Baicalein reduces cardiac inflammatory infiltration in EAM mice by blocking the CCL2-CCR2 signaling axis through its binding with TNF-α and CCR2.

Abstract

Background: Myocarditis refers to localized or diffuse inflammatory lesions of the myocardium. Experimental autoimmune myocarditis (EAM) in mice is commonly utilized as an animal model for studying the pathogenesis of myocarditis. Baicalein (BAI), the main active component extracted from Scutellaria baicalensis root, has been proven to possess diverse effects such as anti-inflammatory, anti-tumour, and antioxidant activities. However, further investigation is warranted to elucidate the mechanism of action underlying BAI's efficacy in EAM.

Purpose: The aim of this study is to the potential of BAI in combination with TNF-α to downregulate the TNF-α/TNFR1-AP-1 signaling pathway. Furthermore, we will explore whether BAI exhibits an inhibitory effect on the CCL2/CCR2-ROCK1 signaling pathway.

Study design and methods: In this study, we employed the EAM animal model to investigate the inhibitory effect of BAI on macrophage and Th1 cell chemotaxis towards cardiac tissue in EAM mice. Techniques such as HE staining, immunofluorescence, and other methods were utilized for assessment. Additionally, computer-simulated molecular docking, Streptavidin pull-down, and co-immunoprecipitation experiments were conducted to explore the potential binding of BAI with TNF-α and CCR2. Furthermore, real-time quantitative polymerase chain reaction (qPCR), western blotting, and flow cytometry were employed to elucidate the impact of BAI on the TNF-α/TNFR1-CCL2/CCR2 signaling pathway.

Results: BAI suppressed the expression of chemokine CCL2 in EAM mouse myocardial tissue and attenuated the infiltration of macrophages and Th1 cells. In vitro, BAI exhibited binding affinity to TNF-α, leading to downregulation of the TNF-α/TNFR1-AP-1 signaling pathway and subsequent inhibition of CCL2 secretion by macrophages and vascular endothelial cells. Additionally, BAI demonstrated binding capability to CCR2, resulting in downregulation of the CCL2/CCR2-ROCK1 pathway and consequent inhibition of chemotactic migration of macrophages and Th1 cells.

Conclusion: This study demonstrates that BAI can downregulate the secretion of CCL2 and the CCL2/CCR2-ROCK1 signaling pathway by binding with TNF-α and CCR2, thereby inhibiting the migration of macrophages and Th1 cells to the lesion site, thus alleviating the inflammation severity in EAM.