영화Odyssey형광성상계통검측기구단백경련2 (MLC2)적린산화수평.용FITC-귀필배태특이성염기동단백사,관찰Sal B대HSC중기동단백골가적영향.량조간비교용t검험,다조간비교용방차분석,Q검험. 결과 대조조응효면적위76.89％±3.84％,ET-1조응효여대조조상비명현수축,면적위37.10％±5.10%(q=25.51,P＜ 0.01).104mol/L Sal B혹10-5 mol/L Y-27632예처리능현저억제HSC수축,응효면적분별위67.01％±4.14％화77.28％±2.00％,여ET-1조상비,q치분별위16.97、25.76,P치균＜0.01,차이균유통계학의의.재기출상태하MLC2린산화수평위(0.35±0.05) mol PO4/mol MLC2; ET-1자격후,MLC2린산화수평신속상승,재ET-1자격5min시위(0.87±0.04) molPO4/mol MLC2,30min시체도고봉[(0.96±0.04)mol POJmol MLC2].SalB능현저억제ET-1유도적MLC2린산화,가사ET-1자격30min시MLC2린산화수평하강63.1％(q=26.67,P＜0.01),병차사기동단백사해취,세포골가송이. 결론 Sal B능유효억제ET-1유도적대서HSC수축.저충효응시기우SalB억제ET-1유도적MLC2린산화이급억제HSC중기동단백섬유적취합.
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.