Department of Life Sciences and Biotechnology, University of Ferrara, I‑44121 Ferrara, Italy.
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, I‑35131 Padova, Italy.
Int J Mol Med. 2023 Sep;52(3). doi: 10.3892/ijmm.2023.5279. Epub 2023 Jul 21.
Since its spread at the beginning of 2020, the coronavirus disease 2019 (COVID‑19) pandemic represents one of the major health problems. Despite the approval, testing, and worldwide distribution of anti‑severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) vaccines, the development of specific antiviral agents targeting the SARS‑CoV‑2 life cycle with high efficiency, and/or interfering with the associated 'cytokine storm', is highly required. A recent study, conducted by the authors' group indicated that sulforaphane (SFN) inhibits the expression of IL‑6 and IL‑8 genes induced by the treatment of IB3‑1 bronchial cells with a recombinant spike protein of SARS‑CoV‑2. In the present study, the ability of SFN to inhibit SARS‑CoV‑2 replication and the expression of pro‑inflammatory genes encoding proteins of the COVID‑19 'cytokine storm' was evaluated. SARS‑CoV‑2 replication was assessed in bronchial epithelial Calu‑3 cells. Moreover, SARS‑CoV‑2 replication and expression of pro‑inflammatory genes was evaluated by reverse transcription quantitative droplet digital PCR. The effects on the expression levels of NF‑κB were assessed by western blotting. Molecular dynamics simulations of NF‑kB/SFN interactions were conducted with Gromacs 2021.1 software under the Martini 2 CG force field. Computational studies indicated that i) SFN was stably bound with the NF‑κB monomer; ii) a ternary NF‑kB/SFN/DNA complex was formed; iii) the SFN interacted with both the protein and the nucleic acid molecules modifying the binding mode of the latter, and impairing the full interaction between the NF‑κB protein and the DNA molecule. This finally stabilized the inactive complex. Molecular studies demonstrated that SFN i) inhibits the SARS‑CoV‑2 replication in infected Calu‑3 cells, decreasing the production of the N‑protein coding RNA sequences, ii) decreased NF‑κB content in SARS‑CoV‑2 infected cells and inhibited the expression of NF‑kB‑dependent IL‑1β and IL‑8 gene expression. The data obtained in the present study demonstrated inhibitory effects of SFN on the SARS‑CoV‑2 life cycle and on the expression levels of the pro‑inflammatory genes, sustaining the possible use of SFN in the management of patients with COVID‑19.
自 2020 年初爆发以来，2019 年冠状病毒病（COVID-19）大流行是主要的健康问题之一。尽管已经批准、测试并在全球范围内分发了抗严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）疫苗，但仍迫切需要开发针对 SARS-CoV-2 生命周期的高效、具有特异性的抗病毒药物，以及/或干扰相关的“细胞因子风暴”。作者所在团队最近的一项研究表明，萝卜硫素（SFN）可抑制 IB3-1 支气管细胞用 SARS-CoV-2 重组刺突蛋白处理后诱导的 IL-6 和 IL-8 基因的表达。在本研究中，评估了 SFN 抑制 SARS-CoV-2 复制和 COVID-19“细胞因子风暴”编码蛋白的促炎基因表达的能力。在支气管上皮细胞 Calu-3 中评估 SARS-CoV-2 复制。此外，通过逆转录定量液滴数字 PCR 评估 SARS-CoV-2 复制和促炎基因的表达。通过蛋白质印迹法评估 NF-κB 的表达水平。使用 Gromacs 2021.1 软件在 Martini 2 CG 力场下进行 NF-κB/SFN 相互作用的分子动力学模拟。计算研究表明：i）SFN 与 NF-κB 单体稳定结合；ii）形成三元 NF-κB/SFN/DNA 复合物；iii）SFN 与蛋白质和核酸分子相互作用，改变后者的结合模式，并削弱 NF-κB 蛋白与 DNA 分子的完全相互作用。这最终稳定了无活性的复合物。分子研究表明，SFN i）抑制感染 Calu-3 细胞中的 SARS-CoV-2 复制，减少 N 蛋白编码 RNA 序列的产生，ii）降低感染 SARS-CoV-2 的细胞中 NF-κB 的含量，并抑制 NF-κB 依赖性 IL-1β 和 IL-8 基因表达。本研究获得的数据表明 SFN 对 SARS-CoV-2 生命周期和促炎基因表达水平具有抑制作用，支持 SFN 可用于 COVID-19 患者的治疗。
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