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Acute lung injury induced by recombinant SARS-CoV-2 spike protein subunit S1 in mice

Respiratory Research volume 26, Article number: 59 (2025) Cite this article

Abstract

Background

The intricacies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing acute lung injury (ALI) and modulating inflammatory factor dynamics in vivo remain poorly elucidated. The present study endeavors to explore the impact of the recombinant SARS-CoV-2 spike protein S1 subunit (S1SP) on ALI and inflammatory factor profiles in mice, aiming to uncover potential therapeutic targets and intervention strategies for the prevention and management of Coronavirus Disease 2019 (COVID-19).

Methods

To mimic COVID-19 infection, K18-hACE2 transgenic mice were intratracheally instilled with S1SP, while C57BL/6 mice were administered LPS to form a positive control group. This setup facilitated the examination of lung injury severity, inflammatory factor levels, and alterations in signaling pathways in mice mimicking COVID-19 infection. Histopathological assessment through HE staining, along with analysis of lung wet/dry ratio and ultrasound imaging, revealed severe lung injury.

Results

After molding, K18-hACE2 mice exhibited a pronounced reduction in body weight and showed more significant lung injury (P < 0.05). Notably, there was a significant elevation in vascular permeability, total protein, and total white blood cells in bronchoalveolar lavage fluid (BALF) (P < 0.05), indicative of tissue damage. Additionally, the tight junction of lung tissue was compromised (P < 0.05), accompanied by intense oxidative stress marked by decreased SOD activity and elevated MDA content (P < 0.05). Cytokine levels, including IL-6, IL-1β, TNF-α, and MIG, were significantly upregulated in both BALF and serum of S1SP + K18 mice (P < 0.05). Furthermore, S1SP prominently augmented the expression of p-p65/P65 and attenuated IκBα expression in the NF-κB signaling pathway of humanized mice (P < 0.05), corroborating a heightened inflammatory response at the tissue level (P < 0.05).

Conclusion

The administration of S1SP to K18-hACE2 mice resulted in severe lung injury, enhanced vascular permeability, and compromised epithelial barrier function in vivo. This was accompanied by disruption of lung tight junctions, the manifestation of severe oxidative stress and a cytokine storm, as well as the activation of the NF-κB signaling pathway, highlighting key pathological processes underlying COVID-19-induced lung injury.

Background

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It is still a serious threat to human health and can lead to respiratory failure in severe cases [1,2,3]. Current studies have shown that acute lung injury caused by SARS-CoV-2 infection in humans or animals may be fatal. SARS-CoV-2 binds to angiotensin-converting enzyme 2(ACE2) receptors on the surface of target organs or target cells through its Spike protein (SP) [4]. After entering the host, a large number of viruses replicate in the host body, leading to cell death [5]. A large number of dead cells activate inflammatory cells in the body through a positive feedback mechanism, secrete a large number of cytokines, and form cytokine storm [6, 7]. Excessive cytokines have obvious toxicity to respiratory epithelial cells and vascular endothelial cells, and seriously affect lung gas exchange and ventilation function [8].

Khan found that SARS-CoV-2 S protein induced the production of a large number of cytokines in vitro [9]. This suggested that S protein alone, despite having no activity, causes cell damage and inflammation. S1SP can damage peritoneal macrophages and human bronchial epithelial cells in vitro [10,11,12]. Colunga study found that S1SP can lead to endothelial barrier dysfunction [13]. Relevant studies found that alcohol-fed mice were more likely to have acute lung injury under the intervention of S1SP [14]. Zhang found that SARS-CoV-2 spike RBD protein combined with LPS aggravated ALI in mice [15]. Whether S1SP can cause lung injury and cytokine storm in vivo is still poorly elucidated. Therefore, in this study, we intratracheally instilled the S1SP in K18-hACE2 mice that overexpress human ACE2 to mimic COVID-19, and examined signs of acute lung injury 72 h later, which could provide potential therapeutic targets and intervention strategies for the prevention and management of COVID-19.

Materials and methods

Materials

Rabbit monoclonal antibodies ZO-1 (ab276131) and Occludin (ab216327) were acquired from Abcam Biotechnology Ltd. (United Kingdom). Rabbit polyclonal antibodies IL-6 (DF6087), IL-1β (AF5103), TNF-α (AF7014), Phospho-NF-kB p65 (AF2006), and claudin 5 (AF5216) were purchased from Affinity Biosciences Ltd. (USA). Goat Anti-Rabbit IgG H&L, BF488 conjugated (bs-0295G-BF488), and Goat Anti-Rabbit IgG H&L, BF594 conjugated (bs-0295G-BF594) were purchased from Bioss Co. Ltd. (USA). Polyvinylidenefluoride (PVDF) (ISEQ00010) was purchased from Merck Millipore Ltd. (USA). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit (P0012A), EDTA Antigen Retrieval Solution (P0085), BCA protein assay kit (P0010S), and protein loading buffer were acquired from Beyotime Biotechnology Ltd. (China). Protease Inhibitor Cocktail (HY-K0010), and Phosphatase Inhibitor Cocktail III (HY-K0023) were purchased from MedChemexpress Ltd. (USA).

Animals

K18-hACE2 mice (22–26 g; 8–10 weeks old) on a C57BL/6 background were purchased from Cyagen Biosciences Inc. (Jiangsu, China) and male C57BL/6 mice (WT) (22–26 g; 8–10 weeks old) from Jinan Pengyue Laboratory Animal Breeding Co., ltd. (Shangdong, China). The mice were kept in a controlled environment with a temperature range of 24 ± 2 ℃ and a humidity level of 50 ± 10%. Food and water were provided in unlimited quantities. All animal experiments and feeding methods complied with the guidelines for the Care and Use of Laboratory Animals established by the US National Institutes of Health and approved by the Binzhou Medical University Hospital Institutional Review Board (No. 20220128-76).

Experimental mice were randomly divided into four groups: wild type control group (PBS + WT), LPS positive control group (LPS + WT), S1SP negative control group (S1SP + WT), and S1SP interferes with K18-hACE2 mice group (S1SP + K18). S1SP + WT and S1SP + K18 were established by exposing tracheal instillation of S1SP (400 μg/kg, 2 mL/kg) [10, 14, 16]. LPS (5 mg/kg, 2 mL/kg) was instilled into trachea to establish LPS positive control group [17]. A wild-type control group was established by intratracheal instillation of 2 mL/kg PBS (Fig. 1A). Mice in each group were anesthetized by intraperitoneal injection of avertin (tribromoethanol and tert-amyl alcohol) 72 h after modeling, and specimens were collected for various biochemical or histological tests.

Fig. 1
figure 1

S1SP stimulation aggravated the degree of ALI in mice. A The mouse model was established by exposure tracheal instillation. B Body weight of mice was monitored at 0 h, 24 h, 48 h, and 72 h after modeling to evaluate the change in body weight n = 6. C, D 72 h after intratracheally instilled LPS and S1SP, lung tissues were evaluated histologically (magnification 200 × , scale bar: 100 µm n = 3). E lung W/D ratio n = 6. F The degree of lung injury in mice was qualitatively analyzed by ultrasound. G Quantitative analysis of Evans blue content in lung tissue was performed by tail vein injection of Evans blue n = 6. H, I Bronchoalveolar lavage fluid (BALF) was collected, and the total cell number and total protein concentration in BALF were determined n = 6. All data are presented as the mean SD of three independent experiments. * p < 0.05, ** p < 0.01

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Weight changes

The mice were weighed every 24 h, and the changes in body weight were recorded at 0 h, 24 h, 48 h, and 72 h after modeling.

Histopathology and lung injury score

Mouse lung tissues were collected and fixed with 4% paraformaldehyde for 48 h. The tissues were processed, embedded in paraffin, sectioned into 4 µm-thick slices, stained with hematoxylin and eosin (H&E) (Solarbio, China), and visualized under an optical microscope (Olympus Optical, Japan) for histological analysis. For each sample, six randomly selected high-magnification fields of view were scored for characteristics, and the mean of the scores obtained for each field of view represented the pathology score for each sample on the lung injury scoring scale [18].

Wet/dry (W/D) lung weight ratio

Lung tissue was removed from anaesthetized mice, the surface of the lung tissue was blotted dry, the wet weight was weighed and placed in an oven at 60 °C. After baking for at least 48 h, the weight of the lung tissue was measured to a constant weight, at which point the weight was weighed as the dry weight, and the degree of lung oedema was assessed by the W/D ratio.

Lung ultrasound

After the experimental mice were anaesthetized by mouse intraperitoneal injection, an Aplio i900 ultrasound machine (Canon, Japan) was used to probe the chest of the mice, and an i24LX8 line-array probe was used, with a probe frequency of 24 MHz, and the probe was fine-tuned forward and backward until a clear image of the heart and lung tissue was obtained, and the distribution of A- and B-lines in the lung tissue was observed.

Alveolar permeability assay

Mice were injected with Evans blue (20 mg/kg, 5 mg/mL) in the tail vein and anaesthetized after 1 h. 0.9% saline was used to flush the circulating blood in the lungs adequately, and the lung tissues were homogenized in 1 mL of formamide solution, and then centrifuged for 24 h in a water bath at 60 °C. Supernatants were taken by centrifugation, and then put in microtiter plates, and the absorbance was measured at 620 nm, and the absorbance was measured at 620 nm.

Bronchoalveolar lavage fluid (BALF) analysis

After the mice were anesthetized, the trachea was exposed, and the lungs were rinsed three times with sterile precooled PBS, 0.5 ml each. Aliquots of BALF were collected for determination of cell counts and protein concentrations. BALF was collected and immediately centrifuged, and the supernatant was used to determine protein concentration by the bisquinolinic acid assay. The cell pellet was resuspended, and the total cell count was determined by an automated cell counter (Countstar, China). Cells in BALF were stained with Wright-Giemsa staining, and 100 nucleated white blood cells were sorted and counted.

Oxidation and antioxidant indicators

Oxidative stress indicators SOD, MDA, XOD in serum and lungs were determined using kits following the manufacturer's instructions. SOD (BC0175), MDA (BC0025) and XOD (BC1095) was obtained from (Solarbio, China).

Western blot analysis

Total protein was extracted from cells and lung tissues using RIPA buffer (Solarbio, China) plus protease inhibitor cocktail (MCE, USA). Protein concentration was determined using the enhanced BCA assay kit (Boster, China). The concentration was calculated and 20 µg of sample was added to each well. Samples were then separated on 8% to 12% SDS-PAGE gels. Subsequently, proteins within the gel were transferred to a PVDF membrane. PVDF membranes were blocked with 5% skim milk and incubated for 2 h at room temperature. Membranes were placed in primary antibodies, including anti-p65 (1:1000), p-P65 (1:1000), IκBα (1:1000), IL-6 (1:2000), IL-1β (1:1000), TNF-α (1:1000), and GAPDH (1:2000) overnight at 4 °C. Finally, the membranes were incubated with horseradish peroxidase conjugated secondary antibody (1:500) for 1 h at room temperature. Protein bands were visualized using an electrochemiluminescence kit. Densitometric analysis was performed using ImageJ software.

Immunofluorescence

Lung tissues were cut into 4 μm thick slices, antigenically repaired with EDTA (Solarbio, China), closed with goat serum and treated with ZO-1 (1:200), Occludin (1:200), Claudin1 (1:200), IL-6 (1:200), IL-1β (1:200), and TNF-α (1:200) antibodies were blocked for 6 h. Subsequently, drops of fluorescent secondary antibody (1:200) were added and incubated in the dark for 1 h. Then, nuclei were treated with DAPI for 8 min. Finally, images were obtained by using a fluorescence microscope (Olympus BX53, Japan).

ELISA

Cytokines levels in the serum and BALF were determined with an enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions. IL-6 (EK0499), IL-1β (EK0502), TNF-α (EK0497), IL-10 (EK0503), IL-17 (EK4288), and IFN-γ (EK0496) were purchased from Signalway Antibody (USA). IP10 (1212153) was obtained from Thermo Fisher (USA). MIG (EK0733) and MCP-1 (EK0568) were obtained from BOSTER (China).

Statistical analysis

All data are presented as mean ± standard deviation. Independent sample t test was used to analyze continuous variables in independent sample data, and one-way analysis of variance was used to analyze multiple groups. A P < 0.05 was considered statistically significant. All data were statistically analyzed using IBM SPSS 26.0, Image analysis using Image J, and graphing using GraphPad Prism 9.

Results

Body weight changes and lung injury in mice under S1SP intervention

The body weights of mice in each group were weighed at 0 h, 24 h, 48 h, and 72 h, respectively. Different from the control group, the S1SP + K18 mice showed continuous weight loss (Fig. 1B). Increased pulmonary vascular permeability is the basic pathological feature of ALI. To investigate the effect of S1SP on lung injury and vascular permeability in K18-hACE2 mice, HE staining of lung tissue showed significant lung injury in the S1SP + K18 group, with severe damage to alveolar morphological structure, increased inflammatory cell infiltration, significantly thickened septum (Fig. 1C, D), and significantly increased lung W/D ratio (Fig. 1E). The lung ultrasound results showed that the A line was also visible around the heart in the S1SP + K18 group, and the B line was visible behind the heart with a wide width and a strong echo (Fig. 1F). The results of Evans blue detection of lung permeability showed that the content of Evans blue in the S1SP + K18 group was significantly higher than that in the other control groups (Fig. 1G). The total protein (Fig. 1H) and total white blood cells in BALF of the S1SP + K18 group were significantly higher than those of the control group (Fig. 1I). Further differential counting of white blood cells showed that the proportion of neutrophils in BALF of S1SP + K18 group mice did not increase significantly, but the proportion of lymphocytes decreased significantly, and the neutrophil-to-lymphocyte ratio (NLR) decreased significantly (Fig. 2A‒C). The results suggested that the S1SP + K18 mice had obvious lung injury, increased alveolar permeability and significantly reduced lymphocytes.

Fig. 2
figure 2

Changes in white blood cells, tight junction proteins and oxidative stress levels after S1SP intervention. A–C Changes of neutrophils, lymphocytes and NLR in BALF of mice n = 6. D–I Immunofluorescence was used to detect the levels of ZO-1, Occludin, and Claudin1 in each group (magnification 200 × , scale bar: 100 µm), and the quantitative of ZO-1, Occludin, and Claudin1 n = 3. The oxidative stress status in lung tissue and serum of mice was detected. J–O The activity of SOD, the content of MDA and the activity of XOD in lung tissue and serum were detected n = 6. *P < 0.05, **P < 0.01

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Effect of S1SP intervention on alveolar epithelial tight junction proteins ZO-1, occludin and claudin5

The results of immunofluorescence assay for alveolar epithelial tight junction proteins ZO-1, Occludin and Claudin1 showed that the fluorescence intensity of ZO-1, Occludin and Claudin1 in the S1SP + K18 group was significantly lower than that of the control group (Fig. 2D‒I), which indicated that the expression of alveolar epithelial tight junction proteins was significantly decreased under the intervention of S1SP, and the endothelial barrier was severely damaged.

Effect of S1SP intervention on oxidative antioxidant status in mice

The results of antioxidant SOD activity showed that SOD activity in lung tissue and serum was significantly lower in the S1SP + K18 group, and MDA in lung tissue and serum was significantly higher in the S1SP + K18 group than in the rest of the groups, and XOD content in lung tissue and serum was higher in the S1SP + K18 group (Fig. 2J‒O). The results suggested that the S1SP + K18 mice showed significant oxidative stress and lipid oxidation, and the antioxidant capacity was significantly reduced.

S1SP stimulation triggers cytokine changes in BALF and serum

It was found that the levels of cytokines IL-6, IL-10, IL-1β, IL-17, TNF-α, IFN-γ, and chemokines IP10, MIG, and MCP-1 within the BALF of the S1SP + K18 group mice intervened by S1SP were significantly higher than those in the rest of the control group (Fig. 3A‒I). The levels of cytokines IL-6, IL-10, IL-1β, IL-17, TNF-α, IFN-γ and chemokines IP10, MIG, MCP-1 within their serum were also significantly higher (Fig. 4A‒I). The results indicated that inflammatory factors and chemokines were significantly elevated in the BALF and serum of the mice in the S1SP + K18 group, suggesting that a severe cytokine storm existed in the S1SP + K18 mice.

Fig. 3
figure 3

Changes in inflammatory factors in BALF after S1SP intervention. A–I The levels of inflammatory cytokines IL-6, IL-10, IL-1β, IL-17, TNF-α and IFN-γ and chemokines IP10, MIG and MCP-1 in BALF were detected n = 6. *P < 0.05, **P < 0.01

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Fig. 4
figure 4

Changes of inflammatory factors in serum following S1SP intervention. A–I The serum levels of inflammatory cytokines IL-6, IL-10, IL-1β, IL-17, TNF-α and IFN-γ and chemokines IP10, MIG and MCP-1 were detected n = 6. *P < 0.05, **P < 0.01

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Effects of S1SP intervention on IL-6, IL-1β and TNF-α in lung tissues

To further detect the expression of inflammatory factors, we used Western blotting analysis to detect the expression of IL-6, IL-1β and TNF-α in each group. We observed that the expression of IL-6, IL-1β and TNF-α in the S1SP + K18 group was significantly increased (Fig. 5A‒D). The results of immunofluorescence were consistent with those of Western blotting (Fig. 5E‒J). In summary, the data showed that the expression of inflammatory factors IL-6, IL-1β and TNF-α was significantly increased in the S1SP + K18 mice.

Fig.5
figure 5

S1SP intervention resulted in increased levels of inflammatory factors in lung tissue. A Western blotting analysis of the expression levels of IL-6, IL-1β and TNF-α in the lung tissue of mice in each group at 72 h after modeling n = 3. B–D GAPDH was used as an internal control. Quantitative analysis of IL-6, IL-1β and TNF-α n = 3. E–G The results of immunofluorescence were basically consistent with those of Western blot (magnification 200 × , scale bar: 100 µm n = 3). H–I Quantitative analysis of IL-6, IL-1β and TNF-α n = 3. All data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

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S1SP intervention activated the NF-κB signaling pathway in the lung of mice

Under the stimulation of S1SP, the ratio of phosphorylated P65 to total P65 (p-P65/P65) was increased, the level of IκBα was decreased (Fig. 6A‒C), and the classical NF-κB signaling pathway was activated in the S1SP + K18 mice. The results showed that the activation of NF-κB signaling pathway was closely related to the increased expression of inflammatory factors in the S1SP + K18 mice.

Fig. 6
figure 6

Effect of S1SP intervention on NF-κB signaling pathway. A Western blotting analysis of the protein expression levels of p-P65, P65 and IκBα in lung tissue of each group at 72 h after modeling n = 3. GAPDH was used as an internal control. B, C Quantitative analysis of p-P65/P65 and IκBα n = 3. All data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

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Discussion

Acute lung injury and respiratory distress syndrome are important causes of high mortality in clinical COVID-19 patients, which are mainly closely related to pulmonary ventilation and gas exchange dysfunction, oxidative stress disorder and severe cytokine storm [19]. Relevant studies have found that the levels of inflammatory factors and chemokines such as IL-6 and MCP-1 in human bronchial epithelial cells increased under the intervention of S1SP [12].

In this study, a potential mouse model of SARS-CoV-2 infection was established by exposed tracheal instillation of S1SP. In fact, this mouse model recapitulates certain aspects of lung injury observed in COVID-19, but does not fully replicate the complex immunopathogenic features of SARS-CoV-2 infection. Seventy-two hours after modeling, the mice with SARS-CoV-2 infection showed continuous weight loss. HE staining and W/D ratio measurement of the lung tissue of mice showed that the S1SP + K18 mice had significant lung injury, with severe damage to the alveolar morphology and structure, increased inflammatory cell infiltration, and obvious thickening of the septum. These results suggest that S1SP protein stimulation can cause severe lung injury in mice, which is basically consistent with the clinical symptoms of patients [20]. Peixoto found that lung ultrasound (LUS) is an effective and safe method to detect lung injury in severe COVID-19 patients [21]. After 72 h of modeling, we found that the lungs of the humanized mice treated with S1SP showed wide and strong echo B-lines, suggesting that the mice had lung injury and pulmonary edema. In this study, it was found that the pulmonary vascular permeability of the S1SP + K18 mice was increased, the total protein and total white blood cells in BALF were significantly increased, and the number of lymphocytes and NLR were significantly decreased. It was demonstrated that the binding ability of S1SP to hACE2 receptor could alter the pulmonary vascular permeability, cause obvious damage to epithelial cells and decrease the immune function even if the virus replication ability was removed. To provide new insights for the further study of coronavirus spike protein. Epithelial cell damage is the pathological basis of severe COVID-19 patients. Previous studies have found that S protein RBD can cause endothelial and epithelial cell damage [22]. We investigated whether S1SP stimulation affected the tight junction of endothelial cell barrier in vivo, which is composed of Occludin, Claudins and ZO-1. We found that the protein expression of ZO-1, Occludin and Claudin1 was significantly decreased in the S1SP + K18 mice. Previous studies have found that the gene expression of Claudin1 is significantly reduced in patients with COVID-19 infection [23]. In vivo studies in patients with COVID-19 have found that SARS-CoV-2 replication leads to transient decline in epithelial barrier function and disruption of tight junctions [24]. This suggests that both S1SP and SARS-CoV-2 envelope proteins can lead to the destruction of tight junctions, which helps to explain how the virus causes extensive lung damage and extrapulmonary organ damage in critically ill patients.

Severe patients may progress to septic shock after novel coronavirus infection [25], and severe multiple organ dysfunction is also the main manifestation [26]. Uncontrolled inflammatory response and oxidative stress are the core factors [27]. The results of this study showed that mice showed obvious oxidative stress and lipid oxidation, decreased SOD activity, increased MDA content, and significantly decreased antioxidant capacity. These results indicated that S1SP intervention could lead to oxidative stress and increased lipid oxidation levels in mice. XOD activity in serum and alveolar lavage fluid was increased in S1SP + K18 group mice, and xanthine oxidase was involved in the catalytic production of uric acid and superoxide anion free radicals [28]. A large amount of superoxide accumulation can further cause tissue damage in the organism, which is closely related to multiple organ system dysfunction after COVID-19 infection.

The main reason for the higher fatality rate of COVID-19 than common coronavirus infection is the cytokine storm caused by the infection. The multiple organ failure caused by the cytokine storm maybe fatal. Clinical studies have found that severe cytokine storm can be observed in critically ill patients [29]. A large number of cytokines produce exogenous stimulation and autoimmunity in the body [30], and the body secrets cytokines to form a positive feedback mechanism [31], which leads to an abnormal increase in the level of inflammatory factors in the body and the formation of cytokine storm. Eventually, it leads to multiple organ damage and functional failure and death. In this study, we constructed a model of S1SP intervention in K18-hACE2 mice to investigate whether S1SP causes cytokine increase or cytokine storm. We found that both the anti-inflammatory cytokines IL-6 and IL-10, the pro-inflammatory cytokines IL-1β, IL-17, TNF-α, IFN-γ in the bronchoalveolar lavage fluid and serum of the S1SP + K18 mice, and the proinflammatory cytokines Il-1β, IL-17, TNF-α, IFN-γ in the BALF and serum of the S1SP + K18 mice. The levels of chemokines IP10, MIG and MCP-1 were significantly increased, and Western blotting and immunofluorescence experiments also confirmed the infiltration of inflammatory factors in vivo. This is basically consistent with the results of relevant studies on clinical COVID-19 patients [32]. Similarly, in related animal experiments, the levels of TNF-α, IFN-γ and other cytokines and chemokines in SARS-CoV-2 infected mice increased [33]. Relevant studies have found that it produces a large number of cytokines such as IL-6, IL-1β and MIG in vitro and in vivo experiments [9]. Our study further confirmed that S1SP intervention can also induce cytokine storm in mice, which provides new evidence for in vivo experimental study.

NF-κB signaling pathway is a classical inflammatory signaling pathway, which plays a key role in the regulation of gene expression induced by exogenous stimuli and cytokines. NF-κB signaling pathway is the main pathway that drives the inflammatory response of COVID-19 patients. This study explored the effect of S1SP intervention on NF-κB signaling pathway in mice. Previous studies have shown that SARS-CoV-2 capsid protein can promote the molecular mechanism of excessive activation of host NF-κB signaling pathway and inflammatory response [34]. The present study found that S1SP intervention caused an increase in p-P65/P65 and a decrease in IκBα protein level in K18-hACE2 mice, indicating that S1SP could also activate the NF-κB signaling pathway, which was basically consistent with the above hypothesis. The results of this study provide new in vivo experimental evidence for the study of S1SP. Activation of the NF-κB signaling pathway induces the secretion of a large number of inflammatory factors and participates in the generation of cytokine storm.

Based on this study, it has been established that S1SP intervention can induce an inflammatory factor storm in humanized mice to some extent and activate the NF-κB signaling pathway. Consequently, in clinical settings, the pathophysiological changes in critically ill patients can be mitigated by modulating oxidative stress levels. Targeting this pathway presents a potential therapeutic strategy; by using inflammatory factor receptor antagonists, it is possible to disrupt the positive feedback loop of inflammatory factors and their signaling pathways, thereby reducing patient morbidity. This approach holds significant clinical application value.

This study has certain limitations. First of all, this is a mouse animal study, which cannot fully mimic the pathophysiological changes of COVID-19 patients and does not fully replicate the complex immunopathogenic features of SARS-CoV-2 infection. Secondly, the PBS + K18-hACE2 group was not set up in this study, and the differences in lung pathology between PBS + K18-hACE2 group and wild-type mice were not analyzed. Finally, this study was mainly a preliminary study of lung injury induced by S1SP in mice, and the pathogenic mechanism was not explored in depth.

In summary, we have shown that administering a single component of SARS-CoV-2, namely S1SP, via intratracheal instillation in K18-hACE2 transgenic mice elicits inflammatory responses that mimic both local (lung) and systemic manifestations of COVID-19. This animal model provides a platform for preclinical studies to explore potential countermeasures and to investigate the long-term consequences of the inflammatory response triggered by SARS-CoV-2.

Availability of data and materials

Data and materials may be made available upon written request to the corresponding author.

Abbreviations

COVID-19:

Coronavirus Disease 2019

SARS-CoV-2:

Severe acute respiratory syndrome coronavirus 2

ACE2:

Angiotensin-converting enzyme 2

SP:

Spike protein

S1SP:

Spike protein S1 subunit

PVDF:

Polyvinylidenefluoride

SDS-PAGE:

Sulphate–polyacrylamide gel electrophoresis

BALF:

Bronchoalveolar lavage fluid

ELISA:

Enzyme-linked immunosorbent assay

NLR:

Neutrophil-to-lymphocyte ratio

LUS:

Lung ultrasound

ZO-1:

Zona Occludens 1

SOD:

Superoxide dismutase

MDA:

Malondialdehyde

XOD:

Xanthine oxidase

LPS:

Lipopolysaccharide

PBS:

Phosphate buffer saline

NF-κB:

Nuclear factor kappa-B

hACE2:

Human angiotensin-converting enzyme 2

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Acknowledgements

Sincerely thank Professor Xiao Huang for her help in the study.

Funding

This study was supported by National Natural Science Foundation of China (CN, 82370092), the Natural Science Foundation of Shandong Province (ZR2021MH360), Science and Technology Innovation Project of Binzhou Social Development (CN, 2023SHFZ033), the “Clinical + X” Scientific Innovation Project of Binzhou Medical University (CN, BY2021LCX22), Research Project on COVID-19 Prevention and Control, Binzhou Medical University (CN, BY2021XGFY05).

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Author notes

  1. Jiwei Zhu, Jinglin Wu and Manlu Lu have authors contributed equally to this work.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, 661 Yellow River Road, Binzhou, 256603, China

    Jiwei Zhu, Jinglin Wu, Manlu Lu, Qianqian Jiao, Xiaojing Liu, Lu Liu, Mingzhen Li, Bin Zhang, Yan Yu & Lei Pan

  2. Department of Ultrasound Medicine, Binzhou Medical University Hospital, 661 Yellow River Road, Binzhou, 256603, China

    Junhong Yan

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  1. Jiwei Zhu
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Contributions

P.L. conceived this project. J.Z., J.W. and M.L. performed the major animal experiments. J.Z. contributed to the design of animal experiments and animal operations. J.Z., Q.J. and X.L. contributed to the western blot and qPCR analysis and corresponding analysis of data. M.L., L.L. and M.L. contributed to the breeding of animal, animal treatment and lung tissue collection. B.Z., Y.Y. and P.L. contributed to the overseen and supervision of the whole project. B.Z., Y.Y. and P.L. Provided the fund for this study. J.Z, J.Y, and P.L. wrote and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Junhong Yan, Yan Yu or Lei Pan.

Ethics declarations

Ethics approval and consent to participate

All animal experiments were formally approved by the Institutional Review Board of Binzhou Medical University Hospital under acceptance number 20220128–76. This study was performed in accordance with the Declaration of Helsinki and approved by research ethics board of Binzhou Medical University Hospital.

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The authors declare no competing interests.

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Zhu, J., Wu, J., Lu, M. et al. Acute lung injury induced by recombinant SARS-CoV-2 spike protein subunit S1 in mice. Respir Res 26, 59 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-025-03143-7

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Keywords

Respiratory Research

ISSN: 1465-993X

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