As the global outbreak of COVID-19 caused by SARS-CoV-2 is rapidly evolving and expanding, its effects is becoming evident—from mild, self-limiting respiratory tract illness to severe acute respiratory distress syndrome (ARDS), multiple organ failure, and death(14). Most patients with COVID-19 have mild symptoms like low-grade fevers and mild fatigue, but no symptoms suggestive of pneumonia. About 5% develop severe symptoms, which can include ARDS, septic shock, and severe metabolic acidosis and coagulation disorders(15).
Innate immune sensing serves as the first line of antiviral defense and is essential for immunity to viruses. Based on recent transcriptional and high dimensional cytometry studies on COVID-19 patients, crucial roles for impaired innate antiviral response, high pro-inflammatory response and dysfunction of adaptive immune response in disease severity of SARS-CoV-2 infection has been established(16, 17). In this present study, immune cell abundance analysis showed significant increases of adaptive immune cells including CD4 + T cells, B cells, CD8 + Tem and iDC, as well as of pro-inflammatory neutrophils, macrophages and mast cells among COVID-19 patients. And we also found an increase of NK cells in the lung of COVID-19 patients, which are identified as vital mediators of early eradication of virus(18). Interferons (IFNs) are considered the most important for antiviral defense. Study demonstrated that SARS-CoV-2 suppresses IFN release in vitro and in vivo, as evidenced by significantly decreasing the production of type I/III IFN signatures in infected cell lines and a ferret model(19). In the meantime, compared to mild or moderate cases, patients with severe COVID-19 present remarkably impaired IFN-I signatures(20). Numerous studies also demonstrated that SARS-CoV-2 is sensitive to IFN-I/III pretreatment in vitro(19, 21, 22). By evaluating the transcriptomic alterations in COVID-19, we identified 136 genes associated with SARS-CoV-2 infection. Functional enrichment analysis of the 136 genes revealed that they were involved in response to type I interferon, type I interferon signaling pathway, response to interferon gamma, interferon alpha/beta signaling. In addition, these genes were significantly related to regulation of cytokine production, cytokine-mediated signaling pathway and signaling by interleukins, which supports the immunopathology of excessive inflammatory cytokine release in systemic infection patients.
It has been found that SARS-CoV-2 is able to bind to the ACE2 receptor on the surface of cells. Zhou et al. demonstrated that ACE2 was the cell entry receptor for SARS-CoV-2 in in vitro studies(23). Our previous study also analyzed the importance of ACE2 in the susceptibility of SARS-CoV-2 infection(24). Therefore, we employed ACE2 as the therapeutic target of COVID-19 to explore the related TCM. Unexpectedly, we obtained five TCMs, including Radix Cyathulae, Flos Puerariac, Radix Bupleuri, Radix Puerariac and Radix Hemerocallis. We also found that puerarin was the common active compound across the five TCMs, which targeted ACE2. Besides mediation of the virus entry, it has been documented that ACE2 is related to the regulation of inflammatory cytokine response in coronavirus infected cells. In this current study, we found that 6 puerarin target proteins were significantly involved in our previously constructed PPI network of inflammatory cytokine production which was crucial in ACE2-associated inflammation. This is consistent with previous studies. Puerarin was also able to reduce inflammation by regulation of neutrophils and eosinophils in rabbit models(25). In addition, puerarin had no toxic effect on host cells but only had a moderate ability to reduce viral production(26). Our functional enrichment analysis also suggested that the puerarin target genes were principally participated in COVID-19-associated gene sets. Therefore, we speculated that puerarin would have a potential effect on COVID-19.
However, due to the complex targets by puerarin, the detailed mechanisms need to be further addressed. To figure it out, we intersected the potential targets of puerarin with COVID-19 associated genes. Several genes, ACE2, STAT1, RNASE3, LCK, TNF, FOS, PDE5A, IMPDH2, MAPK8 and MAPKAPK2, were identified as potential therapeutic targets of COVID-19 by puerarin. Molecular docking simulation indicated that puerarin with effective score when docking with these potential therapeutic targets, which is in agreement with the above integrated analysis results. These target genes were significantly enriched in T-cell-associated pathway, such as virus indued T cell response, T Cell Receptor Signaling, Cell Differentiation of Th1, Th2 and Th17. A wealth of studies confirmed that Th1 cells are the leading Th cells in T cell immunity during SARS-CoV infection. Th1 cells generally secret IL-2, IFN-γ, IL-12, and TNF-a in the site of viral infection(27). However, recent studies on COVID-19 patients proposed that the production of Th2 cytokines along with Th17-specific cytokines are also increased. TCR and cytokines released from Th cells are indispensable for activation of CD8 + T cells. Cheng MH and his colleague analyzed the TCR repertoire in adult SARS-CoV-2 infected patients, and found that SARS-CoV-2 spike glycoprotein as a superantigen induces cytokine storm via binding to TCR followed by TCR skewing(28). Indeed, puerarin has been shown to regulate expression of Th1/Th2 cytokines and inhibit Th17 cells in rats(29). Thus, we speculated that puerarin probably exert a protective effect on COVID-19 through modulation of T cell immunity.
Of all the targets, TNF, STAT1 and RNASE3, in addition to ACE2, were mainly up-regulated after SARS-CoV-2 infection. According to our GSEA analysis, high expression of TNF mainly associated with virus response and cytokine secretion related immune cell response. And STAT1 was primarily correlated with virus defense response, cytokine production and humoral immunity. It has been previously reported that serum TNF-α levels in severe COVID-19 patients are significantly higher than patients who have mild symptoms(30). Karki et al. identified that TNF-α is a key factor, working together with IFN-γ, to induce immune cell death and cytokine shock syndrome during SARS-CoV-2 infection through the of JAK/STAT1/IRF1 axis(31). Meanwhile, RNASE3 was highly related to virus defense, macrophage activation and mast cell mediated cytokine response based on our GSEA analysis. Earlier, KEGG pathway analysis of the up-regulated differentially expressed genes conducted by Lu and co-workers suggested that TNF signaling pathway are significantly enriched in RNase3 treated macrophages. STAT1 are also activated in RNase3 exposed macrophages, and this activation is followed by the hyper-production of chemokines and cytokines including TNF-α(32). Moreover, uncontrolled activation of mast cells can rapidly release large amounts of TNF-α and proteases stored in granules to further aggravate cytokine secretion storm and exacerbate disease severity(33). Additionally, our PPI analysis also showed that TNF was the hub protein in the regulation network, indicating that this molecule was critical in COVID-19. As ARDS with cytokine storm might be the main cause of death due to severe viral infection, anti-inflammatory active compounds could be effective options for COVID-19. It has been established that chloroquine, a classic antimalarial compound, can inhibit production of TNF-α on the mRNA level, the pretranslational level and the post-transitional level, as well as block the receptor induced TNF pathway during virus infection(34). Early study demonstrated that puerarin greatly suppresses systemic inflammation by inhibiting the transcription of inflammatory factor TNF-α, IL-6, and IL-1β in sepsis mouse(35). Additionally, puerarin significantly inhibited inflammation through the down-regulation of nuclear factor-κB (NF-κB), MAPK activation and the secretion of pro-inflammatory mediators (36, 37). Puerarin administration also reduces the expression of TNF-α and STAT1 in bone mesenchymal stem cells by regulation of miR-155-3p(38). From this point, we speculated that puerarin may against COVID-19 by mainly mediation of proinflammatory cytokine response, especially targeting the TNF pathway.