巴瑞替尼在新型冠状病毒感染中对Ⅰ型干扰素信号通路的影响

Baricitinib inhibits type Ⅰ IFN-signaling during SARS-Co V-2 infection in vitro

目的 探讨巴瑞替尼在新型冠状病毒感染中的抗病毒作用及对细胞因子表达的影响。方法 首先使用SARS-CoV-2(MOI=0.1)感染Calu-3细胞系,通过RT-qPCR分析新冠病毒感染过程中炎症因子(IL-6、IL-8、TNF-α和IL-1β)、干扰素β(IFN-β)和干扰素刺激基因(IFIT2)mRNA表达水平。下一步用巴瑞替尼预处理Calu-3细胞2 h,之后感染SARS-CoV-2(MOI=0.1),分别在感染后0、24、36、48 h时间点收集细胞,比较药物处理组和未处理组中上述基因mRNA表达水平及对病毒复制的影响作用。结果 SARS-CoV-2感染Calu-3细胞后,可诱导促炎因子(IL-6、TNF-a、IL-1β)和干扰素及干扰素刺激因子(IFN-β和IFIT2)mRNA水平发生不同程度的显著上调,病毒感染组在感染48 h相对于未感染组上调了近100倍或以上(P<0.000 1),上述因子的mRNA表达水平随感染时间延长持续增加(P<0.001或P<0.000 1)。IL-8 mRNA水平上升幅度尽管无上述基因显著,但也呈现2~4倍的增加。巴瑞替尼预处理Calu-3细胞后,对胞内SARS-CoV-2的复制水平无显著抑制作用(P>0.05)。然而,该药物可显著抑制SARS-CoV-2感染诱导的IL-6和TNF-α水平的上调(分别下调5.25倍和3.90倍,P<0.01),对IL-8和IL-1β的水平影响不大。另外,该药物也可明显下调病毒感染对IFN-β和IFIT2水平的增加(分别下调10.51倍和90.78倍,P<0.000 1)。结论 巴瑞替尼药物尽管对新冠病毒在细胞内病毒载量水平无显著抑制效应,但可不同程度抑制新冠病毒感染诱导的炎症因子的大量释放,尤其对干扰素和干扰素刺激基因表达的抑制作用更加显著。考虑到IFN-Ⅰ反应在病毒感染过程中发挥重要作用,临床必须谨慎使用巴瑞替尼治疗COVID-19患者。

Objective To explore the antiviral effect of baricitinib in the SARS-Co V-2 infection and influence oncytokine levels.Methods Calu-3 cells were infected with SARS-Co V-2 at MOI of 0.1, and the levels of inflammatorycytokines(IL-6, IL-8, TNF-α and IL-1β), interferon β(IFN-β) and interferon-stimulated gene, IFIT2 in the infected cellswere analyzed by q RT-PCR methods. At the same time, Calu-3 cells were infected with SARS-Co V-2(MOI=0.1) after beingtreated with baricitinib for 2 hours. Cells were collected at 0, 24, 36, and 48 hours, and analyzed for the m RNA of the abovegenes in the drug-treated and untreated groups.Results The mRNA levels of IL-6, TNF-a, IL-1β, IFN-β and IFIT2 inCalu-3 infected by SARS-Co V-2 cells were increased significantly. These cytokines were increased by nearly 100-fold post-infection 48 h compared with the control(P<0.000 1), and continued to increase with the infection time(P<0.001 or P<0.0001). The increase of IL-8 m RNA level was not as significant as IL-6, TNF-α, IL-8, IL-1β, but it also showed a 2-4 foldsincrease. Baricitinib does not affect the level of viral RNA in Calu-3 cells after SARS-Co V-2 infection(P>0.05). However,baricitinib can significantly inhibit the up-regulation of IL-6 and TNF-α levels induced by SARS-Co V-2 infection(5.25-foldand 3.90-fold down-regulation, respectively, P<0.01), and has little effect on the levels of IL-8 and IL-1β. In addition, thedrug could also significantly down-regulate the increase in IFN-β and IFIT2 levels caused by viral infection(10.51-fold and 90.78-fold down-regulation, respectively, P<0.000 1).Conclusions Baricitinib inhibits the release of inflammatorycytokines to some extent, but it drastically down-regulates the expression of interferons and interferon-stimulated genes(ISGs),and has limited antiviral effect on SARS-Co V-2. Considering that interferon signal pathways play important roles on viralinfection, caution should be exercised when using baricitinib to treat COVID-19 patients.