新型冠状病毒刺突蛋白对单核巨噬细胞分泌炎症因子的影响

Effect of novel coronavirus spike protein on the secretion of inflammatory factors by mononuclear macrophages

目的 探讨SARS-CoV-2对单核巨噬细胞分泌炎症因子的作用及机制。方法 纳入石家庄市第五医院2020年1月21日至2月10日以及2021年1月3日至1月7日住院的COVID-19确诊患者(病例组)与健康对照者(对照组)各53例,收集病例资料与血常规检测结果。采用SARS-CoV-2刺突蛋白刺激单核巨噬细胞,细胞增殖-毒性检测细胞活力,荧光定量PCR法检测炎症因子IL-6、IL-1β和CCL2与信号通路分子JAK1、STAT1和NF-κB的表达。结果 COVID-19患者的单核细胞比值与中性粒细胞比值高于对照组,而淋巴细胞比值、淋巴细胞计数、嗜酸性粒细胞比值、嗜酸性粒细胞计数与红细胞压积低于对照组。SARS-CoV-2刺突蛋白刺激单核巨噬细胞后,在100ng/ml与300ng/ml的浓度下细胞活力高于对照组(P=0.046),在浓度为500ng/ml时IL-6、IL-1β与CCL2的mRNA表达含量最高(P<0.01,P=0.036,P <0.001),在浓度100ng/ml时JAK1与STAT1的mRNA表达明显升高(P <0.001),在300ng/ml与500ng/ml刺激下,NF-κB表达增高(P<0.001)。结论 COVID-19患者的单核细胞升高,SARS-CoV-2刺突蛋白可能通过JAK1/STAT1信号通路促进单核巨噬细胞分泌炎症因子,在细胞因子风暴中发挥作用。

Objective The aim of this study was to investigate the role and mechanism of SARS-CoV-2 virus on the secretion of inflammatory factors by mononuclear macrophages.Method Fifty-three patients with confirmed COVID-19 hospitalized from January 21 to February 10,2020 and January 3 to January 7,2021 were selected from Shijiazhuang Fifth Hospital,fifty-three healthy controls each were included,and the results of medical records and routine blood test were collected.Mononuclear macrophages were stimulated with SARS-CoV-2 virus spiked protein,cell viability was detected by CCK-8 assay.The level of pro-inflammatory cytokines IL-6,IL-1βand CCL2 and the expression of signaling pathway molecules JAK1,STAT1 and NF-κB were detected by quantitative RT-PCR.Result The monocyte ratio and neutrophil ratio were higher,while the lymphocyte ratio,lymphocyte count,eosinophil ratio,and eosinophil count,and hematocrit were lower in COVID-19 patients than those in controls.After stimulation of mononuclear macrophages by SARS-CoV-2 virus spiked protein,cell viability was higher at concentrations of 100ng/ml and 300ng/ml(P=0.046).The highest mRNA expression levels of IL-6,IL-1βand CCL2 at a concentration of 500ng/ml(P<0.01,P=0.036,P<0.001).The expression of JAK1 and STAT1 at a concentration of 100ng/ml(P<0.001),the expression of NF-κB expression was increased at 300 and 500ng/ml stimulation(P<0.001).Conclusion SARS-CoV-2 viral spike protein may promote inflammatory cytokine secretion by mononuclear macrophages through the JAK1/STAT1 signaling pathway,which play a role in the cytokine storm.