卡维地洛抑制血小板衍生因子BB诱导的人肝星状细胞活化和纤维化的作用机制

丁茜; 李振; 刘滨; 凌立平; 张春清

doi:10.3969/j.issn.1001-5256.2017.03.018

卡维地洛抑制血小板衍生因子BB诱导的人肝星状细胞活化和纤维化的作用机制

DOI: 10.3969/j.issn.1001-5256.2017.03.018

山东大学附属山东省立医院消化科

基金项目:

国家自然科学基金资助项目(81370590)；

详细信息

中图分类号: R575.2
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出版历程
- 出版日期: 2017-03-20

Inhibitory effect of carvedilol on human hepatic stellate cell activation and fibrosis induced by platelet-derived growth factor-BB and related mechanisms of action

Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong University

Research funding:

摘要

摘要: 目的研究卡维地洛对人肝星状细胞迁移、侵袭及纤维化作用的影响,并进一步探讨其信号通路机制。方法人肝星状细胞系（LX-2）加入不同浓度（0、1、2、5、10μmol/L）的卡维地洛处理一定时间后,加入血小板衍生因子（PDGF）BB刺激细胞使其激活,分别采用CCK-8检测细胞增殖水平、划痕实验检测细胞迁移能力、Transwell小室进行侵袭能力检测、Real-time PCR和Western Blot检测纤维化相关指标及通路相关蛋白的表达水平。包括空白对照组、PDGF-BB组（只加入PDGF-BB）和4个不同卡维地洛浓度的混合组（1μmol/L组、2μmol/L组、5μmol/L组、10μmol/L组,且均加入了PDGF-BB）。计量资料多组间比较采用单因素方差分析,两组间比较采用Dunnett-t检验。结果卡维地洛可抑制LX-2増殖且呈浓度依赖性,半抑制浓度为28.42μmol/L;和PDGF-BB组比较,10μmol/L组可明显抑制LX-2的迁移能力[（59.780±8.898）%vs（17.270±2.668）%,t=4.576,P=0.010];PDGF-BB可诱导LX-2侵袭能力增...
- 卡维地洛 /
- 肝星状细胞 /
- 肝硬化 /
- 血小板源性生长因子
Abstract: Objective To investigate the effect of carvedilol on the migration, invasion and fibrosis of human hepatic stellate cells, as well as related signaling pathways and mechanisms.Methods Human hepatic stellate cell line LX-2 was treated with different concentrations of carvedilol (0, 1, 2, 5, and 10 μmol/L, and platelet-derived growth factor-BB (PDGF-BB) was added to activate the cells.CCK-8 assay was used to measure cell proliferation, wound healing assay was used to measure migration, Transwell chamber assay was used to measure invasion, and Western blot and real-time PCR were used to measure the protein and mRNA expression of fibrosis markers and pathway proteins.The cells were divided into blank control group, PDGF-BB group (only PDGF-BB was added) , and four carvedilol groups (with 1, 2, 5, or 10 μmol/L carvedilol, as well as PDGF-BB) .A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett-t test was used for comparison between experimental groups and control group.Results Carvedilol inhibited the proliferation of LX-2 cells in a concentration-dependent manner, with a half-inhibitory concentration of28.42 μmol/L.Compared with the PDGF-BB group, the 10 μmol/L carvedilol group had significantly inhibited migration of LX-2 cells (59.780%±8.898% vs 17.270%±2.668%, t=4.576, P=0.010) .PDGF-BB increased the invasion of LX-2 cells, and carvedilol inhibited the invasion of LX-2 cells in a concentration-dependent manner;the invasion of LX-2 cells was reduced from 157.00%±10.52% to 85.15%±13.50% in the 2 μmol/L carvedilol group (t=4.198, P=0.014) , to 55.67%±9.54% in the 5 μmol/L carvedilol group (t=7.133, P<0.01) , and to 45.37%±10.70% in the 10 μmol/L carvedilol group (t=7.438, P<0.01) .The mRNA expression of type I collagen was reduced from 1.068±0.128 to 0.453±0.085 in the 5 μmol/L carvedilol group (t=3.997, P<0.05) and to 0.151±0.019 in the 10 μmol/L carvedilol group (t=7.091, P<0.01) .The mRNA expression of fibronectin was reduced from 1.285±0.042 to 0.879±0.063 in the 1 μmol/L carvedilol group (t=5.345, P<0.01) , to 0.768±0.010 in the 2 μmol/L carvedilol group (t=4.773, P<0.01) , to 0.742±0.117 in the 5 μmol/L carvedilol group (t=4.385, P=0.012) , and to 0.591±0.049 in the 10 μmol/L carvedilol group (t=10.76, P<0.01) .The expression of fibronectin was reduced from 2.103±0.414 to 0.739±0.132 in the 5 μmol/L carvedilol group (t=3.137, P=0.035) and to 0.600±0.114 in the 10 μmol/L carvedilol group (t=3.499, P=0.025) , and the expression of α-smooth muscle actin was reduced from 1.418±0.241 to 0.543±0.215 (t=2.710, P=0.035) and 0.343±0.118 (t=4.005, P <0.01) , respectively.Y751 phosphorylation was reduced from 2.309±0.181 to 1.278±0.304 in the 2 μmol/L carvedilol group (t=2.912, P=0.044) , to 0.555±0.038 in the 5 μmol/L carvedilol group (t=9.476, P<0.01) , and to 0.175±0.039 in the 10 μmol/L group (t=11.51, P<0.01) .Akt phosphorylation was reduced from 1.106±0.185 to 0.335±0.132 in the 5 μmol/L carvedilol group (t=3.386, P=0.015) and to 0.137±0.110 in the 10 μmol/L carvedilol group (t=4.494, P<0.01) .Conclusion Carvedilol can inhibit the proliferation, migration, invasion, and fibrosis of LX-2 cells induced by PDGF-BB, mainly by blocking the PDGF-BB/PDGFR-β/Akt signaling pathway.
- carvedilol /
- hepatic stellate cells /
- liver cirrhosis /
- platelet-derived growth factor

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