In silico analysis of the potential mechanism of a preventive Chinese medicine formula on coronavirus disease 2019

Abstract

Ethnopharmacological relevance: With the spread of Coronavirus Disease (2019) (COVID-19), combination with traditional Chinese medicine (TCM) has been widely used as a prevention and therapy strategy in China. Xin guan No.1 (XG-1) prescription is a preventive formula recommended by the Hunan Provincial Administration of TCM to prevent the pandemic of COVID-19.

Aim of the study: To explore the potential preventive mechanisms of XG-1 against COVID-19 in the combination of network pharmacology approach, single-cell RNA expression profiling analysis, molecular docking and retrospective study.

Materials and methods: Encyclopedia of Traditional Chinese Medicine (ETCM) database was used to determine the meridian tropism, active components and target genes of XG-1. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were conducted by R Cluster Profiler package (3.14.3). Single cell RNA sequencing (scRNA-seq) data of human lung (GSE122960) was downloaded from Gene Expression Omnibus (GEO) database and analyzed by R Seurat package (3.1.2). Cytoscape (3.7.2) was used to construct the interaction network. The main ingredients in XG-1 were identified by HPLC- Q-TOF- MS and used for molecular docking with COVID-19 3CL hydrolytic enzyme and angiotensin converting enzyme II (ACE2). A retrospective study of 47 close contact participants from Dongtang Community of Hunan Province was conducted to evaluated the preventive effect of XG-1.

Results: According to the network pharmacology analysis, XG-1 formula was closely related to lung-, spleen- and stomach-meridians and include a total of 206 active components and 853 target genes. GO and KEGG pathway enrichment revealed that XG-1 mainly regulated cellular amino acid metabolism process and neuroactive ligand-receptors interaction. The scRNA-seq profiling showed that angiotensin converting enzyme 2 (ACE2) was principally expressed in alveolar type 2 epithelial cells (AT2). 153 genes were up-regulated in AT2 cells expressing ACE2 and 12 genes were obtained by intersecting with XG-1 target genes, of which 3 were related to immunity. Five main chemical ingredients were detected in XG-1 sample by HPLC-Q-TOF-MS. The molecular docking showed that Rutin, Liquiritin and Astragaloside Ⅳ had a good affinity with COVID-19 3CL hydrolytic enzyme and ACE2. Compared with participants who didn't take XG-1, preventive treatment with XG-1gradules resulted in a significant lower rate of testing positive for SARS-CoV-2 nucleic acid (P < 0.0001).

Conclusion: The present study showed that XG-1 exerts a preventive effect in close contacts against COVID-19. The underlying mechanism may be related to modulate immunity response through multiple components, pathways, and several target genes co-expressed with ACE2. These findings provide preliminary evidences and methodological reference for the potential preventive mechanism of XG-1 against COVID-19.

Keywords: Chinese medicine preventive formula; Close contacts; Coronavirus disease 2019; Molecular docking; Network pharmacology; Single-cell RNA Sequencing.

Graphical abstract

Fig. 1

The major chemical components contained in XG-1 preparation identified by HPLC- Q-TOF -MS. A: Chlorogenic acid, the retention time occurs at 7.9 min; B: Rutin, the retention time occurs at 21.1 min; C: Liquiritin, the retention time occurs at 23.9 min; D: Phillyrin, the retention time occurs at 31.6 min; E: Astragaloside Ⅳ, the retention time occurs at 32.2 min.

Fig. 2

Meridian network of XG-1 (A) and numbers of targeted Chinese medicines-genes (B). The green arrow represents the herbs, and the yellow nodes represents the meridians. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The top 20 pathways for GO enrichment analysis of XG-1. x-axis represents count, indicating the number of target genes belonging to a pathway and the number of the annotated genes located in the pathway. y-axis represents pathway. The color of the column reflects the different P adjust. values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

The top 20 KEGG pathway enrichment results of XG-1. x-axis represents gene riao, indicating the ratio of the number of target genes belonging to a pathway and the number of the annotated genes located in the pathway. The size of the dot in-dicates the number of target genes in the pathway and the color of the dot reflects the different P adjust. value. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

tSNE classification (A) and cell clusters statistics (B) of human lung scRNA-Seq. Single cell clustering diagram of ACE2 expression (C).

Fig. 6

Heat map of single cell gene expression and eight cell clusters were divided. (0: AT2; 1: Macrophages; 2: Monocytes; 3: Endothelial Cells; 4: Club Cells; 5: AT1; 6: Cilited Cells; 7: Dendritic Cells). Representative high expressed marker genes in each cell cluster are displayed.

Fig. 7

VlnPlot of 12 intersecting target genes expressed in a single cell.

Fig. 8

The interaction network of the intersection target genes. The yellow diamond represents the intersection target genes; the blue and red circles represent the interaction genes of the intersection target genes. The red ones also indicate that they are the target gene of XG-1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

3D-interaction molecular docking diagram of the three active ingredients with 6LU7 and 1R42.

Fig. 10

2D-interaction diagram of the three active ingredients with 6LU7 and 1R42.