Objective

IL-6-induced STAT3 activation is associated with various chronic inflammatory diseases. In this study, we investigated the anti-STAT3 mechanism of the dietary polyphenol, biochanin A (BCA), in IL-6-treated macrophages.

Methods

The effect of BCA on STAT3 and p38 MAPK was analyzed by immunoblot. The localization of both these transcription factors was determined by immunofluorescence and fractionation studies. The impact on DNA-binding activity of STAT3 was studied by luciferase assay. To understand which of the isoforms of p38 MAPK was responsible for BCA-mediated regulation of STAT3, overexpression of the proteins, site-directed mutagenesis, pull-down assays and computational analysis were performed. Finally, adhesion-migration assays and semi-quantitative PCR were employed to understand the biological effects of BCA-mediated regulation of STAT3.

Results

BCA prevented STAT3 phosphorylation (Tyr⁷⁰⁵) and increased p38 MAPK phosphorylation (Thr¹⁸⁰/Tyr¹⁸²) in IL-6-stimulated differentiated macrophages. This opposing modulatory effect of BCA was not observed in cells treated with other stress-inducing stimuli that activate p38 MAPK. BCA abrogated IL-6-induced nuclear translocation of phospho-STAT3 and its transcriptional activity, while increasing the cellular abundance of phospho-p38 MAPK. BCA-induced phosphorylation of p38δ, but not α, β, or γ was responsible for impeding IL-6-induced STAT3 phosphorylation. Interestingly, interaction with phospho-p38δ masked the Tyr⁷⁰⁵ residue of STAT3, preventing its phosphorylation. BCA significantly reduced STAT3-dependent expression of icam-1 and mcp-1 diminishing IL-6-mediated monocyte adhesion and migration.

Conclusion

This differential regulation of STAT3 and p38 MAPK in macrophages establishes a novel anti-inflammatory mechanism of BCA which could be important for the prevention of IL-6-associated chronic inflammatory diseases.

中文翻译：

---

Biochanin A通过上调IL-6刺激的巨噬细胞中的p38δMAPK磷酸化来阻止STAT3激活。

目的

IL-6诱导的STAT3激活与各种慢性炎症性疾病有关。在这项研究中，我们调查了IL-6处理的巨噬细胞中饮食多酚生物chanin A（BCA）的抗STAT3机制。

方法

通过免疫印迹分析了BCA对STAT3和p38 MAPK的影响。通过免疫荧光和分级研究确定了这两个转录因子的定位。通过荧光素酶测定研究了对STAT3的DNA结合活性的影响。为了了解p38 MAPK的哪些同工型负责BCA介导的STAT3调节，进行了蛋白的过表达，定点诱变，下拉测定和计算分析。最后，通过黏附迁移试验和半定量PCR来了解BCA介导的STAT3调节的生物学效应。

结果

BCA阻止了IL-6刺激的分化巨噬细胞中STAT3磷酸化（Tyr ⁷⁰⁵）并增加了p38 MAPK磷酸化（Thr ¹⁸⁰ / Tyr ¹⁸²）。在用其他激活p38 MAPK的应激诱导刺激物处理的细胞中未观察到BCA的相反调节作用。BCA废除了IL-6诱导的磷酸STAT3的核易位及其转录活性，同时增加了磷酸p38 MAPK的细胞丰度。BCA诱导的p38δ磷酸化，而不是α，β或γ负责阻止IL-6诱导的STAT3磷酸化。有趣的是，与磷酸-p38δ的相互作用掩盖了STAT3的Tyr ⁷⁰⁵残基，阻止了其磷酸化。BCA显着降低STAT3依赖性 icam-1和 mcp-1 减少了IL-6介导的单核细胞粘附和迁移。

结论

巨噬细胞中STAT3和p38 MAPK的这种差异调节建立了BCA的新型抗炎机制，这对于预防与IL-6相关的慢性炎性疾病可能很重要。