Early intervention with TCM can reduce conversion from mild disease to severe and critical disease[20]. The TCM dialectical formula corresponding to each syndrome in the diagnosis and treatment plan has been widely used in clinical practice. However, no clinical research has further explored the pharmacological mechanisms of HSZF in the treatment of COVID-19. In this study, we initially used the TCMSP database to screen the active TCM constituents of HSZF, the SwissTargetPrediction database to predict targets, and Cytoscape software to construct a pharmaceutical active-compound–potential-target network and a PPI network. GO and KEGG pathway enrichment analysis were performed to construct a target–signal pathway network diagram so as to discover the main active constituents of the formula, as well as its main targets and regulatory pathways. Our research results are summarized as follows.
Based on analysis of the HSZF active-compound–potential-target network, we found that 10 constituents—pectolinarigenin, genkwanin, 5-Hydroxy-7,4'-dimethoxyflavanon, diosmetin, wogonin, quercetin, kaempferol, pachypodol, luteolin, and herbacetin—were the main active constituents of HSZF. This indicated that flavonoids are important for the mechanism by which HSZF treats this disease. ARDS and acute lung injury (ALI) are the main clinical manifestations of critical COVID-19 illness; their symptoms are dyspnea and hypoxemia. Sepsis caused by Gram-negative bacterial infections often cause ALI, and the main component of endotoxins in the outer membranes of Gram-negative bacteria cell walls is lipopolysaccharide (LPS). LPS is a common endotoxin and inflammation-triggering factor that can activate neutrophils to produce a large number of inflammatory factors. After LPS enters the lung tissue of rats, it can cause edema in the pulmonary interstitium and symptoms such as dyspnea and hypoxemia[21]. Difficulty breathing and MOF, which occur in COVID-19, might be related to endotoxins. Studies have shown that quercetin can protect mice from LPS-induced ALI by inhibiting the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and reducing production of NF-κB and intercellular adhesion molecule 1 (ICAM-1) to achieve anti-inflammatory effects[22]. In addition, quercetin mainly prevents the virus receptor complex from entering cells by antagonizing the calcium ion channel and interrupting its life cycle, thereby causing the virus to die and achieving antiviral effect[23]. Diosmetin can reduce LPS-induced ALI by activating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway and inhibiting the nucleotide-binding domain–like receptor protein 3 (NLRP3) inflammatory response[24]. Genkwanin has excellent anti-inflammatory effects in vitro. It mainly inhibits the production of pro-inflammatory mediators by regulating the micro–ribonucleic-acid (miR101), microtubule-associated protein kinase phosphatase-1 (Mkp-1), and mitogen-activated protein kinase (MAPK) pathways in LPS-activated macrophages [25]. Wogonin can inhibit excessive activation of the complement system in vivo and improve ALI induced by influenza A virus[26]. Luteolin has a variety of pharmacological activities, such as anti-inflammatory, anti-allergic, anti-tumor, anti-bacterial, and anti-viral. It is mainly used in clinical practice for resolving cough, eliminating phlegm, and combating inflammation[27]. Kaempferol has anti-oxidant activity and can scavenge oxygen free radicals and reduce tissue damage caused by hypoxia[28], as well as inhibit the expression of tumor necrosis factor alpha (TNF-α), IL-6, IL-10, IL-1β, vascular cell adhesion protein 1 (VCAM-1), and ICAM-1 by reducing the activity of the MAKP, NF-κB and other pathways to exert an anti-inflammatory effect[29].
In terms of potential targets, studies have shown that SARS-CoV-2, like SARS-CoV, binds to ACE2 in the body through its expressed S protein, then invades the body and causes illness, leading to severe pneumonia and high mortality[30]. Other potential targets, such as kininogen-1 (KNG1), epidermal growth factor receptor (EGFR), Caspase-3 (CASP3), signal transducer and activator of transcription 3 (STAT3), and myeloperoxidase (MPO), have all been confirmed to be involved in pulmonary inflammation. For example, KNG1 is an inflammatory mediator; triptolide can reduce chronic obstructive pulmonary disease (COPD) by inhibiting the cellular inflammation caused by cigarette smoke through the KNG1 signaling pathway[31]. EGFR transactivation can induce airway epithelial cells to produce mucus and promote inflammatory-cytokine secretion[32], and studies have shown that EGFR is associated with airway inflammation in asthmatic rats[33]. CASP3 participates in the activation cascade of caspases and is responsible for initiating apoptosis. In a study involving a mouse model of allergic airway inflammation, quercetin improved chronic histopathological changes by regulating the processes of epithelial-derived cytokines and epithelial-cell apoptosis. In addition to basement membrane thickness in lung tissue, it also had beneficial effects on inflammation[34]. STAT3 transcription factors can activate macrophages and neutrophils and enhance the inflammatory response. Research has shown that inhibition of STAT3 activity can protect against ALI caused by LPS[35]. After hemorrhagic-shock resuscitation in rats, MPO activity is enhanced, decomposing extracellular fibers and matrix, thereby causing inflammatory reactions in lung parenchyma and ALI. Inhaling 2% hydrogen can reduce such MPO activity as well as infiltration of inflammatory cells into lung tissue, thereby minimizing the degree of lung injury[36]. The abovementioned findings elucidate the potential action mechanisms of the active constituents and targets of HSZF in the treatment of COVID-19, but there are still some highly relevant targets that previous studies did not prove to have antiviral or anti-inflammatory effects. For example, the biological effect of estrogen is currently thought to be mainly regulated by estrogen receptor α (ERα), and the potential target estrogen receptor 1 (ESR1) combined with estrogen can exert antitumor effects[37]. However, there are no reports on ESR1 having anti-inflammatory or lung-protective effects. Our findings provide new predictions for the molecular basis of HSZF in treating COVID-19 and new ideas for further research.
From the results of GO enrichment analysis, it can be seen that the potential targets of HSZF were mainly distributed across cell membranes, neural axons, lysosome cavities, and other areas, with biological functions such as endopeptidase activity; G protein–coupled peptide receptor activity; nuclear receptor activity; oxidoreductase activity; steroid hormone receptor activity; and participation in biological processes such as cellular metal ion homeostasis, calcium ion homeostasis, cellular divalent inorganic-cation homeostasis, fatty acid–derivative metabolism, unsaturated fatty acid metabolism, positive regulation of phospholipase activity, ion channel regulation, immune regulation, and chemical-signal transmission. Studies have shown that the hydroxyl group can exert its cytotoxic effect by changing the homeostasis of the cell’s calcium ion, causing alveolar macrophages to produce excessive reactive oxygen free radicals, which leads to oxidative lung injury[38]. Inflammation causes disease through fluid transfer across cell membranes and cell layers, which leads to changes in muscle function and pain, and changes in ion channels have been detected in ALI[39]. Neutral endopeptidase is an enzyme that cleaves inflammatory bioactive peptides and plays a protective role in the pathogeneses of ALI and ARDS[40].
From the results of KEGG pathway enrichment analysis, we saw that of the pathways involved in the treatment process, arachidonic acid metabolism was an important inflammatory pathway. It mainly synthesizes inflammatory mediators and mediates the production of multiple inflammatory factors such as monocyte chemotactic protein-1 (MCP-1), TNF, interleukins, and interferon, which are closely related to the development, progression, and resolution of inflammation[41]. Studies have found that SARS-CoV-2 can bind to ACE2 and cause depletion through the receptor-binding domain (RBD) of the S protein, leading to an imbalance in RAS, upregulation of the ACE/angiotensin II (Ang II)/Ang II type 1 receptor (AT1R) pathway, and severe pneumonia[30]. Phospholipase D is closely related to inflammation; PLD1 and PLD2 cause increased leukocyte chemotaxis and inflammation[42]. Platelet activation is also one of the main pathways involved in the treatment process. Platelets play a central role in maintaining coagulation homeostasis, as they are also involved in immune responses and host defenses[43]; and that chemokine (C-X3-C motif) ligand 7 (CXCL7), which induces chemotaxis and activation of neutrophils, and chemokine (C-X3-C motif) ligand 4 (CXCL4), which induces neither, promote ALI via complementary effects and activation of vascular permeability[44]. TRP channel proteins regulate various physiological activities of cells, such as autophagy and apoptosis, by regulating Ca2+ [45]. It is speculated that HSZF treats COVID-19 by inhibiting inflammatory response and regulating both immune function and apoptosis.
In summary, we used network pharmacology techniques and methods to retrieve active TCM constituents and potential targets of HSZF, screened active constituents of drugs and common targets between active constituents and COVID-19, and performed GO and KEGG pathway enrichment analysis on the targets. We speculated that the active constituents of HSZF in COVID-19 treatment might be genkwanin, diosmetin, wogonin, quercetin, kaempferol, and luteolin; and the potential targets might be ACE2, KNG1, EGFR, CASP3, MPO, and STAT3. The abovementioned active components regulate signaling pathways such as arachidonic acid metabolism, platelet activation, phospholipase D, RAS, and inflammatory-mediator regulation of TRP channels to inhibit inflammatory response, regulate immune function, regulate cell apoptosis, and reduce lung injury, so as to achieve the purpose of treating COVID-19.