Tong Huimin, Wang Shuang, Liu Meng, Wang Tiantian, Jia Xihui, Li Yuanyuan, Yu Dongyu, Li Ling
Department of Human Anatomy, Histology and Embryology, School of Basic Medical, Qingdao University, Qingdao, China.
Department of Biochemistry, School of Basic Medical, Qingdao University, Qingdao, China.
J Nutr Biochem. 2025 Jul 11:110029. doi: 10.1016/j.jnutbio.2025.110029.
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.
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.
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.
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.
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.
心肌炎是指心肌的局限性或弥漫性炎性病变。小鼠实验性自身免疫性心肌炎（EAM）常被用作研究心肌炎发病机制的动物模型。黄芩苷（BAI）是从黄芩根中提取的主要活性成分，已被证明具有抗炎、抗肿瘤和抗氧化等多种作用。然而，有必要进一步研究BAI在EAM中发挥作用的机制。
本研究旨在探讨BAI联合肿瘤坏死因子-α（TNF-α）下调TNF-α/TNFR1-AP-1信号通路的潜力。此外，我们将探究BAI是否对CCL2/CCR2-ROCK1信号通路具有抑制作用。
在本研究中，我们采用EAM动物模型来研究BAI对EAM小鼠心肌组织中巨噬细胞和Th1细胞趋化性的抑制作用。使用苏木精-伊红（HE）染色、免疫荧光等技术进行评估。此外，进行计算机模拟分子对接、链霉亲和素下拉和免疫共沉淀实验，以探究BAI与TNF-α和CCR2的潜在结合。此外，采用实时定量聚合酶链反应（qPCR）、蛋白质印迹法和流式细胞术来阐明BAI对TNF-α/TNFR1-CCL2/CCR2信号通路的影响。
BAI抑制EAM小鼠心肌组织中趋化因子CCL2的表达，并减轻巨噬细胞和Th1细胞的浸润。在体外，BAI对TNF-α表现出结合亲和力，导致TNF-α/TNFR1-AP-1信号通路下调，随后抑制巨噬细胞和血管内皮细胞分泌CCL2。此外，BAI对CCR2具有结合能力，导致CCL2/CCR2-ROCK1通路下调，从而抑制巨噬细胞和Th1细胞的趋化迁移。
本研究表明，BAI可通过与TNF-α和CCR2结合下调CCL2的分泌及CCL2/CCR
