基于人工智能技术的治疗非重症新型冠状病毒感染中药方剂研发及其作用机制研究

The Development of Chinese Herbal Formulae for Non-severe COVID-19 Based on Artificial Intelligence Technology and Investigation of Its Action Mechanisms

目的 研发治疗非重症新型冠状病毒感染(COVID-19)的中药方剂并验证其抗炎作用机制。方法基于COVID-19患者支气管肺泡灌洗液中巨噬细胞的表达谱和新型冠状病毒(SARS-CoV-2)体外感染的肺上皮细胞表达谱分别确定了COVID-19在这两类细胞中失调的信号通路。通过文献收集临床治疗非重症COVID-19的中药方剂102首,基于特征信号通路评价102首方剂在肺上皮细胞和巨噬细胞中的通路逆转率,并根据中医理论确定“基础方”。通过临床常用方剂总结得到治疗非重症COVID-19的常用药,结合《中华人民共和国药典》中药筛选结果得到COVID-19中药方剂研发的“药物池”。通过靶向转录组获得每味药物对信号通路的调控作用,以逆转COVID-19失调的信号通路为目标展开计算,基于中药智能组方方法采用穷举法和平行运算进行组方得到若干个候选复方,参考中医专业经验最终得出治疗非重症COVID-19的守正创新方。采用RT-qPCR法检测该组方醇提物对脂多糖(LPS)处理的RAW264.7细胞中白细胞介素1b (IL1b)、C-X-C基序趋化因子配体2 (Cxcl2)、C-X-C基序趋化因子配体10 (Cxcl10)、C-C基序趋化因子配体2 (Ccl2)、一氧化氮合成酶2 (Nos2)和前列腺素内过氧化物合成酶2 (Ptgs2) mRNA的表达,对抗炎效果进行验证。结果在巨噬细胞和肺上皮细胞中,确定了COVID-19相关的各34条失调信号通路。基于失调信号通路和中药靶向转录组,对临床治疗非重症COVID-19的102首中药复方的功效进行评估,结果显示银翘散合平胃散在巨噬细胞及肺上皮细胞上的通路逆转率分别为91.18%和100%,效果最佳;且该复方具有疏风清热、解毒化湿的功效,符合COVID-19风热夹湿的病机,故选用银翘散合平胃散作为守正创新方研发的基础方。利用中药智能组方方法结合中医专业经验对银翘散合平胃散进行加减化裁,得到守正创新方翘蒡术皮饮。该方逆转了巨噬细胞和肺上皮细胞上COVID-19相关全部失调信号通路,逆转率均为100%。翘蒡术皮饮中的君药连翘、豨莶草和牛蒡子可下调多条与病毒感染、免疫及损伤增值相关的信号通路。RT-qPCR结果表明,与模型组比较,翘蒡术皮饮组下调了LPS诱导RAW264.7细胞中的IL1b、Tnf、Cxcl2、Cxcl10、Ccl2、Nos2和Ptgs2mRNA表达(P<0.05或P<0.01)。结论 基于靶向转录组、中医专业经验和人工智能研发得到治疗非重症COVID-19的翘蒡术皮饮,该方剂的乙醇提取物在细胞炎症模型上能够抑制多种促炎基因的表达。

Objective To develop traditional Chinese medicine(TCM)formulae for the treatment of nonsevere coronavirus disease 2019(COVID-19)and to explore its anti-inflammatory mechanism.Methods The dysregulated signaling pathways were determined in macrophages from bronchoalveolar lavage fluid of COVID-19 patients and in lung epithelial cells infected with SARS-CoV-2 in vitro based on transcriptome analysis.A total of 102 TCM formulae for the clinical treatment of nonsevere COVID-19 were collected through literature.The pathway-reversing rates of these formulae in macrophages and lung epithelial cells were evaluated based on signature signaling pathways,and the basic formula was determined in conjunction with TCM theory.The commonly used Chinese materia medica for nonsevere COVID-19 were summarized from the 102 TCM formulae as abovementioned.And together with the screening results from the Pharmacopoeia of the People's Republic of China,a“Chinese materia medica pool”was esta-blished for the development of TCM formulae for COVID-19.The regulatory effects of each herb on signaling pathways were obtained based on targeted transcriptome analysis.Oriented at reversing dysregulated signaling pathways of COVID-19,the calculation was carried out,and the artificial intelligent methods for compositing formulae,that are exhaustive method and parallel computing,were used to obtain candidate compound formulas.Finally,with reference to professional experience,an innovative formula for the treatment of nonsevere COVID-19 was developed.The ethanol extract of the formula was evaluated for its anti-inflammatory effects by detecting the mRNA expression of interleukin 1b(Il1b),C-X-C motif chemokine ligand 2(Cxcl2),C-X-C motif chemokine ligand 10(Cxcl10),C-C motif chemokine ligand 2(Ccl2),nitric oxide synthase 2(Nos2),and prostaglandin-endoperoxide synthase 2(Ptgs2)using reverse transcription-quantitative polymerase chain reaction(RT-qPCR)in RAW264.7 cells treated with lipopolysaccharide(LPS).Results In macrophages and lung epithelial cells,34 dysregulated signaling pathways associated with COVID-19 were identified respectively.The effects of the 102 formulae for clinical treatment of nonsevere COVID-19 were evaluated based on the dysregulated signaling pathways and targeted transcriptome,and the result showed that Yinqiao Powder and Pingwei Powder(银翘散合平胃散,YQPWP)ranked first,reversing 91.18%of the dysregulated signaling pathways in macrophages and 100%of the dysregulated signaling pathways in lung epithelial cells.Additionally,YQPWP had the function of scattering wind and clearing heat,resolving toxins and removing dampness in accordance with the pathogenesis of wind-heat with dampness in COVID-19.It was selected as the basic formula,and was further modified and optimized to develop an innovative fomula Qiaobang Zhupi Yin(翘蒡术皮饮,QBZPY)based on expert experience and artificial intelligence in composing formulae.QBZPY can reverse all the dysregulated signaling pathways associated with COVID-19 in macrophages and lung epithelial cells,with the reversing rates of 100%.The chief medicinal of QBZPY,including Lianqiao(Fructus Forsythiae),Xixiancao(Herba Siegesbeckiae)and Niubangzi(Fructus Arctii),can down-regulate multiple signaling pathways related with virus infection,immune response,and epithelial damage.RT-qPCR results indicated that compared with the model group,the QBZPY group down-regulated the mRNA expression of Il1b,tumor necrosis factor(Tnf),Cxcl2,Cxcl10,Ccl2,Nos2 and Ptgs2 induced by LPS in RAW264.7 cells(P<0.05 or P<0.01).Conclusion Based on targeted transcriptome analysis,expert experience in TCM and artificial intelligence,QBZPY has been developed for the treatment of nonsevere COVID-19.The ethanol extract of QBZPY has been found to inhibit mRNA expression of several pro-inflammatory genes in a cellular inflammation model.