Zheng Yanjun, Lin Jingrong, Wan Guoqing, Gu Xuefeng, Ma Jian
Shanghai Key Laboratory of Molecular Imaging, Zhoupu Hospital, Shanghai University of Medicine and Health Sciences, Shanghai, 201318, China.
Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital, Pudong New Area, Shanghai, 201318, China.
Biol Direct. 2025 May 26;20(1):65. doi: 10.1186/s13062-025-00657-4.
Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, the function of Notch1 signaling in macrophages activation and its underlying molecular mechanism during sepsis has not been fully elucidated.
We evaluated Notch1 expression in a lipopolysaccharide (LPS)-induced model of septic cardiac dysfunction. Using macrophage-specific Notch1 knockout mice (NOTCH1) in conjunction with AAV-F4/80-mediated NICD1 overexpression, we investigated the impact of Notch1 on septic cardiac injury. LPS-stimulated bone marrow-derived macrophages (BMDMs) were analyzed by flow cytometry and ELISA to assess mitochondrial damage and inflammasome activation. Mitophagy flux and related protein levels were quantified, and a mitophagy inhibitor was applied to further delineate Notch1's in vivo role. Downstream targets of Notch1 were identified and validated via ChIP-qPCR and luciferase reporter assays.
Intraperitoneal injection of LPS markedly impaired cardiac function, increased macrophage infiltration, and elevated Notch1 expression compared with PBS-treated controls. Notch1 expression was inversely correlated with cardiac performance in LPS-treated mice. Notably, macrophage-specific deletion of Notch1 significantly improved cardiac function, whereas NICD1 overexpression worsened LPS-induced cardiac injury. NOTCH1 macrophages showed reduced mitochondrial damage and diminished activation of NLRP3-dependent caspase-1. Moreover, LPS induced mitophagy, an effect that was further enhanced by Notch1 knockout. Mechanistically, ChIP-seq and qPCR analyses revealed that NICD1 upregulates Mst1 transcription. Furthermore, overexpression of Mst1 counteracted the increased mitophagy in Notch1-deficient macrophages, resulting in elevated mitochondrial reactive oxygen species production, inflammatory cytokine secretion, and caspase-1 activation during prolonged LPS stimulation.
Our study uncovers a novel role for Notch1 in exacerbating LPS-induced septic cardiac dysfunction by suppressing mitophagy in macrophages. These findings suggest that targeting Notch1 may offer a promising therapeutic strategy to mitigate sepsis-induced inflammation by restoring proper mitophagy.
脓毒症是一种危及生命的疾病，治疗选择有限，其特征为过度的全身炎症反应和多器官功能衰竭。巨噬细胞在脓毒症发病机制中起关键作用。尽管众多研究支持Notch信号在大多数炎症性疾病中的关键作用，但Notch1信号在巨噬细胞激活中的功能及其在脓毒症期间的潜在分子机制尚未完全阐明。
我们评估了在脂多糖（LPS）诱导的脓毒性心脏功能障碍模型中Notch1的表达。使用巨噬细胞特异性Notch1基因敲除小鼠（NOTCH1）并结合AAV-F4/80介导的NICD1过表达，我们研究了Notch1对脓毒性心脏损伤的影响。通过流式细胞术和ELISA分析LPS刺激的骨髓来源巨噬细胞（BMDM），以评估线粒体损伤和炎性小体激活。对线粒体自噬通量和相关蛋白水平进行定量，并应用线粒体自噬抑制剂进一步阐明Notch1在体内的作用。通过染色质免疫沉淀-定量聚合酶链反应（ChIP-qPCR）和荧光素酶报告基因测定法鉴定并验证Notch1的下游靶点。
与用磷酸盐缓冲盐水（PBS）处理的对照组相比，腹腔注射LPS显著损害心脏功能，增加巨噬细胞浸润，并提高Notch1表达。在LPS处理的小鼠中，Notch1表达与心脏功能呈负相关。值得注意的是，巨噬细胞特异性缺失Notch1可显著改善心脏功能，而NICD1过表达则加重LPS诱导的心脏损伤。NOTCH1巨噬细胞显示线粒体损伤减少，NLRP3依赖性半胱天冬酶-1的激活减弱。此外，LPS诱导线粒体自噬，Notch1基因敲除进一步增强了这种作用。从机制上讲，ChIP-seq和qPCR分析表明，NICD1上调Mst1转录。此外，Mst1过表达抵消了Notch1缺陷型巨噬细胞中线粒体自噬的增加，导致在长时间LPS刺激期间线粒体活性氧生成增加、炎性细胞因子分泌增加和半胱天冬酶-1激活。
我们的研究揭示了Notch1通过抑制巨噬细胞中的线粒体自噬在加重LPS诱导的脓毒性心脏功能障碍中的新作用。这些发现表明，靶向Notch1可能提供一种有前景的治疗策略，通过恢复适当的线粒体自噬来减轻脓毒症诱导的炎症。
