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目的研究心肌营养素-1(cardiotrophin-1,CT-1)对心肌转录因子GATA4的表达影响以及其3条主要信号通路,即JAK-信号传导子和转录激活子3(JAK-STST3)通路、细胞外信号调节激酶1/2(ERK1/2)及磷脂酰肌醇3-激酶(PI3-K)在其中发挥的作用。方法应用半定量RT-PCR及EMSA技术,测定CT-1对大鼠心肌细胞GATA4 mRNA及结合活性表达的时间、剂量依赖影响。通过单独或联合应用Parthenolide(STAT3通路阻断剂)、U-0126(ERK通路阻断剂)及LY-294002(PI3-K通路阻断剂),分析GATA4两指标的表达变化阐述3条信号通路在该过程中发挥的作用。结果时间依赖实验,0.1nmol/L的CT-1刺激后,GATA4 mRNA表达在3h出现显著性增加,刺激6h以后均表现为极显著性差异(P<0.01),6h以后各组间差异无统计学意义。GATA4结合活性在刺激10min后开始增加,到60 min时达到最大值,180 min下降至刺激前水平。剂量依赖实验,GATA4 mRNA表达在CT-1的刺激浓度为0.01 nmol/L以上时各组均表现为极显著性增加(P<0.01)。GATA4结合活性在0.01nmol/L时,灰度值较无刺激组略增高,0.1 nmol/L时灰度值达到最高值,以后强度略减弱并维持该水平。通路实验结果显示,U-0126可以增加CT-1刺激后GATA4 mRNA(P<0.05)的表达和结合活性的增加,Parthenolide可以显著降低GATA4 mRNA(P<0.01)和结合活性的表达,应用LY- 294002后GATA4相关指标与对照组差异无统计学意义。此外,Parthenolide可以抑制由U-0126引起的GATA4 mRNA表达的增加(P<0.01)及结合活性的增高。结论CT-1可引起大鼠心肌转录因子GATA4转录及结合活性的表达增强。STAT3通路在CT-1刺激GATA4表达中起关键性作用,ERK通路则通过增强STAT3通路来促进CT-1对GATA4的表达影响,P13-K通路在两者的相关关系中不发挥作用。
Objective To investigate the effects of cardiotrophin-1 (CT-1) on the expression of myocardial transcription factor GATA4 and its three main signal pathways, JAK-STAT3 and JAK-STST3, The role of extracellular signal-regulated kinase 1/2 (ERK1 / 2) and phosphatidylinositol 3-kinase (PI3-K) in this role. Methods The semi-quantitative RT-PCR and EMSA techniques were used to determine the time-and dose-dependent effects of CT-1 on the expression of GATA4 mRNA and its binding activity in rat cardiomyocytes. The changes of GATA4 expression were analyzed by using Parthenolide (STAT3 pathway blocker), U-0126 (ERK pathway blocker) and LY-294002 (PI3-K pathway blocker) alone or in combination. The role played in the process. Results After time-dependent experiments, the expression of GATA4 mRNA increased significantly at 3 h after stimulated with 0.1 nmol / L CT-1, and the difference was significant after 6 h (P <0.01) Significance of learning. GATA4 binding activity began to increase 10min after stimulation, reached the maximum at 60min, and decreased to pre-stimulation level at 180min. In a dose-dependent manner, GATA4 mRNA expression showed a highly significant increase (P <0.01) when CT-1 stimulation was above 0.01 nmol / L. When GATA4 binding activity was 0.01nmol / L, the gray value of GATA4 was slightly higher than that of the non-stimulated GATA4. The gray value of GATA4 reached the highest value at 0.1 nmol / L. Pathway experiments showed that U-0126 could increase the expression of GATA4 mRNA (P <0.05) and increase the binding activity after CT-1 stimulation. Parthenolide could significantly decrease the expression of GATA4 mRNA (P <0.01) and the binding activity. 294002 after GATA4 related indicators and the control group, the difference was not statistically significant. In addition, Parthenolide can inhibit the increase of GATA4 mRNA expression (P <0.01) and the increase of binding activity induced by U-0126. Conclusion CT-1 can induce the expression of transcriptional and binding activity of GATA4 in rat myocardium. STAT3 pathway plays a key role in the expression of GATA4 induced by CT-1, and ERK pathway promotes the expression of GATA4 by enhancing the STAT3 pathway. P13-K pathway does not play a role in the relationship between the two.