转录因子E4BP4通过AMPK-TGF-β1/SMAD3信号转导途径调控病理性心肌纤维化

Regulatory effect of transcription factor E4BP4 on pathological myocardial fibrosis through the AMPK-TGF-β1/SMAD3 signaling pathway

摘要

目的 探讨转录因子腺病毒E4启动子结合蛋白（adenovirus E4 promoter-binding protein，E4BP4）通过腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase，AMPK）-转化生长因子（transforming growth factor，TGF）-β1/SMAD同源物3（Smad homolog 3，SMAD3）通路在调控病理性心肌纤维化的作用。 方法 建立小鼠心脏纤维化模型，分别于模型组和假手术组中检测E4BP4的表达差异。分离和培养原代心脏成纤维细胞，血管紧张素Ⅱ（angiotensin Ⅱ，Ang Ⅱ）刺激增殖活化，分别转染E4BP4过表达质粒（Ang Ⅱ+E4BP4组）、E4BP4干扰质粒（Ang Ⅱ+siE4BP4组）、Ang Ⅱ组和未经Ang Ⅱ处理的对照组。免疫荧光检测α-肌动蛋白（α-smooth muscle actin，α-SMA）荧光强度，细胞增殖检测试剂盒测定细胞活力，聚合酶链式反应检测E4BP4、α-SMA、Ⅰ型胶原蛋白（collagen type Ⅰ，Collagen Ⅰ）和Ⅲ型胶原蛋白（collagen type Ⅲ，Collagen Ⅲ）的表达，蛋白质印迹检测TGF-β1、AMPK和SMAD3的蛋白表达。 结果 与假手术组比较，模型组心肌纤维化程度（38.46±1.21 vs. 3.39±0.39，t=-78.564，P=0.000）、E4BP4蛋白表达量（0.96±0.03 vs. 0.75±0.03，t=-11.480，P=0.000）均明显增加。体外实验发现，与Ang Ⅱ+E4BP4组比较，Ang Ⅱ+siE4BP4组在平均荧光强度（0.05±0.01 vs. 0.42±0.03，F=677.591，P=0.000）、细胞活力（91.30±2.39 vs. 123.74±2.60，F=132.696，P=0.000）、α-SMA（1.26±0.09 vs. 3.59±0.86，F=52.274，P=0.000）、Collagen Ⅰ（1.16±0.11 vs. 3.79±0.89，F=55.336，P=0.000）、Collagen Ⅲ（1.23±0.13 vs. 2.92±0.36，F=119.929，P=0.000）、TGF-β1（0.66±0.04 vs. 0.96±0.02，F=142.954，P=0.000）和p-SMAD3/SMAD3（0.81±0.03 vs. 1.37±0.02，F=739.609，P=0.000）的水平明显降低，而p-AMPK/AMPK的表达量在Ang Ⅱ+siE4BP4组明显高于Ang Ⅱ+E4BP4组（0.89±0.01 vs. 0.58±0.02，F=284.541，P=0.000）。 结论 E4BP4是纤维化调控的关键因子，抑制其表达可通过激活AMPK进而抑制TGF-β1/SMAD3通路发挥抗纤维化作用。

Abstract

Objective To explore the effects of transcription factor adenovirus E4 promoter-binding protein（E4BP4） in regulating pathological myocardial fibrosis through the adenosine monophosphate-activated protein kinase（AMPK）-transforming growth factor （TGF）-β1/Smad homolog 3（SMAD3） pathway. Methods A mouse model of myocardial fibrosis was established，and the expression of E4BP4 was determined in the model group and the sham-operation group. Primary cardiac fibroblasts were isolated，cultured，activated by angiotensin Ⅱ（Ang Ⅱ），and divided into the following groups：Ang Ⅱ+E4BP4 group （transfected with E4BP4 overexpression plasmids），Ang Ⅱ+siE4BP4 group （transfected with E4BP4 interfering plasmids），Ang Ⅱ group，and control group（without Ang Ⅱ treatment）. The fluorescence intensity of ɑ-smooth muscle actin（α-SMA） was determined by the immunofluorescence assay，the cell viability by the cell counting kit，the expression of E4BP4，α-SMA，collagen type Ⅰ（collagen Ⅰ），and collagen type Ⅲ（collagen Ⅲ） by polymerase chain reaction，and the protein expression of TGF-β1，AMPK，and SMAD3 by Western blot. Results Compared with the sham-operation group，the model group showed significantly increased myocardial fibrosis degree（38.46±1.21 vs. 3.39±0.39，t=-78.564，P=0.000） and E4BP4 protein expression（0.96±0.03 vs. 0.75±0.03，t=-11.480，P=0.000）. In vitro experiments found that the mean fluorescence intensity（0.05±0.01 vs. 0.42±0.03，F=677.591，P=0.000），cell viability（91.30±2.39 vs. 123.74±2.60，F=132.696，P=0.000），and the levels of α-SMA（1.26±0.09 vs. 3.59±0.86，F=52.274，P=0.000），collagen Ⅰ（1.16±0.11 vs. 3.79±0.89，F=55.336，P=0.000），collagen Ⅲ（1.23±0.13 vs. 2.92±0.36，F=119.929，P=0.000），TGF-β1（0.66±0.04 vs. 0.96±0.02，F=142.954，P=0.000），and p-SMAD3/SMAD3（0.81±0.03 vs. 1.37±0.02，F=739.609，P=0.000） in the Ang Ⅱ+siE4BP4 group were significantly lower than those in the Ang Ⅱ+E4BP4 group. The expression of p-AMPK/AMPK in the Ang Ⅱ+siE4BP4 group was significantly higher than that in the Ang Ⅱ+E4BP4 group（0.89±0.01 vs. 0.58±0.02，F=284.541，P=0.000）. Conclusion E4BP4 plays a crucial role in the regulation of fibrosis. Inhibition of E4BP4 expression exerts an anti-fibrotic effect by activating AMPK and inhibiting TGF-β1/SMAD3 pathway.

关键词

心肌纤维化 / 转录因子腺病毒E4启动子结合蛋白 / 腺苷酸活化蛋白激酶 / 转化生长因子-β / SMAD同源物3

Key words

myocardial fibrosis / adenovirus E4 promoter-binding protein / adenosine monophosphate-activated protein kinase / transforming growth factor-β / Smad homolog 3

基金资助

贵州省科技计划资助项目(黔科合基础-ZK（2024）一般327)

遵义市科技计划资助项目(遵市科合HZ字（2023）245号)

遵义医科大学博士科研启动基金资助项目(院字（2022）6号)