Coronavirus disease 2019 (COVID-19) was a public health emergency of international concern, causing over 632 million confirmed cases and 7 million deaths worldwide. Furthermore, COVID-19 has had a serious socioeconomic impact globally in the last three years (1). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19 and directly binds to the SARS-CoV-2 entry receptor, angiotensin-converting enzyme-2 (ACE2) (2, 3). Infected patients have clinical features ranging from asymptomatic to severe disease. Most critically ill patients deteriorated suddenly in the later stages of the disease and died in a short period of time due to acute respiratory distress syndrome (ARDS), which arises from cytokine release syndrome (CRS) and a complex immune-inflammatory response (4). Global efforts were made to rapidly develop specific anti-SARS-CoV2 antiviral drugs. However, the long process of validating the clinical efficacy and safety limited the drug’s development. Therefore, the current agents proved efficient and safe and required further evaluation in COVID-19 treatment. Emerging evidence has shown that excessive inflammation is a key driver of poor COVID-19 outcomes. The coagulation markers, proinflammatory cytokines, such as interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF‐α), and lymphopenia are associated with the severity of COVID-19 (5–7). Therapeutic immunomodulation is effective in the management of inflammatory diseases. Currently, the immunomodulatory agents include therapies targeting the IL-6/interleukin 6 receptor (IL-6R), TNF-α, and IL-1β pathways (8, 9). These agents have been suggested for treating the advanced stage of COVID-19 by reducing inflammatory responses (10, 11). The clinical outcomes of patients with COVID-19 were improved by these immunomodulatory therapies (4). However, multiple clinical trials of these agents have shown limited benefits (12, 13). Thus, effective, convenient, and inexpensive therapeutic strategies are required to treat SARS-CoV-2 infections.

Natural food products are excellent candidates for use in drug discovery. Medicinal plant extracts have been used to treat various diseases, including viral infection (14–16). Garlic has been consumed by humans for a thousand years as a functional food and a traditional remedy for preventing and treating various diseases since ancient times (17–19). Garlic and its active organosulfur compounds alleviate several viral infections in preclinical and clinical investigations (16). The World Health Organization considers it an essential component of any balanced diet with immunomodulatory properties (20). Ajoene [(E)-(Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxiade], a major bioactive compound extracted from garlic (21), is synthetically produced by the decomposition of allicin (22, 23). Ajoene has several biological functions, including anti-thrombotic, antioxidant, anti-microbial, anti-viral, anti-cancer, and anti-inflammatory activities, regulating the immune response (24–26). Ajoene exhibits a broad spectrum of in vitro and in vivo activities against several DNA and RNA viruses (18, 27, 28), including inhibitory activity against human immunodeficiency virus 1 (HIV-1) by blocking HIV-1 virus-cell attachment (29). Sulforaphane (SFN), which is extracted from cruciferous vegetables, including cabbage, broccoli, and Brussels sprouts, induces the activation of nuclear factor-erythroid 2-related factor 2 (NRF2), with acknowledged anti-inflammatory and anti-oxidant properties (30). However, whether ajoene-enriched garlic extract (AGE) and SFN play therapeutic roles in SARS-CoV-2 infection remains unclear.

In this study, we established an effective and biologically safe mouse model that mimicked the pathological processes of ARDS and CRS during SARS-CoV-2 infection (31). We utilized a SARS-CoV-2 acute lung injury mouse model to investigate the role of two immunomodulatory agents, AGE and SFN, in anti-inflammatory and immune response modulation. We examined whether interventions with these two agents could mitigate the inflammatory response and restore the immune system in SARS-CoV-2 in vivo. Here, for the first time, we observed that AGE and SFN significantly decreased the level of lung injury, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated the number of lymphocytes, such as CD4⁺T and CD8⁺T cells, in a SARS-CoV-2 ARDS mouse model. AGE and SFN treatment had the same effect as an antagonist of IL-6R, the neutralizing antibody suggested for treating advanced COVID-19. These results provide a theoretical basis for applying AGE and SFN in the treatment of patients with COVID-19.

The SARS-CoV-2 pandemic has substantially impacted global health, leading to a cumulative burden on medical resources, society, and the economy. Although numerous drug discovery efforts have led to the authorization of anti-viral drugs and monoclonal antibodies by the Food and Drug Administration (FDA), inexpensive therapeutics that can be rapidly and conveniently translated into clinical use are urgently required. The immunopathogenesis of COVID-19 involves acute respiratory distress syndrome (ARDS) and organ failure (39). We established a SARS-CoV-2 acute lung injury mouse model to mimic the pathological characteristics of SARS-CoV-2 infection. The mouse model exhibited obvious pathological changes, including alveolar pneumonia, characterized by stronger inflammatory responses, including typical alveolar and peribronchiolar inflammation, increased infiltration of neutrophils in BALF and blood, and serious CRS in the lung tissue, resulting in tissue damage and acute lung injury. A decreased percentage of lymphocytes, such as CD3⁺, CD4⁺, and CD8⁺ T cells, was observed in this model.

The immune system plays a pivotal role in the control and immunopathogenesis of SARS-CoV-2 infection. Lymphopenia is commonly observed in the blood of patients with COVID-19, and the degree of lymphopenia correlates with disease severity (40). Our results are consistent with those of patients with COVID-19 (41). Furthermore, this model provides greater safety and convenience for studying the pathogenic mechanisms and pharmacodynamics of agents against COVID-19 (31).

Garlic has been a common herb consumed as a functional food for thousands of years worldwide. Various garlic extracts have been reported to exhibit therapeutic efficacy against various diseases. Ajoene is a synthetic product of the decomposition of allicin. Growing evidence has demonstrated that allicin and ajoene have effective activities as anti-viral, anti-bacterial, anti-inflammatory, and anti-tumor agents (42, 43). Preclinical studies, both in vitro and in vivo, have shown that active organosulfur compounds derived from garlic, such as ajoene, allitridin, garlicin, and diallyl sulfide, possess potential anti-viral (28, 44–47), immune-enhancing, and other therapeutic activities (48–50). Ajoene shows a broad spectrum of in vitro and in vivo activities against zoopathogenic fungi, many yeast strains, zoopathogenic parasites, and several DNA and RNA viruses. Furthermore, in this study, we observed for the first time that AGE could restrain the inflammatory response and recover the immune system, leading to decreased levels of lung injury in SARS-CoV-2 acute lung injury mouse models. Garlic extract reduces neutrophil migration and blocks the biosynthesis of inflammatory cytokines, such as IL-6 and TNF-α (51–54), which supports our results.

The dual anti-viral and anti-inflammatory properties of ajoene against viral infections have been previously described. Ajoene prevents HIV-induced destruction of CD4⁺T cells and enhances cellular immunity in vitro. Additionally, it inhibits viral attachment to host cells and the reverse transcriptase of HIV-1 (29). Additionally, a non-organosulfur proteinaceous compound derived from garlic showed anti-viral activity against SARS-CoV by inhibiting early viral attachment and viral activity, which provides evidence for the potential anti-viral activity of ajoene in SARS-CoV-2 (45).

NF-κB activation is crucial for the inflammatory response to multiple viral infections, including COVID-19 (55). Ajoene and other garlic extracts directly inhibit NF-κB activation (56). A previous study showed that garlic extract exerts anti-inflammatory activity by inhibiting lipopolysaccharide (LPS)-induced toll-like receptor-4 dimerization, followed by the suppression of cyclooxygenase (COX)-2 (57). In addition, ajoene can target and covalently modify the signal transducer and activator of transcription 3 (STAT3) and COX-2, leading to an attenuated inflammatory response in macrophages (24). COX-2 catalyzes the synthesis of prostaglandins (PGs), which play a major role in mediating inflammatory responses. Coronaviruses, including SARS-CoV-2, enhance the expression of genes of COX-1, COX-2, and cytosolic prostaglandin E synthase (PTGES) (58). SARS-CoV-2 infection upregulates COX-2 expression in vivo in K18-hACE2 mice and in vitro in human cells (59). Similarly, COX-1, COX-2, and PTGES were upregulated in peripheral blood mononuclear cells isolated from patients with COVID-19 compared with healthy controls (60, 61). Therefore, these findings support the evidence that ajoene can treat SARS-CoV-2. However, further studies are required to investigate the cellular pathways involved in the antiviral and anti-inflammatory activities of ajoene against SARS-CoV-2.

In recent years, there is a growing evidence show that the gut microbiota, which plays a pivotal role in shaping both the innate and adaptive immune systems, may significantly contribute to the pathogenesis of COVID‐19 (62). The clinical researches found that a notable aspect of COVID‐19 is the prevalence of severe gastrointestinal symptoms reported by approximately 50.5% of affected individuals (63). Furthermore, clinical analyses have demonstrated that individuals with gastrointestinal involvement tend to experience a more severe disease course (64). A clear collection between SARS-CoV-2 infection and notable abnormalities in the gut microbiota, which marked as a decreased bacterial diversity, an increased opportunistic pathogens, a depletion of beneficial commensal organisms, and impaired biosynthesis of essential metabolites was established (65–70). In the humanized ACE2 knock‐in (hACE2‐KI) mouse model, a notable reduction in intra‐individual bacterial richness (α‐diversity) and distinct variations in intergroup microbiota composition (β‐diversity), was observed following SARS-CoV-2 infection (62). In our study, we found that α-diversity and β‐diversity were recovered with the AGE treatment. Garlic has shown the physiological function of balancing intestinal microbiota in other diseases. This is the first report about the effect of AGE on intestinal microbiota in the SARS-CoV-2 acute lung injury mouse model. However, the change of intestinal microbiota following the SARS-CoV-2 infection with the AGE treatment is still unknown and still needs further study.

SFN is an orally accessible dietary phytochemical observed in high amounts in cruciferous vegetables (71). SFN is commercially available at a low cost and has limited side effects (72, 73). SFN has several activities, such as anti-viral, anti-bacterial, anti-inflammatory, and anti-tumor. Ordonez et al. (74) reported that administration of SFN to K18-hACE2 mice before intranasal SARS-CoV2 infection significantly reduced the viral load in the respiratory tract, lung injury, and pulmonary pathology, suggesting that SFN is a potential agent for preventing COVID-19. However, whether SFN is therapeutic in the SARS-CoV-2 ARDS mouse model remains unclear. In this study, we observed that SFN restrained the inflammatory response and restored the immune system, leading to decreased levels of lung injury in a SARS-CoV-2 ARDS mouse model. SFN is an activator of NRF2, which is suppressed in the lung biopsies obtained from patients with COVID-19 (75). Under pathological conditions, reactive oxygen species (ROS) could damage the body through various pathways. When SARS‐CoV‐2 binds to ACE2, the angiotensin II receptor type 1 (AT1R) axis associated with oxidative stress is upregulated, leading to lung and endothelial damage. NRF2 can enhance the ability of cells to scavenge ROS and reduce oxidative damage. The therapeutic effects of NRF2 activation have been demonstrated in animal models of several lung disorders, including respiratory infections and ARDS (76). Sun et al. (30) reported that SFN exerts a significant anti-inflammatory effect on ARDS in rabbits by upregulating NRF2 expression, which is consistent with our results. NF-κB activation and its consequent release of cytokines contribute to the CRS in patients with COVID-19 (77). SFN inhibited NF-κB activation, endowing the anti-inflammatory properties of SFN, which can be another mechanism that explains the beneficial effects of SFN in patients with COVID-19. In addition, NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation is downstream of SARS-CoV2 infection, which is important in inducing inflammatory responses in patients with COVID-19. NLRP3 is inhibited by SFN, independent of the NRF2 pathway. Gasparello et al. (78) reported that downstream cytokines of NLRP3 inflammasome activation, such as IL-6 and IL-8, which are involved in the cytokine storm, were inhibited by SFN. Therefore, targeting the NLPR3 inflammasome with SFN in bronchial cells had beneficial effects. These results are consistent with those of this study.

This study expanded the indications for the clinical application of AGE and SFN and demonstrated that AGE and SFN can inhibit the inflammatory response and recover the immune response in a SARS-CoV-2 acute lung injury model. Given that AGE and SFN are naturally extracted, orally bioavailable, commercially available, and have limited side effects, our results demonstrate that these two immunomodulatory agents can be promising alternative approaches for treating patients with COVID-19 and ARDS.

The data presented in the study are deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences that are publicly accessible at https://ngdc.cncb.ac.cn/gsa. The accession number CRA016284.

The animal study was approved by Ethics Review Commission of the Laboratory Animal Center of Zhengzhou University. The study was conducted in accordance with the local legislation and institutional requirements.

SL: Writing – original draft, Writing – review & editing. BW: Writing – original draft, Writing – review & editing. TC: Methodology, Writing – review & editing. HW: Writing – review & editing, Methodology. JL: Writing – review & editing, Methodology. XZ: Writing – original draft, Writing – review & editing. YZ: Writing – original draft, Writing – review & editing.