MAPK抑制因子对HSC中Smad2/3磷酸化及Smad4核转位的影响  

Effects of MAPK inhibitors on phosphorylation of Smad2 /3 and nuclear translocation of Smad4 in HSCs

作　　者：江宇峰[1] 伍超[1] 吴佳俊[1] 王极宇 马成骁 陈帆[1] 吴德卫[1] 杨明[1] 郑凌云[1] 杨雁[1] 

机构地区：[1]安徽医科大学药理学教研室,合肥230032

出　　处：《安徽医科大学学报》2015年第9期1243-1247,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81073098;81374012)

摘　　要：目的探究3种丝裂原活化蛋白激酶(MAPK)抑制因子对转化生长因子-β(TGF-β)活化的大鼠肝星状细胞(HSC)中Smad2/3磷酸化及Smad4核转位的影响。方法采用Ⅳ型胶原酶和链酶蛋白酶原位肝灌流法分离正常大鼠HSC并传代培养,分别用细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38抑制因子(PD98059、SP600125、SB203580)与HSC共同培养,再加入TGF-β1活化细胞,采用免疫沉淀和Western blot法检测p Smad2C、p Smad2L、p Smad3L蛋白表达;采用细胞免疫荧光法检测Smad4蛋白表达及细胞内定位情况。结果 Western blot显示静息状态下HSC几乎不表达p Smad2C而低表达p Smad2L、p Smad3L,TGF-β1刺激后显著上调上述磷酸化蛋白表达,p38抑制因子(1、3μmol/L)、ERK抑制因子(1μmol/L)对Smad2C、Smad2L、Smad3L磷酸化无显著影响;p38抑制因子(10μmol/L)降低了p Smad3L蛋白表达;ERK抑制因子(3、10μmol/L)降低了p Smad2C蛋白表达;JNK抑制因子(1、3、10μmol/L)减少了p Smad2C、p Smad2L、p Smad3L蛋白表达。TGF-β1刺激可明显诱导Smad4核转位,而3种MAPK抑制因子不同程度地抑制TGF-β1诱导的Smad4转位入核。结论在HSC细胞中TGF-β1可能通过活化JNK通路促进Smad2/3磷酸化,活化ERK、JNK、p38通路促进Smad4核转位。Objective To investigate the effects of three MAPK inhibitors on phosphorylation of Smad2/3 and nu- clear translocation of Smad4 in TGF-β1-activated hepatic stellate cell （HSC）. Methods HSC was isolated from normal rat liver by using collagenase IV and pronase-E digestion in situ and continuously cultured in vitro. The cells in log phase were pretreated with ERK inhibitor （PD98059）, JNK inhibitor （SP600125） and p38 inhibitor （SB203580） in corresponding group respectively, then activated by TGF-β1. The protein expressions of phospho- rylated （p）Smad2C, pSmad2L and pSmad3L were measured by immunoprecipitation（IP） and western blot assay. The protein expression and intracellular localization of Smad4 were assessed by cell immunofluorescence assay. Results The protein expressions of phosphorylated Smad2C, Smad2L and Smad3L were feeble in quiescent HSC, while markedly increased by TGF-β1 stimulation. The two concentrations of p38-specific inhibitor （1, 3μmol/L） and low concentration ERK-specific inhibitor （ 1 μmol/L） had no significant effect on the phosphorylation of Smad2/3; the high concentration of p38-specific inhibitor （10 μmoL/L） significantly down-regulated elevated pSmad2C by TGF-β1; the two concentrations of ERK-specific inhibitor （3, 10μmol/L） could remarkably de- crease elevated pSmad2C by TGF-β1 ; the three concentrations of JNK-specific inhibitor （ 1,3, 10μmol/L） inhib- ited the phosphorylation of Smad2C, Smad2L and Smad3L stimulated by TGF-β1. All three MAPK-specific inhibi- tors suppressed increased nuclear translocation of Smad4 protein in TGF-β1-stimulated HSCs. Conclusion The phosphorylation of Smad2/3 and nuclear translocation of Smad4 might be induced by TGF-β1 via activating ERK, JNK, p38 pathways in HSCs.

关 键 词：大鼠肝星状细胞 丝裂原活化蛋白激酶抑制剂 SMAD2 Smad3 SMAD4 

分 类 号：R349.5[医药卫生—基础医学]