Wu Chenxi, Xu Xinru, Shi Yueyue, Li Fenfen, Zhang Xiaoxi, Huang Yan, Xia Daozong
School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China.
Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China.
Int J Mol Sci. 2024 Dec 27;26(1):143. doi: 10.3390/ijms26010143.
Neutrophil extracellular traps (NETs) formation is a key process in inflammatory diseases like gout, but the underlying molecular mechanisms remain incompletely understood. This study aimed to establish a model to examine the formation of NETs induced by monosodium urate (MSU) and phorbol 12-myristate 13-acetate (PMA) and to elucidate their molecular pathways. Laser confocal microscopy was used to visualize NET formation, while flow cytometry was employed to detect reactive oxygen species (ROS) production. The microstructure of neutrophils was observed by transmission electron microscopy, and the expression of key proteins was determined by Western blotting. Additionally, the effect of various inhibitors targeting the MAPK signaling pathway on NET formation was evaluated. They include the Ras inhibitor Salirasib, Raf inhibitor Vemurafenib, ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, as well as NADPH oxidase inhibitor DPI and neutrophil elastase inhibitor Alvelestat. The results showed that MSU and PMA triggered significant NET formation, which was accompanied by increased ROS levels, lactate dehydrogenase release, dsDNA, and IL-8. Notably, selective MAPK pathway inhibitors and DPI and Alvelestat, except for SB203580, effectively down-regulated these indicators. These data indicated that the activation of a signaling pathway involving Ras-Raf-ERK, which is dependent on ROS, is crucial for the induction of NET formation by MSU and PMA. Given the involvement of NETs in multiple pathologies, our findings could potentially serve as molecular targets for the intervention and treatment of crystal-related diseases, especially for gout.
中性粒细胞胞外诱捕网（NETs）的形成是痛风等炎症性疾病中的关键过程，但其潜在的分子机制仍未完全阐明。本研究旨在建立一个模型，以检测尿酸钠（MSU）和佛波酯（PMA）诱导的NETs形成，并阐明其分子途径。利用激光共聚焦显微镜观察NETs的形成，同时采用流式细胞术检测活性氧（ROS）的产生。通过透射电子显微镜观察中性粒细胞的微观结构，并通过蛋白质免疫印迹法测定关键蛋白的表达。此外，评估了靶向丝裂原活化蛋白激酶（MAPK）信号通路的各种抑制剂对NETs形成的影响。这些抑制剂包括Ras抑制剂Salirasib、Raf抑制剂维莫非尼、细胞外信号调节激酶（ERK）抑制剂PD98059、p38丝裂原活化蛋白激酶抑制剂SB203580，以及烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂二苯基碘（DPI）和中性粒细胞弹性蛋白酶抑制剂阿伐司他。结果显示，MSU和PMA触发了显著的NETs形成，同时伴随着ROS水平升高、乳酸脱氢酶释放、双链DNA（dsDNA）和白细胞介素-8（IL-8）增加。值得注意的是，除SB203580外，选择性MAPK通路抑制剂以及DPI和阿伐司他有效地下调了这些指标。这些数据表明，依赖于ROS的涉及Ras-Raf-ERK的信号通路激活对于MSU和PMA诱导NETs形成至关重要。鉴于NETs参与多种病理过程，我们的研究结果可能为晶体相关疾病，尤其是痛风的干预和治疗提供潜在的分子靶点。
