COVID-19的特征是过度的炎症反应和巨噬细胞超活化，这在严重病例中导致肺泡上皮损伤和急性呼吸窘迫综合征。最近的研究报道了SARS-CoV-2刺突（S）蛋白与细菌脂多糖（LPS）相互作用，在体外、巨噬细胞和外周血单个核细胞（PBMCs）以及体内增强炎症反应。下丘脑激素生长激素释放激素（GHRH）除了促进垂体生长激素释放外，还具有许多外周功能，在恶性和非恶性细胞中作为生长因子。反过来，GHRH拮抗剂在不同细胞类型中，包括肺和内皮细胞中表现出强大的抗肿瘤作用和抗炎活性。然而，至今尚未探索GHRH拮抗剂在COVID-19中的抗炎作用。在这里，我们检测了GHRH拮抗剂MIA-602减少受到S蛋白和LPS联合刺激的人THP-1源巨噬细胞和PBMC中的炎症的能力。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。将THP-1细胞暴露于S蛋白和LPS联合刺激后，TNF-α和IL-1β的mRNA水平和蛋白质分泌以及IL-8和MCP-1基因表达均增加，而MIA-602抑制了这种效应。同样，MIA-602阻断了PBMC中TNF-α和IL-1β的分泌，减少了MCP-1的mRNA水平。机制上，MIA-602减弱了THP-1细胞中S蛋白和LPS诱导的炎症途径的激活，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核转位和活性的影响。总的来说，这些发现展示了MIA类GHRH拮抗剂的新型抗炎作用，并暗示其潜在用于治疗炎症性疾病，如COVID-19及相关并发症。Copyright © 2023 Granato, Gesmundo, Pedrolli, Kasarla, Begani, Banfi, Bruno, Lopatina, Brizzi, Cai, Sha, Ghigo, Schally and Granata.

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 vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. 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.Copyright © 2023 Granato, Gesmundo, Pedrolli, Kasarla, Begani, Banfi, Bruno, Lopatina, Brizzi, Cai, Sha, Ghigo, Schally and Granata.