胰岛素样生长因子-1通过细胞外信号调节激酶1/2通路下调转录因子基本转录元件结合蛋白表达抗心肌细胞凋亡

Insulin-like growth factor-1 protecting cardiomyocytes from apoptosis by down-regulating transcription factor basic transcription element binding protein through extracellular regulated kinase 1 /2 pathway

目的探讨胰岛素样生长因子-1(IGF-1)对大鼠心肌细胞凋亡保护作用的基因调控机制。方法体外培养新生大鼠心肌细胞,10nmol/L IGF-1刺激的同时,分别加入磷脂酰肌醇-3激酶(PI3K)、细胞外信号调节激酶(ERK)1/2和Raf-1 3条通路抑制剂(20μmol/L),通过RT-PCR及Western blotting方法观察IGF-1调节基本转录元件结合蛋白(BTEB)的基因表达及其通路调控。100μmol/L H2O2处理诱导心肌细胞凋亡,通过DNA梯度分析、Annexin V-FITC/PI双染色法、Caspase-3活性测定、Hoechest33258染色法观察用BTEB特异性siRNA人为下调BTEB基因表达后对心肌细胞凋亡的影响。结果大鼠心肌细胞经IGF-1刺激60min后,BTEB mRNA和蛋白表达均明显下降;与对照组相比,加入ERK1/2通路抑制剂PD98059组BTEB的mRNA和蛋白表达均明显增高(P<0.01);H2O2诱导的大鼠心肌细胞于下调BTEB表达后,DNA片段化改善,心肌细胞凋亡率下降(P<0.05),Caspase-3活性降低(P<0.05),凋亡小体减少,与IGF-1的抗心肌细胞凋亡效果相似。结论 IGF-1可以通过ERK1/2通路下调转录因子BTEB基因表达而发挥抗心肌细胞凋亡的作用。

Objective To investigate gene regulation mechanism of insulin-like growth factor-1 （IGF-1） anti- apoptotic effect on rat cardiomyocytes. Methods Primary neonatal rat cardiomyocytes （NRCMs） were cultured in vitro, IGF-1 （10nmol/L） was added with different signal transduction pathway inhibitors [ phosphatidylinositol 3-kinase（PI3K） , extracellular regulated kinase （ERK） 1/2 and Raf-11 respectively （20μmol/L）. The gene expression of basic transcription element binding protein （BTEB） was detected by RT-PCR and Western blotting, by which the pathway of IGF-1 down- regulateyl BTEB gene expression was judged. NRCMs were treated with 100umol/L hydrogen peroxide （H202 ） to induce apoptosis. BTEB specific siRNA was transfected into the cells by Lipofectamine 2000. Myocardial cells apoptosis was detected by DNA-ladder analysis, Annexin V-FITC/PI dual staining,Caspase-3 activity assay and Hoechst33258 staining. Results The mRNA and protein expression levels of BTEB gene in NRCMs were down-regulated significantly after IGF-1 had stimulated for 60 minutes. Compared with control groups, BTEB mRNA and protein expression in ERK1/2 pathway inhibitor PD98059 group was significantly higher （P 〈 0.01 ）. The apoptosis of NRCMs was induced by H202. Artificiallyinhibited BTEB gene expression with BTEB specific siRNA, BTEB mRNA and protein expression decreased obviously （P 〈 0.05）. Compared with control group, the apoptotic rates of NRCMs induced by H202in IGF-1 group and BTEB specific siRNA groups were declined （ all P 〈 0.05 ） , decreased Caspase-3 activity （ all P 〈 0.05 ） , attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these groups. The anti-apoptotic effect of BTEB gene silencing on NRCMs was similar with that of IGF-1 treatment. Conclusion IGF-1 protects cardiomyocytes from apoptosis by down- regulating transcription factor BTEB through ERK1/2 pathway.