丹参酚酸B盐对转化生长因子β1活化的大鼠肝星状细胞内p38丝裂原活化蛋白激酶通路及其转录因子的影响  

Effects of salvianolic-acid B on the p38MAPK signaling pathway and its transcriptional factor induced by transforming growth factor β1 in activated rat hepatic stellate eells

作　　者：宋雅芳[1] 吕志刚[1] 徐列明[1,2] 

机构地区：[1]上海中医药大学附属曙光医院上海中医药大学肝病研究所,201203 [2]肝肾疾病病证教育部重点实验室上海市中医临床重点实验室

出　　处：《中华肝脏病杂志》2012年第12期902-907,共6页Chinese Journal of Hepatology

基　　金：基金项目：国家自然科学基金（30271657）、国家中医药管理局中医肝胆病重点学科（2010sh）、上海市教育委员会重点学科（第五期）建设项目（J50307）、上海市高校E-研究院项目（E03008）、上海市高校创新团队建设项目（第一期）（沪教委科C200936号）

摘　　要：目的探讨丹参酚酸B盐（SA—B）对转化生长因子β1（TGFβ1）活化的大鼠肝星状细胞（HSC）内p38丝裂原活化蛋白激酶（MAPK）信号传导通路的影响。方法分离并培养正常大鼠HSC，将TGFD1和SA-B直接添加于原代HSC的无血清培养液中，用p38信号通路特异性阻断剂SB203580和细胞外信号调节激酶（ERK）信号通路特异性阻断剂PD98059分别阻断HSC内p38MAPK和ERK信号通路。细胞内总的P38蛋白、MKK3／6蛋白及MEF2A、MEF2C的测定分为空白对照组、SA—B组、SA-B＋TGFβ1组和TGFβ1组磷酸化P38蛋白、MKK3／6蛋白和α-SMA蛋白的测定分为空白对照组、SA-B组、SA-B＋TGFβ1组、TGFβ1组、PD98059、PD98059＋SA—B组、PD98059＋TGFβ1组和SA—B＋PD98059＋TGFβ1组SA—B对TGFβ1刺激的HSC内MEF2和Ⅰ型胶原报道基因的影响分为突变型（mt）对照组、野生型（wt）对照组、TGFβ1组、SA-B＋TGFβ1组、SA-B组、SB203580＋TGFβ1组、SB203580组。Westem blot法检测HSC内磷酸化和总P38蛋白、MAPK激酶3／6（MKK3／6）蛋白、肌细胞增强因子2（MEF2）A、MEF2C、α-平滑肌肌动蛋白（α-SMA）的表达，荧光素酶报道基因测定法检测MEF2报道基因和Ⅰ型胶原启动子的活性。多组间数据的多重比较用q检验。结果SA-B组磷酸化P38蛋白相对表达量为0．334-0．05，明显低于空白对照组（q=7．08，（P〈0．01）；SA-B＋TGFβ1组的磷酸化P38蛋白相对表达量为0．46±0．04，明显低于TGFD1组（q=10．45，P〈0．01）;SA-B组磷酸化MKK3／6蛋白相对表达量为0．11±0．07，明显低于空白对照组（q=3．944，JP〈0．05）；SA-B＋TGFβ1组磷酸化MKK3／6蛋白相对表达量为0．28±0．07，明显低于TGFβ1组（q=7．91，P〈0．01）;SA-B＋TGFD1组和SB203580＋TGFβ1组MEF2报道基因的相对荧光素酶活性分别为2．93±0．09和2．50±0．05，均明显低于TGFβ1组（q值分别为35．35和37．2，P值均〈0．01）；SA-B组MEF2C及MEF2A的�Objective To investigate the effects of Salvianolic-acid B on p38MAPK signaling pathway and its transcriptional factor activated by Transforming growth factor β1 in rat hepatic stellate cells. Methods Hepatic stellate cells were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method.TGFβ1 （10 ng/ml）, PD98059（50 μmol/L）, SB203580（10 μmol/L） and SA-B （10μmol/ L） were direαly added to the medium of the isolated HSCs. Groups： （1）The detection of total p38, MKK3/6, MEF2A and MEF2C induced by TGFβ1 in HSC： include control group, SA-B group, SA-B ＋ TGFβ1 group and TGFβ1 group. （2）The detection of the phosphorylation of p38, MKK3/6 and α-SMA induced by TGFβ1 in HSC： include control group, SA-B group, SA-B ＋ TGFβ1 group, TGFβ1 group, PD98059 group, PD98059 ＋ SA-B group, PD98059 ＋ TGFβ1 group and SA-B ＋ PD98059 ＋ TGFβ1 group. （3）The effects of SA-B on activity of MEF2 reporter and collagen α l（I） reporter induced by TGFβ1 in HSC： include mt group, wt group, TGFβ1 group, SA-B ＋ TGFβ1 group, SA-B group, SB203580 ＋ TGFβ1 group and SB203580 group. Total and phosphorylated p38 and MKK3/6, MEF2A, MEF2C and α-SMA were assayed by Western blot. HSCs were transfected with either MEF2 or collagen α l（I） luciferase reporter gene by Lipofectamine 2000 transfection method, Cellular extracts were assayed for both MEF2 and collagen αl（I） luciferase activities. Comparisons between groups were performed with Student-Newman-Keuls test. Results The relative expression level of the phosphorylation of p38 of SA-B group is 0.33 ±0.05,obviously lower than control group（q = 7.08, P 〈 0.01）; SA-B ＋ TGFβ1 group is 0.46 ± 0.04, obviously lower than TGF 131 group（q = 10.45, P〈 0.01）; The relative expression level of the phosphorylation of KK3/6 of SA-B group is 0.11 0.07, obviously lower than control group（q = 3.944, P 〈 0.05）; SA-B ＋ TGF β1 group is 0.28 ＋ 0.07, obviously lower than TGFβ1 group

关 键 词：转化生长因子Β1 丝裂原活化蛋白激酶类 信号传导 丹参酚 肝星状细胞 

分 类 号：R285.5[医药卫生—中药学]