三羟基异黄酮对增生性瘢痕成纤维细胞受体型酪氨酸蛋白激酶-丝裂原活化蛋白激酶信号通路的影响

目的 了解5,7,4'-三羟基异黄酮(genistein)对增生性瘢痕成纤维细胞(HSFb)内受体型酪氨酸蛋白激酶-丝裂原活化蛋白激酶(TPK-MAPK)信号转导通路的影响,探讨genistein抑制瘢痕增生的分子机制. 方法体外分离培养HSFb,以不同浓度genistein(25、50、100μmol/L)处理细胞,再加入10 μg/L碱性成纤维细胞生长因子(bFGF)刺激,用γ-32P]腺苷三磷酸底物掺入法检测细胞TPK活性;用蛋白质印迹法检测TPK-MAPK通路中主要信号蛋白分子c-Raf、丝裂原激活蛋白激酶激酶(MEK)、细胞外信号调节激酶(ERK)、p38 MAPK、c-Jun氨基末端激酶(JNK)磷酸化蛋白的表达变化.以二甲亚砜溶剂处理的HSFb为对照组. 结果 25、50、100μmol/L genistein作用后,HSFb内TPK活性分别为(7.15±0.35)、(5.62±0.88)、(3.17±0.94)×105 pmol·min-1·mg-1,与对照组(8.92±0.28)×105 pmol·min-1·mg-1比较,差异有统计学意义(P<0.05或P<0.01);细胞内磷酸化c-Raf、MEK1/2、ERK1/2、p38蛋白的含量均有不同程度降低,与对照组比较,差异有统计学意义(P<0.05或P<0.01).genistein各剂量组磷酸化JNK蛋白含量与对照组近似.在genistein预处理前提下,加入bFGF刺激的细胞内TPK活性及各信号蛋白的表达亦呈下降趋势. 结论 genistein可通过抑制细胞受体TPK信号转导途径影响HSFb的增殖与活化,主要信号通路可能为TPK→Raf→MEK→ERK/p38途径.

The effects of genistein on tyrosine protein kinase-mitogen activated protein kina8e signal transduction pathway in hypertrophic scar fibroblasts

Objective To investigate the inhibitory effects of genistein on tyrosine Drotein kinase (TPK)-mitogen activated protein kinase(MAPK)signal transduction pathway in hypertrophic scar fibroblasts (HSFb), in order to explore the molecular mechanism of inhibition of scar hyperplasia by genistein.Methods HSFbs were isolated from human hypertrophic scar tissues and cultured in vitro.The cells were treated by genlstem in different concentrations(25,50,100μmol/L,respectively),followed by basic fibroblast growth factor(bFGF)stimulation.The activity of TPK was assessed withγ-32 P]ATP substrate incorporatmn.The phosphorylation protein expression levels of main signal molecules in TPK-Ras-MAPK pathway including c-Raf,MEK1/2,extracellular signal regulated kinase(ERK),p38 MAPK,c-Jun N-terminal kinase (JNK)were determined by Western blot.HSFbs treated with dimethylsulfoxid(DMSO)were used as control group. Results After being treated with genistein in concentration of 25,50,100 μmol/L,the activity of TPK in HSFbs was depressed significantly(7.15±0.35)×105,(5.62±0.88)×106,(5.62±0.88)×sion levels of c-Raf,MEK1/2,ERK1/2 and p38 MAPK were lowered in different degree(P<0.05 or P<0.01 )after genistein treatment.The phospho-JNK levels after treatment with genistein were similar to that of control group. Under the condition of pretreatment with genistein,the activities of TPK and signal pathway protein expressions in HSFb showed a downward trend after stimulation with bFGF. Conelusion Genistein can effect the proliferation and activation of HSFb by inhibiting the phosphorylation of receptor of TPK signal transduction pathway(TPK→Raf→MEK→ERK/p38).