4.2 Antiviral action of H2S and its underlying mechanisms in relation to COVID-19 infection
Burgeoning evidence shows that H2S donors such as NaHS and GYY4137 exhibit excellent effects against the family of enveloped RNA viruses of which SARS-CoV-2 is a member (Li et al., 2015; Ivanciuc et al., 2016; Bazhanov et al., 2017; Bazhanov et al., 2018) Also, natural sources of exogenous H2S such as diallyl sulfide, diallyl disulfide and diallyl trisulfide, which are derived from garlic, have been reported to reduce viral load of cytomegalovirus (another enveloped virus) in infected organs of humans and rodents (Fang et al., 1999). Furthermore, sinigrin, a precursor of the H2S donor allyl isothiocyanate, and obtained from the root extract of Isatis indigotica plant for Chinese traditional medicine (Martelli et al., 2020), inhibited the function of 3-chymotrypsin-like protease, the main protease of SARS-CoV, which caused the 2002–2004 outbreak of severe acute respiratory syndrome (Lin et al., 2005).
There are several mechanisms that underlie the antiviral action of H2S. Firstly, the antiviral activity of H2S has been suggested to be partly linked to its antioxidant property - activating and increasing the levels of other antioxidant enzymes including glutathione (GSH), the most abundant naturally occurring antioxidant in the body, which inhibits overproduction of reactive oxygen species (ROS; a destructive mediator in tissue injury) and its consequent oxidative stress (Palamara et al., 1995; Kim et al., 2020). Interestingly, ROS-induced oxidative stress has been associated with viral infection in the kidney (Horoz et al., 2006), impairing the kidney’s antioxidant defense system. Moreover, Kim et al. (2020) recently predicted in their study involving high-throughput artificial intelligence-based binding affinity that GSH interacts with and possibly inhibits the action of ACE2 and TMPRSS2, the two proteins that facilitate SARS-COV-2 entry into the kidney. Secondly, findings from a recent study also suggested that H2S may also exhibit its antiviral activity against SARS-CoV-2 by interfering with ACE2 and TMPRSS2 and blocking the attachment of the virus to these host proteins (Yang et al., 2019), and thereby inhibiting entry of the virus into the host cell (Fig. 2). Besides, administration of H2S via its donor molecule NaHS has been reported to upregulate carotid ACE2 expression and reduced organ damages in a mouse model of carotid artery ligation (Lin et al., 2017). A recent molecular dynamics simulation study showed that reduction of disulfides in ACE2 and S protein of SARS-CoV-2 into sulfydryl groups impairs the binding of the S protein of SARS-CoV-2 to ACE-2 (Hati et al., 2020). Interestingly, administration of N-acetylcysteine (NAC; an antioxidant H2S donor and a source of cysteine for endogenous GSH production) disrupted the disulfides, leading to inhibition of SARS-CoV-2 into the host cell (Manček-Keber et al., 2021). Moreover, NAC is a known mucolytic agent that breaks disulfide bonds in mucus, making it less viscous and easier to be expelled by other mucoactive agents (expectorants and mucokinetics) together with the action of the ciliary apparatus of the respiratory system. Thus, H2S facilitates elimination of potentially harmful viruses such as SARS-CoV-2, suggesting its antiviral action in COVID-19.
Thirdly, the antiviral action of H2S involves Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs) that initiate innate immune response for early immune recognition of a pathogen. Following release of viral RNA (i.e. pathogen-associated molecular pattern) into host cells, it is recognized by PRRs such as TLRs in the host immune cells, which activates production and secretion of large amounts of proinflammatory cytokines and chemokines responsible for cytokine storm and organ damage as seen in the various kidney conditions discussed in previous sections (Sallenave and Guillot, 2020). Chen and colleagues (2021) recently reported that deficiency in endogenous H2S level contributes to sepsis-induced myocardial dysfunction (SIMD) in humans and mice via increased expression of TLRs. However, administration of NaHS in SIMD mice inhibited TLR pathway and prevented TLR-mediated inflammation. Although this study was not in relation to viruses, it is likely that antiviral action of H2S involves the same mechanism. Besides, H2S has been reported to inhibit activation and nuclear translocation of nuclear factor-kappaB (NF-κB; an inflammatory-related transcription factor) and thereby suppressing the transcription of pro-inflammatory genes, leading to inhibition of the secretion of virus-induced chemokines and cytokines (Li et al., 2015). Fourthly, post-mortem examination of transplanted kidney, lungs and heart of COVID-19 deceased patients revealed endotheliitis and accumulation of apoptotic bodies (Varga et al., 2020), suggesting that inflammation of the endothelium (an important gatekeeper of cardiovascular health and homeostasis) and apoptotic cell death contributed to dysfunction or malfunction of these organs in the COVID-19 patients, which resulted in death of these patients. As a potential therapy for COVID-19 patients, there are studies showing the ameliorative effect of H2S endothelial dysfunction in cardiovascular disorders such as hypertension, atherosclerosis, hyperhomocysteinemia as well as in diabetes (Citi et al., 2020; Sun et al., 2020). Besides, overactivation of the sympathetic nervous system has recently been implicated in COVID-19 patients with pre-existing chronic lung diseases, kidney diseases, cardiovascular pathologies, obesity and diabetes mellitus through factors including ACE2 imbalance, which contributes to organ damage in these patients (Porzionato et al., 2020). Interestingly, H2S donors such as NaHS are well-known to suppress sympathetic activation (Kulkarni et al., 2009; Guo et al., 2011; Duan et al., 2015; Salvi et al., 2016), and therefore inhibition of sympathetic outflow could be a potential therapeutic mechanism by H2S donors for COVID-19 patients.
Another mechanism underlying the antiviral action of H2S in relation to COVID-19 involves interaction with endoplasmic reticulum (ER) stress-related proteins. A recent preliminary virtual screening study in patients with COVID-19 pneumonia revealed higher gene expression and serum concentrations of glucose-regulated protein 78 (GRP78; an ER stress protein and the host cell surface protein to which the Spike protein of SARS-CoV-2 binds as revealed by molecular docking) compared to pneumonia patients without COVID-19 (Palmeira et al., 2020). There are studies showing the inhibitory action of H2S donors on GRP78 and other ER stress-related proteins in experimental models of human diseases. Yi et al. (2018) reported that administration of NaHS downregulated the expression of GRP78 and other ER stress-related proteins in uranium-induced rat renal proximal tubular epithelial cells and mitigated ER stress via activation of Akt/GSK-3β/Fyn-Nrf2 pathway, a protective molecular pathway. Thisin vitro result supports a previous result by Wei et al. (2010) who observed attenuation of hyperhomocysteinemia-induced cardiomyocyte injury following H2S administration in rats. Administration of NaHS also markedly inhibited cigarette smoke-induced overexpression GRP78 and other markers of ER stress-mediated apoptosis and prevented lung tissue damage (Lin et al., 2017). These pieces of experimental evidence suggest that H2S donors could be potential antiviral agents that serve to treat COVID-19 patients by preventing entry of SARS-CoV-2 into host cells via inhibition or downregulation of the expression of GRP78 and other ER stress-related proteins, thereby preventing apoptosis and organ damage. In addition to all these mechanisms, we also reported that H2S decreases renal expression of kidney injury molecule (KIM-1; a biomarker of human renal proximal tubular injury) (Dugbartey et al., 2015a; Dugbartey et al., 2015b), which has recently been found to be associated with COVID-19 nephropathy and potential receptor for SARS-CoV-2 entry into renal and lung cells (Wan et al., 2021). Renal and lung epithelial cells of humans and mice co-expressed KIM-1 and SARS-CoV-2 Spike protein (Ichimura et al., 2020), suggesting that KIM-1 could directly bind to SARS-CoV-2 Spike protein following its induction by AKI or other pathological conditions involving the kidney, as this interaction was inhibited by anti-KIM-1 antibodies and the KIM-1 inhibitor, TW-37 (Ichimura et al., 2020). Yang et al. (2021) also implicated KIM-1 and ACE2 in a synergistic interaction which mediated the invasion of SARS-CoV-2 in kidney cells and worsened COVID-19 infection in the kidney. We recently showed that activation of endogenous H2S production by dopamine administration increases renal expression of H2S-producing enzymes (CBS, CSE and 3-MST) and serum H2S level and decreases renal KIM-1 expression, leading to increased kidney protection in a rat model of deep hypothermia/rewarming-induced AKI (Dugbartey et al., 2015a). We also observed decreased expression of KIM-1 in renal tubules and preservation of renal structures following administration of 5’-adenosine monophosphate, which correlated with increased renal H2S-producing enzymes and serum H2S level in a hamster model of therapeutic hypothermia (Dugbartey et al., 2015b). These observations together with other potential mechanisms that decrease KIM-1 expression in kidney and lung tissues suggest that H2S may offer a new therapy for COVID-19-associated nephropathy and pneumopathy. Other mechanisms underlying the antiviral action of H2S or H2S donors include inhibition of gene transcription along with antiviral immunosuppressive effect, as was reported in human cytomegalovirus (Zhen et al., 2006) and alterations of the viral membrane, as XM-01 (an H2S donor) inhibited the activities of enveloped viruses but had no effect on non-enveloped viruses (Pacheco et al., 2017). The findings from all these studies strongly suggest that H2S donors could serve a therapeutic purpose in COVID-19 and its complications including COVID-19-associated nephropathy (Fig. 2).