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

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(P2Y_(2))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P2Y_(2) mRNA的转录水平;小干扰RNA(siRNA)敲低P2Y_(2)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P2Y_(2) 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P2Y_(2)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/P2Y_(2)和ERK信号通路诱导呼吸道上皮细胞发生炎症反应。

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 P2Y_(2) mRNA expression in 16HBE14o-cells after S1 protein stimulation were determined by Real-time quantitative fluorescence PCR(qRT-PCR).Small interfering RNA(siRNA)knockdown P2Y_(2) 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 P2Y_(2) 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 P2Y_(2) 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/P2Y_(2) and ERK signaling pathways.