目的 旨在探讨靶向Ⅰ型干扰素（IFN-I）效应通路的清肺排毒汤入血成分、预防新冠肺炎转重症潜能及其作用机制。方法 通过双荧光素酶报告系统鉴定严重急性呼吸系统综合征冠状病毒2型（SARS-CoV-2）抑制IFN-I效应通路的病毒蛋白。将KM小鼠ig给药清肺排毒汤后，采用UPLC-Q-TOF-MS技术明确复方入血成分，并利用Uniport, Genecard等数据库进行新冠肺炎靶点筛选以及通过DAVID、Gene Ontology等数据库富集复方入血成分对应疾病靶点的相关通路。利用Autodock Vina批量小分子对接，筛选出能靶向IFN-I效应通路关键基因的化合物成分，并通过实时荧光定量PCR和间接免疫荧光实验验证清肺排毒汤及其成分抗SARS-CoV-2的潜在作用机制。结果 双荧光素酶报告系统共鉴定出有16个抑制IFN-I效应通路的SARSCo V-2蛋白，其中，除已经被报道的11个蛋白，结果还发现NSP7和NSP16能显著地抑制IFN-I效应通路。比较复方提取物、给药后含药血清及空白血清，从昆明小鼠血清中发现入血成分56个，网络药理学分析发现P值靠前的10种通路中，清肺排毒汤有5种与免疫及炎症发生相关，其中包括IFN-I效应通路（JAK-STAT通路）。进一步分子对接结果表明黄花败酱苷C和染料木苷2个活性成分与IFN-I效应通路的核心靶点均具有良好的结合活性。清肺排毒汤及其成分干预后不仅能解除SARS-CoV-2Spike蛋白对ISGF3复合体入核的抑制作用，进而上调干扰素刺激基因（ISGs）表达，也能抑制IFN受损的炎症反应。结论 清肺排毒汤及其成分能够靶向IFN-I效应通路发挥抗病毒作用，筛选到的有效成分可为预防新冠肺炎转重症提供新的潜在候选药物。

Objective To explore the blood-entering components of Qingfei Paidu Decoction targeting the IFN-I effect pathway, its potential to prevent the progression to severe disease of COVID-19, and its mechanism of action. Methods To identify the viral proteins of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) that inhibit the IFN-I effector pathway by the DualLuciferase Reporter System. After Qingfei Paidu Decoction was given to Kunming rats by gavage, the blood-entering components were determined by UPLC-Q-TOF-MS. And use the Uniport, Genecard database for COVID-19 targets screening, as well as by DAVID, Gene Ontology database, such as enrichment of compounds into the blood components corresponding to disease targets related pathways. Autodock Vina batch small molecule docking was used to screen compounds that could target key genes of IFN-I effector pathway, and the potential mechanism of Qingfei Paidu Decoction and its components against SARS-CoV-2 was verified by real-time quantitative PCR and Indirect Immunofluorescence Assay. The Dual-Luciferase Reporter System identified a total of 16 SARS-CoV-2 proteins that inhibited the IFN-I effector pathway. Results Among them, in addition to the 11 proteins that have been reported, NSP7 and NSP16 were also found to significantly inhibit the IFN-I effector pathway. By comparing compound extracts, drug-containing serum, and blank serum after administration, 56 blood-entering components were found in the serum of Kunming mice. Network pharmacological analysis found that among the top 10 pathways of P value, five kinds of Qingfei Paidu Decoction were related to immunity and inflammation, including the IFN-I effect pathway(JAK-STAT pathway). Further molecular docking results showed that scabioside C and genistin had good binding activities with the core targets of IFN-I effector pathway. Conclusion Mechanism study has shown that Qingfei Paidu Decoction and its components not only can relieve the inhibitory effect of SARS-CoV-2 Spike protein on the nuclear entry of ISGF3 complex, and thereby upregulate the expression of ISGs, but also inhibit the inflammatory response caused by IFN deficiency. The results showed that Qingfei Paidu Decoction and its components can target the IFN-I effector pathway to play an antiviral role, and the selected effective components can provide new potential candidate drugs for preventing the progression to severe disease of COVID-19.

R285.5

国家自然科学基金资助项目(82470122); 成都医学院“成医英才”登峰计划项目(2024kjTzn01); 成都医学院-成都市青白江区中医医院临床科学研究基金(24LHFBM1-01)