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

doi:10.16098/j.issn.0529-1356.2015.03.007

解剖学报 ›› 2015 ›› Issue (3) : 329-335. DOI: 10.16098/j.issn.0529-1356.2015.03.007

张剑凯¹崔晓军¹许晓玲² 丁碧蓝²李晋菊² 李涛³吴柱国^4*

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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

ZHANG Jian-kai¹CUI Xiao-jun¹XU Xiao-ling²DING Bi-lan² LI Jin-ju²LI Tao³ WU Zhu-guo ^4*

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摘要

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。

Abstract

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-1］ 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-regulated BTEB gene expression was judged. NRCMs were treated with 100umol/L hydrogen peroxide (H2O2) 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 H₂O₂. Artificially inhibited 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 H₂O₂in 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.

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张剑凯崔晓军许晓玲丁碧蓝李晋菊李涛吴柱国*. 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[J]. 解剖学报. 2015(3): 329-335 https://doi.org/10.16098/j.issn.0529-1356.2015.03.007

ZHANG Jian-kai CUI Xiao-jun XU Xiao-ling DING Bi-lan LI Jin-ju LI Tao WU Zhu-guo*. 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[J]. Acta Anatomica Sinica. 2015(3): 329-335 https://doi.org/10.16098/j.issn.0529-1356.2015.03.007

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