Department of Medical Sciences, Division of Endocrinology, Diabetes and Metabolism, University of Turin, Turin, Italy.
Department of Medical Sciences, University of Turin, Turin, Italy.
Front Immunol. 2023 Aug 15;14:1231363. doi: 10.3389/fimmu.2023.1231363. eCollection 2023.
COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses , in macrophages and peripheral blood mononuclear cells (PBMCs), and . The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.
新型冠状病毒病的特征是过度炎症反应和巨噬细胞过度激活，导致严重病例中肺泡上皮损伤和急性呼吸窘迫综合征。最近的研究报告称，SARS-CoV-2 刺突（S）蛋白与细菌脂多糖（LPS）相互作用，从而增强巨噬细胞和外周血单核细胞（PBMC）中的炎症反应，并且，下丘脑激素生长激素释放激素（GHRH）除了促进垂体 GH 释放外，还在外周发挥许多功能，作为恶性和非恶性细胞中的生长因子。反过来，GHRH 拮抗剂在不同类型的细胞中，包括肺和内皮细胞，显示出强大的抗肿瘤作用和抗炎活性。然而，迄今为止，GHRH 拮抗剂在 COVID-19 中的抗炎作用仍未得到探索。在这里，我们研究了 GHRH 拮抗剂 MIA-602 降低 S 蛋白和 LPS 联合刺激的人 THP-1 衍生巨噬细胞和 PBMC 中炎症的能力。Western blot 和免疫荧光分析显示，GHRH 受体及其剪接变体 SV1 存在于 THP-1 细胞和 PBMC 中。S 蛋白和 LPS 联合刺激 THP-1 细胞增加了 TNF-α和 IL-1β的 mRNA 水平和蛋白分泌，以及 IL-8 和 MCP-1 基因表达，而 MIA-602 则阻碍了这种作用。同样，MIA-602 抑制了 PBMC 中 TNF-α和 IL-1β的分泌，并降低了 MCP-1 的 mRNA 水平。在机制上，MIA-602 阻断了 S 蛋白和 LPS 诱导的 THP-1 细胞中炎症途径的激活，如 NF-κB、STAT3、MAPK ERK1/2 和 JNK。MIA-602 还通过降低 ROS 产生、iNOS 和 COX-2 蛋白水平以及 MMP9 活性来减轻 PBMC 中的氧化应激。最后，MIA-602 阻止了 S 蛋白和 LPS 协同作用对 NF-кB 核易位和活性的影响。总的来说，这些发现证明了 MIA 类 GHRH 拮抗剂的新型抗炎作用，并表明它们有可能开发用于治疗炎症性疾病，如 COVID-19 和相关合并症。
