Department of Cardiology, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450000, China.
Department of Clinical Microbiology, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450000, China.
Cell Commun Signal. 2023 Feb 2;21(1):29. doi: 10.1186/s12964-022-01022-y.
The inflammatory cascade and cell death post-myocardial ischemia reperfusion (MI/R) are very complex. Despite the understanding that macrophage inflammation has a pivotal role in the pathophysiology of MI/R, the contribution of macrophage inflammatory signals in tailoring the function of vascular endothelium remains unknown.
In the present study, we analyzed the effects of NEDD4 on the NLRP3 inflammasome activation-mediated pyroptosis in vitro after an acute pro-inflammatory stimulus and in vivo in a MI/R mouse model. TTC and Evan's blue dye, Thioflavin S, immunohistochemistry staining, and ELISA were performed in wild-type and NEDD4 deficiency mice. THP-1 cells were transfected with si-NEDD4 or si-SF3A2. HEK293T cells were transfected with NEDD4 or SF3A2 overexpression plasmid. ELISA analyzed the inflammatory cytokines in the cell supernatant. The levels of NEDD4, SF3A2, and NLRP3/GSDMD pathway were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a fluorescence microscope.
NEDD4 inhibited NLRP3 inflammasome activation and pyroptosis in THP-1 cells treated with lipopolysaccharide (LPS) and nigericin (Nig). Mechanistically, NEDD4 maintained the stability of NLRP3 through direct interaction with the SF3A2, whereas the latter association with NLRP3 indirectly interacted with NEDD4 promoting proteasomal degradation of NLRP3. Deletion of NLRP3 expression further inhibited the caspase cascade to induce pyroptosis. Interestingly, inhibiting NLRP3 inflammasome activation in THP-1 cells could prevent cardiac microvascular endothelial cells (CMECs) injury. In addition, NEDD4 deficiency decreased animal survival and increased myocardial infarct size, no-reflow area, and promoted macrophages infiltration post-MI/R.
NEDD4 could be a potential therapeutic target in microvascular injury following myocardial reperfusion. Video Abstract.
心肌缺血再灌注（MI/R）后的炎症级联反应和细胞死亡非常复杂。尽管人们已经了解到巨噬细胞炎症在 MI/R 的病理生理学中起着关键作用，但巨噬细胞炎症信号在调节血管内皮功能方面的作用仍不清楚。
在本研究中，我们分析了 NEDD4 在急性促炎刺激后体外 NLRP3 炎性小体激活介导的细胞焦亡以及 MI/R 小鼠模型体内的作用。在野生型和 NEDD4 缺陷型小鼠中进行 TTC 和 Evan's 蓝染料、硫黄素 S、免疫组织化学染色和 ELISA 检测。THP-1 细胞用 si-NEDD4 或 si-SF3A2 转染。HEK293T 细胞用 NEDD4 或 SF3A2 过表达质粒转染。ELISA 分析细胞上清液中的炎症细胞因子。Western blot 检测 NEDD4、SF3A2 和 NLRP3/GSDMD 通路的水平。免疫沉淀评估蛋白相互作用。荧光显微镜监测细胞内蛋白共定位。
NEDD4 抑制 LPS 和 Nigericin（Nig）处理的 THP-1 细胞中 NLRP3 炎性小体的激活和细胞焦亡。机制上，NEDD4 通过与 SF3A2 的直接相互作用维持 NLRP3 的稳定性，而后者与 NLRP3 的间接相互作用则促进了 NLRP3 的蛋白酶体降解。NLRP3 表达缺失进一步抑制了半胱天冬酶级联反应，从而诱导细胞焦亡。有趣的是，抑制 THP-1 细胞中 NLRP3 炎性小体的激活可防止心脏微血管内皮细胞（CMECs）损伤。此外，MI/R 后 NEDD4 缺乏降低了动物存活率，增加了心肌梗死面积、无复流区，并促进了巨噬细胞浸润。
NEDD4 可能是心肌再灌注后微血管损伤的潜在治疗靶点。视频摘要。
