SARS-CoV-2 S1 蛋白通过ATP/P2Y2 和ERK1/2 信号通路引起呼吸道上皮细胞IL-6 和IL-8 分泌

SARS-CoV-2 spike S1 protein promotes IL-6 and IL-8 release via ATP/P2Y2 and ERK1/2 signaling pathways in human bronchial epithelial cells

关键词:  SARS-CoV-2 S1 蛋白  胞外ATP  ERK1/2  支气管上皮细胞  炎症

Key words:  SARS-CoV-2 S1 protein  extracellular ATP  ERK1/2  bronchial epithelial cells  inflammation

摘要:

目的　 探究严重急性呼吸综合征冠状病毒 2 （SARS-CoV-2）S1 蛋白诱导呼吸道上皮细胞炎症反应机制 以及嘌呤能受体（P2Y2）参与S1 蛋白引起的炎症反应。方法　酶联免疫吸附实验（ELISA）测量S1 蛋白和/或单克隆抗 体（MAb）刺激的 16HBE14o-细胞培养上清中白介素-6（IL-6）、白介素-8（IL-8）和腺嘌呤核苷三磷酸（ATP）水平，实时 荧光定量聚合酶链反应（qRT-PCR）检测血红素氧合酶 1（HO-1）、双特异性磷酸酶 1（MKP-1）和P2Y2 mRNA的转录水 平；小干扰RNA（siRNA）敲低P2Y2 表达后，ELISA检测S1 蛋白刺激的 16HBE14o-细胞培养上清中IL-6 和IL-8 水平。 结果 S1 蛋白和MAb对细胞活力无显著影响（P>0.05）。S1 蛋白刺激 16HBE14o-细胞引起IL-6 和IL-8 分泌增加，上调 HO-1、MKP-1 和P2Y2 mRNA表达，增加了胞外ATP水平（P<0.001 或 0.01），MAb显著抑制了S1 蛋白引起的IL-6 和IL-8 分泌（P<0.001 或 0.01）。siRNA敲低P2Y2 的表达能够显著降低S1 蛋白诱导的IL-6 和IL-8 分泌，同时ERK1/2 抑制剂 PD98059 也抑制了S1 蛋白引起的IL-6 和IL-8 分泌（P<0.001）。结论 SARS-CoV-2 S1 蛋白能够通过ATP/P2Y2 和ERK 信号通路诱导呼吸道上皮细胞发生炎症反应。

Abstract:

Objective To explore the mechanisms by which SARS-CoV-2 S1 protein induce pro-inflammatory responses in human bronchial epithelial cells and purinegic receptors were involved in the inflammatory by the S1 protein stimulation. Methods The pro-inflammatory cytokine (interleukin-6) IL-6 and (interleukin-8) IL-8 secretion and adenosine triphosphate (ATP) level in16HBE14o- cells induced by S1 protein and/or monoclonal antibody (MAb) were determined by using Enzymelinked immunosorbent assay (ELISA). The (Heme Oxygenase-1) HO-1, (dual specificity phosphatases-1) MKP-1 and P2Y2 mRNA expression in 16HBE14o- cells after S1 protein stimulation were determined by Real-time quantitative fluorescence PCR (qRT-PCR). Small interfering RNA (siRNA) knockdown P2Y2 expression ELISA detected the IL-6 and IL-8 release in 16HBE14o- cells after S1 protein stimulation. Results S1 protein and MAb hadno significant effects on cell viability (P>0.05). S1 protein induced IL-6 and IL-8 secretion, regulated HO-1, MKP-1, and P2Y2 mRNA expression, and increased extracellular ATP levels in 16HBE14o- cells(P<0.001 or 0.01).The MAb significantly inhibited the secretion of IL-6 and IL-8 induced byS1 protein (P<0.001 or 0.01). siRNA knockdown of P2Y2 expression significantly reduced S1 protein-induced IL-6 and IL-8 secretion, while ERK1/2 inhibitor PD98059 also inhibited S1 protein-induced IL-6 and IL-8 secretion (P<0.001). Conclusion SARS-CoV-2 S1 protein induce inflammatory responses in respiratory epithelial cells through the ATP/P2Y2 and ERK signaling pathways.