Liu Luyao, Lan Zhenzhen, Liu Xincan, Chen Yun, Chen Zhihao, Cheng Li, Hu Ting
Heart Center, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, 450000, Henan, China.
The First Clinical Medical College, Henan University of Chinese Medicine, Zhengzhou, 450000, Henan, China.
Mol Cell Biochem. 2025 Aug 26. doi: 10.1007/s11010-025-05375-z.
Neutrophil extracellular traps (NETs) are positively correlated with the severity of calcific aortic valve disease (CAVD). This study aims to elucidate the mechanism by which NETs contribute to CAVD. The CAVD mice model was established by calcification-promoting diets, and NETs formation was modulated via intraperitoneal injection of Cl-amidine. We observed the effect of NETs on Raw264.7 cells by regulating NETs and TLR9 in vitro. Concentrations of TNF-α, MPO-DNA complex, and IL-10 were measured using ELISA. NETs formation was assessed through immunofluorescence assay citrullinated histone H3 (citH3). Expression levels of BMP2, RUNX2, IL-1β, TNF-α, IL-10, and TLR 9 were analyzed by qRT-PCR and Western blotting, while flow cytometry was used to assess the expression of CD86 and CD206 on Raw264.7 cells. Results indicated that compared to the vehicle group, the CAVD group exhibited significant valve thickening and increased calcium deposition, as well as elevated levels of inflammatory factors TNF-α and IL-1β, NET-related markers MPO-DNA complexes and citH3, ossification factors BMP2 and RUNX2, and TLR9. Conversely, IL-10 levels were significantly reduced. Cl-amidine intervention in early CAVD mice significantly improved valve thickness and reduced calcium deposition, inflammatory factors, NETs-related markers, ossification factors, and TLR9 levels, while increasing IL-10 levels. Cl-amidine may delay CAVD progression in mice by reducing NETs. In vitro studies confirmed that serum from CAVD mice induced NETs, promoting the polarization of Raw264.7 cells to the M1 phenotype via TLR9 signaling pathway, thereby releasing pro-inflammatory factors (TNF-α, IL-1β, and IL-6), and inhibiting M2 polarization and IL-10 expression. In summary, our findings suggest that NETs promote Raw264.7 cell polarization to M1 through the TLR9 signaling pathway, contributing to the inflammatory response in CAVD. This study proposes a novel therapeutic strategy targeting NETs to delay CAVD progression.
中性粒细胞胞外诱捕网（NETs）与钙化性主动脉瓣疾病（CAVD）的严重程度呈正相关。本研究旨在阐明NETs促成CAVD的机制。通过促钙化饮食建立CAVD小鼠模型，并通过腹腔注射氯胍来调节NETs的形成。我们在体外通过调节NETs和TLR9来观察NETs对Raw264.7细胞的影响。使用酶联免疫吸附测定法（ELISA）测量肿瘤坏死因子-α（TNF-α）、髓过氧化物酶-DNA复合物（MPO-DNA）和白细胞介素-10（IL-10）的浓度。通过免疫荧光法检测瓜氨酸化组蛋白H3（citH3）来评估NETs的形成。通过实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析骨形态发生蛋白2（BMP2）、 Runt相关转录因子2（RUNX2）、白细胞介素-1β（IL-1β）、TNF-α、IL-10和Toll样受体9（TLR 9）的表达水平，同时使用流式细胞术评估Raw264.7细胞上CD86和CD206的表达。结果表明，与载体组相比，CAVD组表现出明显的瓣膜增厚和钙沉积增加，以及炎症因子TNF-α和IL-1β、NET相关标志物MPO-DNA复合物和citH3、骨化因子BMP2和RUNX2以及TLR9水平升高。相反，IL-10水平显著降低。对早期CAVD小鼠进行氯胍干预可显著改善瓣膜厚度，减少钙沉积、炎症因子、NETs相关标志物、骨化因子和TLR9水平，同时提高IL-10水平。氯胍可能通过减少NETs来延缓小鼠CAVD的进展。体外研究证实，CAVD小鼠血清诱导NETs形成，通过TLR9信号通路促进Raw264.7细胞向M1表型极化，从而释放促炎因子（TNF-α、IL-1β和IL-6），并抑制M2极化和IL-10表达。总之，我们的研究结果表明，NETs通过TLR9信号通路促进Raw264.7细胞向M1极化，促成CAVD中的炎症反应。本研究提出了一种针对NETs的新型治疗策略，以延缓CAVD的进展。
