周期性拉伸通过 p38MAP 激酶和 NF-κB 通路增强培养心肌细胞中 Toll 样受体 4 基因的表达。

Cyclic stretch enhances the expression of toll-like receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-kappaB pathway.

机构信息

Department of Emergency Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.

出版信息

J Biomed Sci. 2010 Mar 5;17(1):15. doi: 10.1186/1423-0127-17-15.

DOI:10.1186/1423-0127-17-15

PMID:20202224

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2844375/

Abstract

BACKGROUND

Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes.

METHODS

Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected.

RESULTS

Cyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-alpha antibody, and p38alpha MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-kappaB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-kappaB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-alpha antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-alpha and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-alpha antibody, or p38alpha siRNA 30 min before Ang II and TNF-alpha stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-alpha.

CONCLUSIONS

Cyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-kappaB pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

摘要

背景

Toll 样受体 4（TLR4）在先天免疫中发挥重要作用。TLR4 在拉伸的心肌细胞中的作用尚不清楚。我们试图研究机械拉伸是否可以调节 TLR4 的表达，以及循环机械拉伸在心肌细胞中调节 TLR4 表达的可能分子机制和信号通路。

方法

在柔性膜基底上生长的新生 Wistar 大鼠心肌细胞通过真空拉伸至最大伸长的 20%，频率为 60 次/分钟。进行 Western blot、实时聚合酶链反应和启动子活性测定。检测体外拉伸的心肌细胞对单核细胞的黏附作用。

结果

循环拉伸在拉伸后 2 小时至 24 小时显著增加 TLR4 蛋白和 mRNA 的表达。在拉伸前 30 分钟加入 SB203580、TNF-α抗体和 p38αMAP 激酶 siRNA 可抑制 TLR4 蛋白的诱导。循环拉伸增加，而 SB203580 则消除了循环拉伸诱导的磷酸化 p38 蛋白。凝胶移位实验显示，拉伸后 NF-κB 的 DNA-蛋白结合活性显著增加，而 SB203580 则消除了循环拉伸诱导的 DNA-蛋白结合活性。由循环拉伸诱导的 DNA 结合复合物可被 p65 单克隆抗体超迁移。循环拉伸增加 TLR4 启动子活性，而 SB203580 和 NF-κB siRNA 则降低 TLR4 启动子活性。循环拉伸增加单核细胞与心肌细胞的黏附，而 SB203580、TNF-α抗体和 TLR4 siRNA 则减弱单核细胞的黏附。TNF-α和 Ang II 显著增加 TLR4 蛋白的表达。在 Ang II 和 TNF-α刺激前 30 分钟加入氯沙坦、TNF-α抗体或 p38αsiRNA 可显著阻断 Ang II 和 TNF-α 对 TLR4 蛋白的增加。