SARS-CoV-2 的刺突蛋白通过 NLRP3 炎症小体和 NF-κB 信号通路激活心脏成纤维细胞纤维化。

BACKGROUND: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood. METHODS: We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor. RESULTS: S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β. CONCLUSION: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner.

背景：严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的刺突蛋白对病毒进入至关重要，并可导致心脏损伤。Toll 样受体 4（TLR4）和 NOD、富含亮氨酸重复序列和 pyrin 结构域 3（NLRP3）炎性小体是参与心脏纤维化的关键免疫系统成分。刺突蛋白通过 TLR4 或血管紧张素转换酶 2（ACE2）受体激活 NLRP3 炎性小体，损害各种器官。然而，刺突蛋白在人类心脏纤维化中的作用及其与 NLRP3 炎性小体和 TLR4 的相互作用仍知之甚少。

方法：我们利用划痕实验、Western blot 和免疫荧光法评估了用刺突（S1）蛋白处理 24 小时后的培养人心房成纤维细胞（CFs）的迁移、纤维化信号、线粒体钙水平、活性氧（ROS）产生和细胞形态，并用 ACE2 中和抗体、TLR4 阻滞剂或 NLRP3 抑制剂处理。

结果：S1 蛋白增强 CFs 的迁移以及胶原蛋白 1、α-平滑肌肌动蛋白、转化生长因子 β1（TGF-β1）、磷酸化 SMAD2/3、白细胞介素 1β（IL-1β）和核因子 kappa-轻链增强子的表达激活 B 细胞（NF-κB）。S1 蛋白增加了 ROS 的产生，但不影响线粒体钙含量和细胞形态。用 ACE2 中和抗体处理可减弱 S1 蛋白对胶原蛋白 1 和 TGF-β1 表达的作用。此外，NLRP3（MCC950）和 NF-κB 抑制剂，但不是 TLR4 抑制剂 TAK-242，可阻止 S1 蛋白增强 CFs 的迁移以及胶原蛋白 1、TGF-β1 和 IL-1β 的过度表达。

结论：S1 蛋白通过 NF-κB 信号通过 ACE2 依赖性方式激活 NLRP3 炎性小体，从而激活人心房成纤维细胞。