XBJ has been approved for the treatment of severe infection (sepsis). For a long period of time in China, it was believed that XBJ could improve prognosis in severe lung infection as well as 28-day mortality rate, Acute Physiologic Assessment and Chronic Health Evaluation (APACHE) II score, white blood cell (WBC) count, and temperature in sepsis patients without causing serious adverse events. A prospective, randomized controlled trial in 33 hospitals in China, published in September 2019, showed that XBJ can significantly improve the primary endpoint of pneumonia severity in patients with severe community-acquired pneumonia, as well as secondary endpoints such as mortality rate, mechanical-ventilation duration, and length of intensive-care unit (ICU) stay. Since the start of the COVID-19 outbreak, XBJ has been recommended as a Chinese patent medicine in local COVID-19 diagnosis and treatment plans released by many provincial health commissions due to its rapid onset and significant efficacy in critically ill patients. This shows that XBJ might have important clinical value in COVID-19 treatment. However, the material bases and molecular effector mechanisms are still unclear. Therefore, analysis of the potential effector mechanisms of XBJ in the treatment of COVID-19, elucidating its potential active ingredients and their potential effector targets, and demonstrating the network effector mechanisms of XBJ on COVID-19, can provide a scientific basis for using XBJ in the clinical treatment of COVID-19.
This study preliminarily demonstrated XBJ’s “multiple component–multiple target–multiple pathway” effector characteristics, and our network topology analysis of potential effector targets identified some critical effector targets of the compound. GO and KEGG enrichment analyses found that the potential targets of XBJ involved multiple inflammation- and immune-related gene functions and signaling pathways, which might be one basis for XBJ treatment in COVID-19.
From a potential active-ingredient perspective, XBJ possesses potential anti-inflammatory and immune-boosting effects. The three active ingredients of Carthamus tinctorius have protective effects in lipopolysaccharide (LPS)-induced acute lung injury (ALI). The potential anti-inflammatory components in XBJ inhibit NF-κB activity; decrease expression of TNF-α, IL-1β, and IL-6; alleviate inflammatory responses; and inhibit secretion of pro-inflammatory cytokines mediated by the high-mobility group box 1 protein (HMGB1)–receptor for advanced glycation endproducts (RAGE) axis, thereby decreasing mortality rate in a mouse model. XBJ also upregulates toll-interacting proteins in septic rats to protect the lungs from permeable leakage and injury. In addition, it prevents cytokine storm, inhibits inflammatory responses, and regulates regulatory T-cell (Treg)–T helper 17 cell (Th17) balance to improve survival in septic shock. In addition, XBJ promotes the expression of annexin A1 to inhibit cleavage of pro-inflammatory cytokines and decreases IL-8 and TNF-α levels to protect rats from damage due to Acinetobacter baumannii sepsis. SARS-CoV-2 replicates in respiratory-tract epithelial cells to cause acute inflammation and severe respiratory disease. During infection, local production of pro-inflammatory cytokines exacerbates disease progression. Therefore, the anti-inflammatory activity and cytokine-inhibitory effects of XBJ might constitute its potential mechanism in COVID-19 treatment.
In GO BP analysis, the 144 potential therapeutic targets of XBJ involved multiple inflammation- and immune-related BPs such as extracellular signal-regulated kinase 1 and 2 (ERK1, ERK2) cascade, the T-cell receptor signaling pathway, activation of MAPK activity, and cellular response to LPS. This suggests that the significant anti-inflammatory effects of XBJ are its therapeutic effects in inflammation. Virus-host interactions are an important aspect of viral replication. Ribonucleic acid (RNA) viruses such as influenza, Ebola virus, and SARS-CoV can induce Raf–mitogen-activated protein kinase kinase (MEK)–ERK signal transduction in the MAPK cascade, which is associated with replication of pathogenic RNA viruses in humans and allows for cell differentiation and proliferation. This is consistent with the fact that XBJ targets the ERK1/ERK2 cascade.
Extensive proteinoid and serous exudates are present in the alveoli of COVID-19 patients. These cases also present bilateral diffuse alveolar damage accompanied by fibromyxoid exudates, and both lungs show apparent pulmonary edema, alveolar epithelial detachment, and hyaline-membrane formation. In terms of infection-related serum markers, studies show that C-reactive protein (CRP), IL-6, and erythrocyte sedimentation rate (ESR) are significantly increased in many patients. Severe cytokine storm can appear in severely to critically ill patients, resulting in excessive immune activation and excess production of IL-7, IL-10, granulocyte colony-stimulating factor (GCSF), interferon gamma inducible protein 10 kD (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1A (MIP1A), and TNF-α. From this, it can be seen that SARS-CoV-2–mediated inflammation plays an important role in COVID-19 progression, and uncontrollable lung inflammation may be the main cause of death in COVID-19. Therefore, intervention measures to reduce inflammation might help decrease the mortality rate.
With regard to effector targets, the degree values of GAPDH, albumin (ALB), TNF, EGFR, MAPK1, CASP3, STAT3, MAPK8, PTGS2, JUN, IL-2, Estrogen Receptor 1 (ESR1), and MAPK14 were all >40, suggesting that in COVID-19 these could be the main therapeutic targets of XBJ’s active ingredients. Then we verified the binding activity of the XBJ key compounds and the COVID-19 key targets via molecular docking. The results indicated that they have potential binding activity and these ingredients may be potential compounds in anti-COVID-19 of XBJ. Wikipathways and KEGG enrichment analysis contained 40 and 94 signaling pathways, showing that the 144 potential XBJ therapeutic targets were involved in many inflammation- and immune-related signaling pathways.
The renin–angiotensin system (RAS), OS and cell death, cytokine storm, and endothelial dysfunction are four main pathways in COVID-19 pathogenesis. ACE is a receptor in the airway, alveoli, and vascular endothelium. COVID-19 uses ACE to enter type II pneumocytes or intestinal epithelial cells in order to induce ACE2 internalization and shedding, resulting in the occurrence and development of acute respiratory distress syndrome (ARDS). Many of XBJ’s active ingredients act on multiple targets in the RAS, which could potentially interfere with ACE receptors. NF-κB activation exacerbates lung inflammation caused by SARS-CoV infection, and inhibition of NF-κB signaling significantly reduces such inflammation and increases the survival rate of SARS-CoV-infected mice[50, 51]. In addition, NF-κB is an important transcription factor that induces expression of viral genes, and inhibition of NF-κB activation is an immune evasion mechanism of SARS-CoV. Therefore, we speculate that XBJ’s active ingredients might interfere with the NF-κB pathway and regulate innate immunity and inflammation during viral infection to alleviate lung inflammation during COVID-19.
One study has shown that XBJ inhibits MAPK and NF-κB expression and has protective effects in ALI. The compound regulates the NF-κB, MAPK, and PI3K–Akt pathways in mouse macrophages and downregulates inflammatory cytokines such as IL-6, TNF-α, MCP-1, MIP-2, and serum IL-10 to increase the survival rate of septic mice . XBJ downregulates toll-like receptor 4 (TLR-4) and NF-κB expression to carry out its anti-inflammatory effects. MAPK activation can promote the expression and release of pro-inflammatory cytokines such as TNF-α and IL-1β, -6, and -8; it is a core factor in inflammation regulation. Viruses usually directly or indirectly affect the PI3K–Akt pathway to control intracellular-signaling pathways. EGFR aggregation and binding of influenza virus to cell surfaces might activate Akt. The PI3K–Akt signaling pathway might synergize with the RAS to promote viral entry, which has significant effects in viral infection in humans. TLR-2, TLR-3, and TLR-4 activation by COVID-19 causes the release of inflammatory cytokines such as IL-1β. The binding of SARS-CoV-2 to TLRs causes release of pro-IL-1β, inflammasome activation, and production of mature IL-1β. Pro-inflammatory cytokines are important mediators of local and systemic inflammation. Viral particles first invade the respiratory mucosa before infecting other cells, thereby inducing a series of immune responses leading to cytokine storm. Therefore, XBJ could be used to treat COVID-19 patients due to its anti-inflammatory effects, anti-immune apoptosis, and alleviation of pneumonia-induced multi-organ damage. In addition, we found that critical nodes on our “XBJ active-compound-potential effector target” network analysis map participated in the aforementioned pathways, suggesting that the predictions made in this study are somewhat accurate.
When compared to other research works on NP related to COVID-19 prevention and treatment, some of our research results and conclusions are consistent with the current similar research in some aspects. Qingfei Paidu Decoction(QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China, has been shown by a recent NP research that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects related to the thrombin and TLR signaling pathway. What’s more, QFPD could protect COVID-19 injury via anti-Viral, anti-Inflammatory activity and metabolic programming. Another research shows that Pudilan (PDL), clinically used as an anti-SARS-CoV-2 infective agent in China. PDL might moderate the immune system to shorten the course of the disease, delay disease progression, and reduce the mortality rate. Hence, QFPD and PDL, together with XBJ may have a therapeutic effect on COVID-19 through three aspects, including the immune system, anti-inflammation, and anti-virus entry into cells. Previous similar study have shown that 8 key compounds and 15 key targets of XBJ were screened and verified by molecular docking, on the basis of this research, we have made improvements in the criteria for screening active ingredients, drug targets, and disease targets. As XBJ is administered intravenously, which does not need to be absorbed through the gastrointestinal tract, therefore, we selected active ingredients of XBJ that had been detected by LC-MS in our study. What’s more, we used the latest update version of Swiss database to predict the targets of XBJ. In terms of the potential core targets of COVID-19, we finally found different results from previous studies.
However, there are several limitations in the current study. The host proteins and existing targets of the relevant coronaviruses are used to be potential therapeutic targets for COVID-19. Although SARS-CoV-2 shared high nucleotide sequence identity with other HCoVs, our predictions are not SARS-CoV-2 specific by lack of the known host proteins on SARS-CoV-2. In terms of COVID-19 treatment targets, we have an adopted alternative strategy that is currently achievable.
SARS is associated with epithelial-cell proliferation and an increase in macrophages in the lung. And diffuse alveolar damage was seen in COVID-19 cases. Regarding the differences between COVID-19 and SARS. Studies reveal that SARS-CoV-2 is very similar in structure and pathogenicity with SARS-CoV, but the most important structural protein, i.e., the spike protein (S), is slightly different in these viruses. Compared to other beta coronaviruses, the presence of a furin-like cleavage site in SARS-CoV-2 facilitates the S protein priming and might increase the efficiency of the spread of SARS-CoV-2[62, 63]. COVID-19 has diverse epidemiological and biological characteristics, making it more contagious than SARS-CoV and MERS-CoV. It has affected more people in a short time period compared to SARS-CoV and MERS-CoV, although the fatality rate of MERS-CoV was higher than SARS-CoV and SARS-CoV.
We explore the molecular mechanism of Xuebijing injection in COVID-19 based on the premise of host and protein interaction. We use coronavirus-related host proteins as potential targets, which aim to produce an indirect intervention against viral targets for the treatment of COVID-19.