Background

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation.

Methods

SARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses.

Results

Angiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.

中文翻译：

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SARS-CoV-2 刺突通过 ROS 抑制的 PI3K/AKT/mTOR 信号通过自噬促进炎症和细胞凋亡

背景

严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 感染引起的炎症反应是导致 2019 年新型冠状病毒病 (COVID-19) 患者死亡的主要原因。然而，SARS-CoV-2 引发炎症反应的机制仍不清楚。在这里，我们旨在探索 SARS-CoV-2 刺突蛋白在感染细胞中的调节作用，并试图阐明 SARS-CoV-2 诱导炎症的分子机制。

方法

SARS-CoV-2 刺突假病毒体 (SCV-2-S) 是使用刺突表达病毒包装系统生成的。进行蛋白质印迹、mCherry-GFP-LC3 标记、免疫荧光和 RNA-seq 以检查 SCV-2-S 在自噬反应中的调节机制。SCV-2-S 对细胞凋亡的影响通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记 (TUNEL)、蛋白质印迹和流式细胞术分析进行评估。进行酶联免疫吸附测定 (ELISA) 以检查 SCV-2-S 在炎症反应中的机制。

结果

血管紧张素转换酶 2 (ACE2) 介导的 SCV-2-S 感染可诱导人支气管上皮细胞和微血管内皮细胞发生自噬和凋亡。从机制上讲，SCV-2-S 通过上调细胞内活性氧 (ROS) 水平来抑制 PI3K/AKT/mTOR 通路，从而促进自噬反应。最终，SCV-2-S 诱导的自噬引发感染细胞的炎症反应和细胞凋亡。这些发现不仅提高了我们对 SARS-CoV-2 感染引起的致病性炎症的潜在机制的理解，而且对开发针对 COVID-19 患者的抗炎疗法（例如 ROS 和自噬抑制剂）具有重要意义。