苦参碱对乙醛活化的肝星状细胞CFSC-8B增殖和胶原合成的影响及机制研究  

Study on the Effects and Mechanism of Matrine on Proliferation and Collagen Synthesis of Hepatic Stellate Cells CFSC-8B Activated by Acetaldehyde

作　　者：王晓丽[1] WANG Xiaoli(School of Pharmacy,Qiqihar Medical University,Heilongjiang Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院药学院,黑龙江齐齐哈尔161006

出　　处：《中国药房》2020年第11期1353-1358,共6页China Pharmacy

基　　金：黑龙江省自然科学基金面上项目(No.H2016098);黑龙江省省属高等学校基本科研业务费科研项目(No.2018-KYYWF-0099)。

摘　　要：目的:研究苦参碱对乙醛活化的大鼠肝星状细胞CFSC-8B增殖和胶原合成的影响,并探讨其可能的作用机制。方法:取体外培养的CFSC-8B细胞,分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱低、中、高浓度组(30、60、120μmol/L)。除空白对照组外,其余各组细胞均加入200μmol/L乙醛溶液诱导活化,并同时加入相应药液(空白对照组和模型组加入等体积空白培养液),共同作用24 h后,采用CCK-8法检测各组细胞的存活率。另取细胞分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱中、高浓度组(60、120μmol/L),同法活化和加药处理。分别采用酶消化法检测细胞培养液中羟脯氨酸(Hyp)含量;采用酶联免疫吸附法检测细胞培养液中Ⅰ型胶原蛋白(Col-Ⅰ)和Ⅲ型胶原蛋白(Col-Ⅲ)含量;采用实时荧光定量-聚合酶链式反应法检测细胞中α-平滑肌激动蛋白(α-SMA)、转化生长因子β(1 TGF-β1)、TGF-β1Ⅰ型受体(TβR-Ⅰ)、TGF-β1Ⅱ型受体(TβR-Ⅱ)、Smad3、Smad4和Smad7 mRNA表达水平;采用Western blotting法检测细胞中α-SMA、TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3、Smad4和Smad7蛋白表达水平。结果:与空白对照组比较,模型组细胞存活率显著升高(P<0.05);细胞培养液中Hyp、Col-Ⅰ、Col-Ⅲ含量和细胞中α-SMA、TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3和Smad4 mRNA及其蛋白表达水平均显著升高(P<0.05),细胞中Smad7 mRNA及其蛋白表达水平均显著降低(P<0.05)。与模型组比较,阳性对照组和苦参碱中、高浓度组细胞存活率和细胞培养液中Hyp、Col-Ⅰ、Col-Ⅲ含量以及细胞中α-SMA、Smad4 mRNA及其蛋白表达水平均显著降低(P<0.05),细胞中Smad7 mRNA及其蛋白表达水平均显著升高(P<0.05);阳性对照组和苦参碱高浓度组细胞中TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3 mRNA及其蛋白表达水平均显著降低(P<0.05)。与苦参碱中浓度组比较,苦参碱高浓度组细胞�OBJECTIVE:To study the effects of matrine on proliferation and collagen synthesis of rat hepatic stellate cells CFSC-8B activated by acetaldehyde,and to investigate its possible mechanism.METHODS:CFSC-8B cells cultured in vitro were divided into blank control group,model group,positive control group(2.5μmol/L colchicine)and matrine low,medium and high concentration groups(30,60,120μmol/L).Except for blank control group,other groups were activated with 200μmol/L acetaldehyde for 24 h;medicine groups were intervened with relevant medicine for 24 h(blank control group and model group were intervened with equal volume blank medium).Survival rate of cell was detected by CCK-8 assay.Cells were divided into blank control group,model group,positive control group(2.5μmol/L colchicine),matrine medium and high concentration groups(60,120μmol/L),then activated and treated with same method.Hydroxyprolin(Hyp)content in cell culture solution was tested by enzyme digestion.The contents of Col-Ⅰand Col-Ⅲin cell culture solution were determined by ELISA.mRNA expressionss ofα-SMA,TGF-β1,TβR-Ⅰ,TβR-Ⅱ,Smad3,Smad4 and Smad7 in cells were detected by RT-PCR.The protein expressions ofα-SMA,TGF-β1,TβR-Ⅰ,TβR-Ⅱ,Smad3,Smad4 and Samd7 in cells were detected by Western blotting.RESULTS:Compared with blank control group,survival rate of cells in model group was increased significantly(P<0.05);the contents of Hyp,Col-Ⅰand Col-Ⅲin cell culture solution,mRNA and its protein expressions ofα-SMA,TGF-β1,TβR-Ⅰ,TβR-Ⅱ,Smad3,Smad4 in cells were increased significantly in model group(P<0.05),while the mRNA and protein expression of Smad7 was decreased significantly(P<0.05).Compared with model group,survival rate of cells,the contents of Hyp,Col-Ⅰand Col-Ⅲin cell culture solution,the mRNA and protein expressions ofα-SMA and Smad4 were decreased significantly in positive control group and matrine medium and high concentration groups(P<0.05),while the mRNA and protein expression of Smad7 was increased significantly(P

关 键 词：苦参碱 肝星状细胞 增殖 胶原合成 转化生长因子β1/Smad通路 机制 

分 类 号：R962[医药卫生—药理学]