IGF-1通过BTEB调控CYPs途径保护心肌细胞免于凋亡
收稿日期: 2014-05-08
网络出版日期: 2015-02-12
基金资助
广东省自然科学基金（No.S201301 0011763）；广东省科技计划项目（No.2011B031600007, No.2013B021800066）
IGF-1 can protect cardiomyocytes from apoptosis through BTEB and CYPs pathway
Received date: 2014-05-08
Online published: 2015-02-12
目的 探讨胰岛素样生长因子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，通过RT-PCR及Werstern-blot观察IGF-1调节BTEB及其下游细胞色素C家族成员CYP1A1、2E1、3A11和11A1的基因表达情况。过氧化氢处理诱导心肌细胞凋亡，通过Annexin V-FITC/PI双染色法、Caspase 3活性测定、Hoechest33258染色法和Tunel法观察下调CYPs的基因表达对心肌细胞凋亡的影响；JC-1线粒体膜电位检测法和透射电镜观察心肌细胞线粒体膜电位和形态的改变。 结果 大鼠心肌细胞经IGF-1刺激60 min后，BTEB mRNA和蛋白表达均明显下降；IGF-1刺激48 h后，CYP1A1、2E1、3A11和11A1的mRNA和蛋白表达均明显下降（均P<0.01）；用BTEB特异性的siRNA人为下调BTEB基因表达后，CYP1A1、3A1 mRNA和蛋白表达明显下降（均P<0.01），CYP2E1、11A1 mRNA和蛋白表达变化不明显。与对照组相比，H2O2诱导的大鼠心肌细胞用CYP1A1、2E1、3A11和11A1特异性的siRNA人为下调上述基因表达后，Annexin V-FITC/PI双染法显示心肌细胞凋亡率下降（均P<0.05）、Caspase 3活性测定显示酶活性降低（均P<0.05）、Hoechest33258染色和Tunel法检测结果显示凋亡小体和凋亡细胞数目减少，与IGF-1的抗心肌细胞凋亡效果相似；且细胞线粒体膜电位下降率明显降低（均P<0.05），线粒体形态明显改善。 结论 IGF-1可以通过下调细胞色素C家族成员CYP1A1、2E1、3A11和11A1的表达改善线粒体功能，其中CYP1A1、3A11受BTEB调控途径发挥抗心肌细胞凋亡作用。
关键词： 心肌细胞; 凋亡; 基本转录元件结合蛋白(BTEB); 细胞色素C; 胰岛素样生长因子1(IGF-1)
张剑凯 , 丁碧蓝 , 许晓玲 , 李涛 , 吴柱国 . IGF-1通过BTEB调控CYPs途径保护心肌细胞免于凋亡[J]. 中国临床解剖学杂志, 2015 , 33(1) : 64 -71 . DOI: 10.13418/j.issn.1001-165x.201
Objective To investigate gene regulation mechanism of the anti-apoptotic effect of the insulin-like growth factor 1 on rat cardiomyocytes. Methods Primary neonatal rat cardiomyocytes (NRCMs) were cultured in vitro，the transcription and translation levels of BTEB and its downstream genes Cytochrome P450 family members (CYP1A1, 2E1, 3A11 and 11A1) were measured by RT-PCR and Western-Blot. NRCMs were treated with 100umol/L Hydrogen peroxide (H2O2) to induce apoptosis. CYP1A1, 2E1, 3A11 and 11A1 specific siRNA were transfected into the cells by Lipofectamine 2000. Myocardial cells apoptosis was detected by Annexin V-FITC／PI dual staining，Caspase-3 activity assay，Hoechst33258 staining and Tunel method. The mitochondrial function was measured by JC-1 mitochondrial membrane potential assay．The mitochondrial morphology was observed by transmission electron microscopy. Results The mRNA and protein expression levels of BTEB gene in NRCMs were down-regulated significantly after IGF-1 had stimulated for 60 minutes with concomitant attenuation of CYP1A1, 2E1, 3A11 and 11A1 expression levels 48 hours later (all P <0.01). Artificial inhibition of BTEB gene expression with BTEB specific siRNA could down-regulate CYP1A1, 3A11 mRNA and protein expression obviously (P<0.01), but CYP2E1, 11A1 mRNA and protein expression was not obvious. Compared with the control group, the apoptotic rates of NRCMs induced by H2O2 in IGF-1 group and CYP1A1, 2E1, 3A11 and 11A1 specific siRNA groups were reduced(all P<0.05)，decreased caspase-3 activity(all P<0.05), reduced apoptotic cells numbers and apoptotic bodies were also observed in these groups. The anti-apoptotic effect of CYP1A1, 2E1, 3A11 and 11A1 genes silencing on NRCMs was similar with that of IGF-1 treatment. And the fall rates of mitochondrial membrane potential in IGF-1 group and CYP1A1, 2E1, 3A11 and 11A1 specific siRNA groups declined significantly (all P<0.05 vs control group), mitochondrial morphology obviously improved. Conclusion IGF-I protects rat cardiomyocytes from apoptosis through down-regulation of Cytochrome P450 family members (CYP1A1, 2E1, 3A11 and 11A1) expression pathways and improving mitochondrial function, of which CYP1A1 and 3A11 were regulated by BTEB.
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