1. Introduction
The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus type 2 infection. Most asymptomatic infected patients and mildly infected patients have a better prognosis, and a few patients become critically ill or even die. Although the COVID-19 vaccine is available, the number of people diagnosed is still rising, and the newly discovered Omicron strain may weaken the vaccine’s protection.[1] On February 11, 2022, China approved the import of Paxlovid, which has been shown to reduce hospitalization and mortality rates in COVID-19 patients.[2] Since the outbreak of COVID-19, traditional Chinese medicine has played an important role in the prevention and treatment of COVID-19.
Luteolin is a flavonoid found in many plants that has a variety of pharmacological activities. Numerous studies have shown that luteolin has anti-inflammatory, antioxidant, and neuroprotective effects in vitro and in vivo and possesses a wide range of anti-cancer activities. As one of the main components of various herbal compounds, luteolin are widely used for the prevention and treatment of various diseases.[3] In the treatment of breast cancer, luteolin can be used not only as a chemopreventive and chemotherapeutic agent but also together with other anticancer drugs, such as cyclophosphamide, doxorubicin, and nonsteroidal anti-inflammatory drugs. Luteolin has been shown to inhibit the Ras/Raf/MEK/ERK signaling pathway by increasing Mir-203 expression, thereby suppressing breast cancer development and progression.[4]
Molecular docking is one of the most commonly used methods to calculate protein-ligand interactions and is widely used in drug discovery, mainly to predict the interaction between ligands and targets at the molecular level. It identifies intermolecular interactions and predicts the best binding mode between 2 molecules according to the principles of geometrical, energy, and chemical environment complementarity. Luteolin has been shown to have multiple pharmacological actions and targets; however, its mechanism of action in the treatment of COVID-19 has not been elucidated. In this study, we analyzed the differences in gene expression in the alveolar tissues of COVID-19 and non–COVID-19 patients and identified 271 differentially expressed genes (DEGs). After analyzing the results of molecular docking, we found that luteolin bound well to 20 of the 22 upregulated DEGs enriched in the COVID-19 signaling pathway; 6 (CFB, EIF2AK2, OAS1, MAPK11, OAS3, STAT1) of these genes had a strong binding activity. This explains, to some extent, the molecular mechanism of luteolin in the treatment of COVID-19.
2. Methods
2.1. Source of data
Based on the GPL29228 NextSeq 550 platform, the alveolar tissue gene expression data set GSE159787 was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), including 21 alveolar tissues from COVID-19 patients and 18 normal alveolar tissues from non–COVID-19 patients. The chemical structure of luteolin was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/).
2.2. Screening the DEGs in COVID-19
Results were evaluated using the “Limma” package in R (v4.1.2). Coronavirus disease 19 alveolar tissue and normal alveolar tissue between the DEGs were evaluated; the standard was | log2FC | ≥ 1 and P < 0.05.
2.3. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes
To further analyze the differences in gene functions and pathways between the 2 groups, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the upregulated genes in DEGs using “clusterprofiler” package in R (v4.1.2).
2.4. Molecular docking
Molecular docking studies were performed on selected proteins using AutoDock Tools 1.5.6 based on the crystal structures of proteins downloaded from the RCSB Protein Database (PDB: https://www.rcsb.org/).
3. Results
3.1. Acquisition of DEGs in COVID-19
Thirty-nine samples downloaded GEO database were divided into 2 groups: the COVID-19 group, which contained 21 COVID-19 alveolar tissue samples, and the normal group, which contained 18 normal alveolar tissue samples. Compared with normal alveolar tissue samples, 271 DEGs were identified in the COVID-19 group, including 186 upregulated genes and 85 downregulated genes.
3.2. Gene Ontology functional and KEGG pathway enrichment analysis
The GO and KEGG pathways of 186 upregulated genes among the DEGs from the COVID-19 and normal groups were further analyzed. Biological processes with high enrichment scores include positive regulation of cell activation, regulation of immune effector processes, activated T cells, and response to viruses. Cellular components include lateral plasma membrane, endocytic vesicles, adherent plaques, and cell-basal junctions. For molecular function, signal receptor activator activity, amide binding, and peptide binding were all related to COVID-19 [Figure 1A]. KEGG analysis showed that these genes were mainly enriched in the signaling pathways of COVID-19, human papillomavirus infection, and Epstein-Barr virus infection [Figure 1B].
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Figure 1: GO and KEGG analysis. (A) GO enrichment analysis of 186 significantly upregulated genes; (B) KEGG pathway enrichment analysis of 186 significantly upregulated genes. BP: Biological process; CC: Cellular component; MF: Molecular function; GO: Gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.
3.3. Molecular docking analysis
Molecular docking results showed that luteolin was well bound to 20 of 22 DEGs enriched in the COVID-19 signaling pathway, which partially explains the therapeutic mechanism of luteolin in COVID-19 [Table 1]. Luteolin inhibited the development of COVID-19 by regulating targets, such as CFB, EIF2AK2, OAS1, MAPK11, OAS3, and STAT1 [Figure 2].
Table 1 -
The results of
molecular docking
| Gene-SYMBOL |
Docked energy |
| CFB |
−8.57 |
| EIF2AK2 |
−8.51 |
| OAS1 |
−8.34 |
| MAPK11 |
−7.66 |
| OAS3 |
−7.59 |
| STAT1 |
−7.21 |
| MAPK3 |
−6.93 |
| IRF9 |
−6.88 |
| DDX58 |
−6.72 |
| CXCL10 |
−6.54 |
| ISG15 |
−6.46 |
| MX1 |
−6.46 |
| STAT2 |
−6.39 |
| C3 |
−6.27 |
| C1R |
−6.15 |
| C1QA |
−5.97 |
| TNFRSF1A |
−5.80 |
| C1S |
−5.77 |
| C1QB |
−5.69 |
| IL6ST |
−5.55 |
| IFIH1 |
−2.52 |
| OAS2 |
– |
Figure 2: Molecular docking data indicate that the binding capacity of luteolin with COVID-19 is significant in the key targets of CFB (A), EIF2AK2 (B), OAS1 (C), OAS3 (D), MAPK11 (E), and STAT1 (F). COVID-19: Coronavirus disease 2019.
4. Discussion
Currently, increasing attention is being paid to research focusing on natural products. Many novel therapeutic drugs derived from natural products can be decoded by molecular biological analysis. This can help clarify disease mechanisms and identify potentially active compounds at the systemic level.
This study integrated publicly available databases with COVID-19 information to predict the potential therapeutic targets and interactions of luteolin. These results show that luteolin can bind to CFB, EIF2AK2, OAS1, MAPK11, OAS3, STAT1, and other targets to regulate the COVID-19 signaling pathway and play a role in the prevention and treatment of COVID-19.
CFB is an important component of the alternative complement activation pathway. It has been found that knockdown of CFB significantly reduces the complement alternative activation pathway in a diabetic mouse model. Under high glucose concentration stimulation, mTOR signaling is activated in mice, STAT1 phosphorylation is enhanced, CFB expression is upregulated, and protein phosphatase 2Aα (PP2Acα) expression is downregulated. In contrast, PP2Acα deficiency enhances high glucose-induced mTORC1/STAT1 activation, CFB upregulation, and podocyte injury, preliminarily elucidating the mechanism of CFB-mediated podocyte injury in diabetic nephropathy.[5] Therefore, the targeted inhibition of CFB expression may play an anti-inflammatory role.
EIF2AK2, also known as protein kinase R, is activated by double-stranded RNA, which blocks the translation of viral mRNA in response to infection and selectively regulates immune responses, signaling, apoptosis, cell proliferation, and differentiation. In studies related to systemic lupus erythematosus, EIF2AK2 was found to regulate the transcription of disease-related histone genes, and it was suggested that this action may be operated mainly by EIF2AK2-targeted transcription factors. EIF2AK2 and EIF2AK2-regulated transcriptional factors were proposed to be a promising antagonist and new therapeutic target for future immune-related diseases.[6] In the COVID-19 signaling pathway, we speculated that this gene may primarily play an immunomodulatory role while stimulating expression in response to drugs, thereby enhancing the immune function of the organism.
OAS1 and OAS3 belong to the OAS family, a family of proteins involved in the immune regulation of chronic inflammation, autoimmune diseases, cancer, and infectious diseases. In addition, OAS1 and OAS3 are members of the interferon-stimulated gene family. Among them, OAS1 negatively regulates the expression of chemokines and interferon-responsive genes in human macrophages, and OAS3, while inducing RNase L cleavage of the RNA virus genome, also activates the type I interferon response to further enhance the immune effect.[7] In tumors, OAS1 and OAS3 were also found to be highly expressed in patients with breast cancer and were associated with poor patient prognosis.[8] These results suggest that the inhibition of OAS1 and OAS3 expression may serve as new drug targets for the treatment of COVID-19.
There are relatively few studies on MAPK11, and most available studies have focused on its interaction with solid tumors. A tumor-related study proposed that a specific knockdown of MAPK11 could lead to a complete loss of radiosensitivity associated with gemcitabine, thus mediating a significant increase in tumor resistance to certain chemotherapeutic agents.[9]
STAT1 can modulate a variety of immune-related diseases by inducing proinflammatory subpopulations. In addition, STAT1 is an important mediator of the anti-tumor response, which can promote T-cell–mediated immune response to tumors by suppressing bone marrow–derived suppressor cells.[10] Research targeting STAT1 may improve the prognosis of COVID-19 patients by regulating the immune response.
5. Conclusions
In our study, we first identified DEGs in COVID-19 and normal patients. Functional enrichment analysis, including GO and KEGG, showed that upregulated genes in DEGs were significantly enriched in COVID-19–related signaling pathways. The results of molecular docking showed that luteolin bound well to 20 upregulated DEGs enriched in the COVID-19 signaling pathway. Six of these genes (CFB, EIF2AK2, OAS1, MAPK11, OAS3, and STAT1) showed strong binding activity. This study provides a theoretical basis for further research on the role of luteolin in COVID-19 pathogenesis.
Author Contributions
Xiaoping Huang and Hechen Rong conceived and designed the article. Xiaoping Huang provided administrative support. Yu Zeng, Nan Zheng, and Zhonghong Guo collected and organized the data. Hechen Rong, Kun Wang, and Yiwei Wang performed the data analysis and interpretation. All authors revised the manuscript. All authors gave final approval of the manuscript.
Conflicts of Interest
None.
Data Availability Statement
The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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