Mesenchymal stem cell treatment alleviates seawater drowning induced lung injury by inhibiting the TNFα/Snail/EMT pathway

Introduction

With the development of the marine industry, the incidence of seawater drowning (SWD) continues to increase every year (1,2). The World Health Organization estimated that drowning leads to 372,000 deaths each year, and ranked drowning as the third leading cause of “unintentional injury deaths” worldwide (3-5). SWD is more severe than freshwater drowning, due to its low temperature, hypertonicity, and rich bacterial content (6). Common complications of SWD include acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (3). The pathogenic mechanisms of SWD-ALI are complex and may involve the inflammatory response, pulmonary edema, decreased alveolar surface-active substances, oxidative stress, apoptosis, and autophagy (7-11). The treatment of SWD-ALI is mainly symptomatic, and includes cardiopulmonary resuscitation, mechanical ventilation, and prophylactic antibiotics (12-14). An absence of previous report or study providing evidence of effective treatments for drowning-related lung injury is a concern (13). Therefore, there is an urgent need for study at the cellular and animal levels to minimize the lethality and disability of SWD-ALI (14).

Tumor necrosis factor (TNF), which is also known as TNFα, is a cellular signaling protein involved in systemic inflammation, is one of the cytokines involved in the acute-phase response, and plays a central role in orchestrating the inflammatory response in mammals (15). In addition, TNFα can disrupt epithelial barrier integrity (16). At the same time, epithelial cells may undergo epithelial-mesenchymal transition (EMT) following injury, further damaging the air-blood barrier and causing lung injury (17). Snail, or SNAI 1, a zinc-finger transcription factor, can bind to E-cadherin to promote EMT (18), and can be stabilized by TNFα, further promoting EMT (19).

Mesenchymal stem cells (MSCs) have antibacterial, anti-inflammatory, and anti-fibrotic properties that promote injury repair, maintain the integrity of the vascular endothelium and barrier epithelium, and have been widely used because of their powerful functions (20-22). MSCs can significantly reduce the level of inflammation in ALI or ARDS (23-27), and they have been shown to be safe and significantly attenuate lung injury in several clinical studies of ARDS (27-30).

In this study, we successfully established mice model of SWD-ALI. Intratracheal administration of MSCs alleviates SWD-ALI by inhibiting the TNFα/Snail/EMT pathway. Further, we found that this treatment managed to effectively limit the progression of pulmonary fibrosis. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1471/rc).

Methods

Artificial seawater was prepared according to the main components of the East China Sea seawater provided by the Oceanic Administration of China (31).

The main reagents and antibody information are listed in Tables S1-S6.

Cell culture

Human umbilical cord MSCs were provided by the Stem Cell and Regenerative Medicine Lab (Beijing, China). The trilineage differentiation potential of the MSCs was confirmed. The expression (of CD29, CD105, CD73, and CD90) and the deletion (of CD45, CD34, CD79a, and HLA-DR) of surface antigens were detected using flow cytometry and analyzed using FlowJo X 10.6.2 software (BD, Franklin Lake, USA).

Human pulmonary alveolar epithelial cells (HPAEpiCs) were provided by the Dalian Institute of Physical Chemistry (Dalian, China) and were cultured in 1640 medium containing 10% fetal bovine serum. After a pre-experimental study, the cells exposed to 30% seawater for 4 h were used as the SWD-ALI model group, and six-generation endogenous MSCs medium was applied to the treatment group.

SWD-ALI model and treatment in mice

Healthy 6–8-week-old male C57BL/6 mice were obtained from the Chinese PLA General Hospital’s Experimental Animal Center (specific pathogen free) (n=87). The random number method was used for grouping. Based on previous studies (9,32-34) or experimental needs, we first completed and characterized the modeling. A small animal nebulizing syringe was then used to nebulize the MSCs via the trachea. All the experimental procedures were approved by the Experimental Animal Welfare Ethics Committee of Chinese PLA General Hospital (No. 2024-X20-69), and adhered to the Guide for the Care and Use of Laboratory Animals (35). A protocol was prepared before the study without registration.

Experimental grouping

SWD-ALI model validation

The control (CON) group comprised normal mice without special intervention. The mice in SWD model (SW) group were treated with 4 mL/kg artificial seawater by laryngoscopy endotracheal injection (LEI) method. Before the experiment, mice were anaesthetized with isoflurane inhalation.

MSCs treatment of SWD induced lung injury

The control (CON) group comprised normal mice who did not undergo any special intervention. After 30 min of SWD, the mice in the SWD + MSC (SW-MSC) group were re-sedated and subsequently injected with 25 µL (5×10⁵ cells) of MSCs by intratracheal inhalation with a nebulizer syringe. The mice in the SWD + phosphate buffer solution (PBS) (SW-PBS) group received an injection of with 25 µL of PBS.

After 6 h from SWD, the animals were euthanized by cervical dislocation and sample collected. If they died, the healthy mice were selected again by random number method (the mortality rate was about 1 in 10).

Micro-CT

The mice were anesthetized via the continuous delivery of isoflurane 2 h after SWD, and scanned using a Quantum FX micro-computed tomography (Micro-CT) imaging system (PerkinElmer, Waltham, USA). Each mouse was scanned for 4 min at 70 kV, 88 µA, and 36 mm field of view parameters. The data acquired from the scans were analyzed and subjected to three-dimensional (3D) reconstruction using Analyze 12.0 software (Analyze Direct, Overland Park, USA) at the same level settings.

Pulmonary function

Pulmonary function was examined 6, 12, 24, 48 h after SWD. The data were collected and analyzed using whole body plethysmography (WBP) produced by Data Sciences International (DSI, St. Paul, USA). Airflow changes (AC) curves were taken from the 2,000 consecutive data between the 5,000^th and 7,000^th consecutive data from the mice after stabilization, and the data were graphed and analyzed. The mean values of the measured data were used to calculate the minute ventilation (MV) and peak inspiratory flow rate (PIFb) changes in each group.

Wet/dry (W/D) weight ratio of lung samples

The samples were extracted after cervical dislocation, and the lung tissue samples were weighed immediately after being obtained to measure the wet (W) weight. The samples were then dried in an oven at 65 ℃, and the stable dry (D) weight was measured after 72 h of drying. The following formula was used to assess the level of lung edema: W/D × 100%.

Vascular permeability assessment

Evans blue was injected intravenously (30 mL/kg) through the tail vein 1 h before cervical dislocation. The samples were extracted after cervical dislocation and the removed lung tissue was placed into 1 mL of formamide at 37 ℃ for 24 hours. Evans blue was extracted by measuring the absorbance at 620 nm.

Histopathologic staining of lungs and scoring of lung injury

The samples were extracted after cervical dislocation, and the left lobe lung was taken for hematoxylin and eosin (HE) staining, and pictures were taken for lung injury scoring. Under the premise of blindness, Smith’s scoring method (36) was used to analyze the degree of lung injury.

Bronchoalveolar lavage fluid (BALF) collection

The mice were anesthetized to death with isoflurane, intubated via the cervical trachea. Bronchoalveolar lavage was performed with ice PBS. The recovered BALF was centrifuged at 4 ℃ for 10 min at 800 g.

Flow cytometry examination

BALF cells and lung tissue grindings were labeled with CD11b, F4/80, and Ly-6G antibodies for 30 min. Flow cytometry was conducted using a CytoFLEX (Beckman Coulter, Pasadena, USA) flow cytometer to detect macrophages (F4/80⁺ cells) and neutrophils (CD11b⁺ Ly-6G⁺ cells), and analyzed using FlowJo X 10.6.2 software. The antibodies used in the flow cytometry are listed in Table S1.

Enzyme-linked immunosorbent assay (ELISA)

An anti-mouse interleukin-1 beta (IL-1β) and TNFα ELISA Kit (Raybiotech, Guangzhou, China) were used to assess the concentration of inflammatory factors in the BALF of the SWD-ALI in mice.

Western blotting

Lung homogenates were collected, protein concentration was determined, and the proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. The antibodies used are listed in Tables S2,S3.

Immunofluorescence analysis

The mouse lung tissues were deparaffinized, dehydrated, closed, and incubated with primary antibody. The tissues were stained using the TSAPLus kit (Servicebio, Wuhan, China). 4,6-Diamidino-2-phenylindole (DAPI) was used to stain cell nuclei, and images were captured using a fluorescence microscope and scanner (Nikon Eclipse C1, 3DHISTECH Pannoramic MIDI, Tokyo, Japan). The antibodies used for the immunofluorescence analysis are listed in Tables S4,S5.

Quantitative real-time polymerase chain reaction (qPCR)

Lung RNA was extracted using TRIzol reagent (Tiangen, Beijing, China) and complementary DNA was synthesized using a reverse transcription kit (Thermo Fisher Scientific, Waltham, USA). QPCR analysis was performed using SYBR (Thermo Fisher Scientific), and the primer sequences are listed in Table S7.

Batch RNA-sequencing

Lung tissue was homogenized using liquid nitrogen milling, avoiding inhomogeneity of damage. RNA from different mouse lungs was extracted separately with TRIzol reagent for evaluation and analysis. Libraries were sequenced and differentially expressed genes (DEGs) were identified with DESeq2 (Bioconductor, Boston, USA) by Annoroad Gene Technology (Beijing, China). Different gene sets were analyzed by Venn diagrams, volcano diagrams, and heat maps, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses that conducted using a Microbiotics-Online Bioinformatics Analysis platform (bioinformatics.com.cn).

Statistical analysis

The data were presented as the mean ± standard error of mean. The between-group comparisons were assessed by the t-test and analysis of variance using SPSS 27 (IBM, Chicago, USA). Bonferroni’s test was used when the variance was homogeneous, and Tamhane’s test was used when the variance was not homogeneous. Correlations were analyzed by Pearson analysis. The significance levels were set as follows: P<0.05, P<0.01, and P<0.001.

Results

SWD-ALI mouse model was successfully established

Following the intratracheal injection of artificial seawater, pulmonary function tests (Figure 1A) showed significant decrease in the respiratory rate and tidal volume, and lung Micro-CT images (Figure 1B) revealed exudative changes (inside the red line). Further, the lung tissue appeared red and swollen at 6 hours post-modeling, and pathological examination revealed hemorrhage, edema, inflammatory cell infiltration, alveolar fracture, and atelectasis with elevated lung injury scores (Figure 1C,1D). Moreover, qPCR testing of the lung tissues revealed decrease in SP-B and SP-C, which maintain alveolar surface tension (Figure 1E), and increase in inflammatory factors IL-1β and IL-6 (Figure 1F). The protein concentration in BALF was also elevated (Figure 1G), and the difference between the CON and SW groups was statistically significant (P<0.01). Therefore, the mouse SWD-ALI model met the evaluation criteria for ALI models in experimental animals proposed by the official symposium of the American Thoracic Society (37), which indicated that the model had been successfully established and thus could be used for further study.

Figure 1 The mouse model of SWD-ALI was successfully established. (A) MV box plot and AC flow of pulmonary function in the two groups, n=6, ***, P<0.001. (B) CT and 3D reconstruction of mouse lung, the red line area indicates the damaged area. (C,D) Representative images of lung tissues and HE staining, the box in the middle figure was the larger area on the right (left scale bar: 2 mm, middle scale bar: 50 μm, right scale bar: 25 μm), and quantitative analysis of the lung injury score, n=6, ***, P<0.001. (E,F) The expression of SP-B, SP-C, IL-1β, and IL-6 mRNA in the mouse lung tissue of each group, n=6, *, P<0.05; **, P<0.01. (G) Total protein concentrations in BALF, n=6, **, P<0.01. CON, control group; SW, seawater drowning model group; MV, minute ventilation; 3D, three-dimensional; TV, tidal volume; AC, airflow changes; HE, hematoxylin and eosin; BALF, bronchoalveolar lavage fluid; SWD-ALI, seawater drowning induced acute lung injury; CT, computed tomography.

The SWD-ALI mechanism in mice was explored using transcriptome sequencing

The above experiments showed that SWD caused significant lung injury in mice. To understand the mechanism of SWD-ALI, we carried out a series of transcriptome sequencing experiments in the CON and SW mice (Figure 2). The two conditions were analyzed by principal component analysis (PCA), and the results revealed a significant difference in their expression profiles (Figure 2A). We then analyzed the genes that were up- or down-regulated in expression using a volcano plot (Figure 2B). This analysis revealed a significant increase in the transcription of the pro-inflammatory genes post-injury. The top 20 genes with the most significant changes in expression were clustered and analyzed as heat maps (Figure 2C). These genes were mainly involved in regulating inflammatory elements and chemokines.

Figure 2 The transcriptome sequencing results showed that the SWD-ALI group exhibited increased inflammation, epithelial damage, and deformation. (A) PCA of both groups was conducted (n=3). (B) Volcano plots showed the up- and down-regulated genes in SW group. (C) Heatmap showed the cluster analysis of the top 20 up- and down-regulated genes. (D) The top 10 KEGG enrichment analysis results of CON and SW groups. (E) GO term circle diagram analysis showed the top 10 in BP, CC, and MF. CON, control group; SW, seawater drowning model group; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function; SWD-ALI, seawater drowning induced acute lung injury; PCA, principal component analysis.

The KEGG pathway enrichment analysis of these genes (Figure 2D) revealed that the most enriched pathways were related to inflammation, including viral infection, and the TNFα, IL-17, NF-κB, and MAPK signaling pathways. Finally, we analyzed the GO terms associated with these genes using a circle plot (Figure 2E). The GO term circle plot analysis of the biological processes, cellular components, and molecular functions, showed that the inflammatory response was mainly concentrated in the cilia assembly, cell surface, and cell basement membrane, which directly led to epithelial cell damage and the alteration of the air-blood barrier, and was also associated with the developmental process of inhalation lung injury. In summary, the main pathology of SWD-ALI is the change in the epithelial cells and basement membrane caused by the inflammatory response in which the TNFα signaling pathway might play an important role.

The TNFα/Snail/EMT pathway mediates SWD-ALI development

The above omics analyses suggested that the TNFα signaling pathway was critical in SWD-ALI. For this study, we used ELISA to measure the TNFα content in BALF of the SWD-ALI mice, and found that the TNFα content was significantly elevated in the SWD-ALI group (Figure 3A).

Figure 3 The TNFα/Snail/EMT pathway was shown to act on SWD-ALI. (A) Detection of TNFα expression by ELISA, n=6, *, P<0.05. (B) The gene expression of Snail and Slug mRNA in the mouse lung tissue of each group, n=6, ns, P>0.05; **, P<0.01. (C) The Snail and Slug protein levels in the mouse lung tissue were detected by Western blotting. (D) Representative immunofluorescence images of lung sections from different groups were stained to detect E-cadherin (green), N-cadherin (red), and DAPI (blue); scale bars: 25 μm. (E) Bright field observation of the effects of seawater stimulation on the morphology of alveolar epithelial cells, scale bar: 50 μm. (F) The expression of TNFα, Snail, and Slug mRNA in the human alveolar epithelial cells of each group, n=9, ***, P<0.001. (G) Claudin-1, E-cadherin, N-cadherin, Snail, and Slug protein levels in the human alveolar epithelial cells were detected by Western blotting. (H) Pearson correlation analysis circle diagram of TNFα, Snail, and EMT pathways (positive correlation in red, negative correlation in green). TNFα, tumor necrosis factor alpha; BALF, bronchoalveolar lavage fluid; CON, control group; SW, seawater drowning model group; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4,6-diamidino-2-phenylindole; EMT, epithelial-mesenchymal transition; SWD-ALI, seawater drowning induced acute lung injury; ELISA, enzyme-linked immunosorbent assay.

A previous study has shown that TNFα can promote the stabilization of Snail, a transcription factor that can bind to E-cadherin on the cell membrane surface to promote EMT (19). Therefore, we examined the expression changes of the Snail gene and protein levels in lung tissue. We found that the expression of Snail (SNAI 1) was significantly increased (P<0.01), while the expression of Slug (SNAI 2) was decreased in the injured group (P>0.05) (Figure 3B,3C).

To further investigate the role of TNFα in SWD-ALI, we performed immunofluorescence analysis of the lung tissue. We found that the expression of the epithelial cell marker, E-cadherin, was decreased, while the expression of the mesenchymal marker, N-cadherin, was increased (Figure 3D). These results suggest that the elevated TNFα content caused by drowning injury may affect the EMT process by regulating Snail family expression, which in turn may trigger severe lung injury.

To validate this mechanism at the cellular level, we constructed an HPAEpiC-level injury model using 30% artificial seawater stimulation. We found that the cells underwent obvious morphological changes after seawater stimulation (Figure 3E). The qPCR analysis showed that TNFα and Snail messenger RNA (mRNA) levels increased, while the expression of Slug mRNA decreased in cells after seawater stimulation (Figure 3F). Western blot analysis revealed that the protein levels of Snail and N-cadherin increased, while the protein levels of Slug, E-cadherin, and Claudin-1 decreased after seawater stimulation (Figure 3G), suggesting that the seawater stimulation induced the EMT.

To further investigate the correlation between the TNFα, Snail, and EMT signaling pathways, we performed correlation coefficient circle plot analysis of the main genes of these pathways using transcriptome sequencing data. We found a significant correlation between TNFα, Snail, and EMT (Figure 3H), which suggesting that the TNFα/Snail/EMT pathway plays an important role in SWD-ALI.

In conclusion, our results suggest that SWD may mediate the activation of the EMT pathway through the TNFα inflammatory response, leading to severe lung injury.

MSCs therapy effectively improves respiratory symptoms and pulmonary function

A previous study has suggested that MSCs have potent anti-inflammatory effects and might suppress lung injury via TNFα suppression (24). To examine the potential effect of MSCs on SWD-ALI, we identified the morphology and surface markers of MSCs (Figure S1A,S1B). We then nebulized the MSCs intratracheally using a mouse nebulizer syringe (Figure S1C). There was no difference in the MSCs activity before and after MSCs nebulization (Figure S1D). We monitored the respiratory function of the mice using WBP at 6, 12, 24, and 48 h post-treatment. Compared to the mice in the CON group, the mice in the SW-PBS group exhibited wheezing, and hypopnea. However, MSCs improved the symptoms of the SW-MSC mice [supplementary pulmonary function video (Video S1)].

From the 6 and 12 h curves, we derived AC flow diagrams (Figure 4A), which showed lower tidal volume in the model group. MSCs treatment improved respiratory function. In SW-PBS group, MV was worse after injury, but improved after MSCs treatment as shown in SW-MSC group. The improvement was more significant at 12 h in SW-MSC group, but was worse at 12 h in SW-PBS group (Figure 4B). MV was worse 24 and 48 h after SWD, significantly improved after MSCs treatment (Figure 4C,4D). In the SW-PBS group, PIFb worsened after injury, but improved after MSCs treatment as shown in the SW-MSC group. From 6 to 12 h, PIFb changes in line with MV (Figure 4E), and the difference was statistically significant.

Figure 4 MSCs improvement of ALI based on non-invasive pulmonary function. (A) Pulmonary function test results of the AC flow for the three groups at 6 and 12 h. (B) MV of the three groups were measured at 6 and 12 h, n=6, *, P<0.05; **, P<0.01; ***, P<0.001. (C,D) The MV of three groups were measured at 24 and 48 h, n=6, *, P<0.05; ***, P<0.001. (E) PIFb of three groups were measured at 6 and 12 h, n=6, **, P<0.01; ***, P<0.001. CON, control group; SW-PBS, seawater drowning + phosphate buffer solution group; SW-MSC, seawater drowning + mesenchymal stem cells group; AC, airflow changes; TV, tidal volume; MV, minute ventilation; PIFb, peak inspiratory flow rate; MSC, mesenchymal stem cell; ALI, acute lung injury.

Therefore, our results suggest that the intratracheal nebulization of MSCs can improve respiratory symptoms and pulmonary function in mice with SWD-ALI.

MSCs treatment alleviates SWD-ALI by suppressing TNFα/Snail/EMT signaling

In vivo non-invasive methods confirmed that MSCs effectively treated the SWD-ALI mice. Further analysis at the cellular and tissue levels was performed to establish the therapeutic efficacy of MSCs in treating SWD-ALI. Nebulized inhalation of MSCs was found to significantly mitigate the pathological lung injury in the SWD mice (Figure 5A) and to reduce the systemic inflammatory response (Figure 5B and Figure S2A). The total protein concentration, cell counts, and inflammatory factor IL-1β in BALF of mice were all reduced (Figure S2B). The flow cytometry analysis of the BALF cells and lung tissue cells revealed decrease in neutrophils and macrophages after MSCs inhalation compared to that the mice of SW-PBS group (Figure 5C and Figure S2C). The lung tissue Evans blue passage rate showed decreasing trend (Figure S2D), but the difference between SW-PBS and SW-MSC was not statistically significant (P>0.05). The lungs W/D ratio in SW-MSC group was also reduced (Figure S2E; P<0.05).

Figure 5 MSCs nebulized inhalation effectively ameliorated SWD-ALI through the TNFα/Snail/EMT pathway. (A) Representative lung tissue images and HE staining, the box in the middle figure was the enlarged area below (up scale bar: 2 mm, middle scale bar: 50 μm, down scale bar: 25 μm), and quantitative analysis of the HE staining results, n=6, ***, P<0.001. (B) Quantification of percentages of neutrophils in the blood, n=11, *, P<0.05; ***, P<0.001. (C) The percentages of BALF and lungs infiltrating neutrophils in each group were analyzed by flow cytometry, and the infiltration of neutrophils into the BALF and lungs were compared among the groups, n=6 (BALF), n=12 (lung), *, P<0.05; **, P<0.01; ***, P<0.001. (D) The expression of TNFα RNA in lung tissues of each group, n=6, ns, P>0.05; **, P<0.01; ***, P<0.001. (E) The TNFα and E-cadherin protein levels in mice lung tissues were detected by Western blotting. (F) The Snail and Slug protein levels in human alveolar epithelial cells were detected by Western blotting. (G) Representative immunofluorescence images of lung sections from different groups were stained to examine TNFα (red), SP-C (yellow), Snail (cyan), Slug (green), and DAPI (blue); scale bar: 25 μm. CON, control group; SW-PBS, seawater drowning + phosphate buffer solution group; SW-MSC, seawater drowning + mesenchymal stem cells group; HE, hematoxylin and eosin; BALF, bronchoalveolar lavage fluid; TNFα, tumor necrosis factor alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MSC, mesenchymal stem cell; SWD-ALI, seawater drowning induced acute lung injury; EMT, epithelial-mesenchymal transition.

Further, qPCR analysis of the lung tissue revealed reduction in the levels of inflammatory factors IL-1β and IL-6 mRNA after MSCs treatment compared to that post-injury (Figure S2F). The reduction in levels of SP-B and SP-C, and the epithelial sodium channel ENaC were also improved (Figure S2G). These results suggest that the mice of SWD-ALI were ameliorated after MSCs inhalation.

Moreover, the TNFα mRNA and protein levels in mice lung tissues were significantly reduced after MSCs treatment, and E-cadherin protein level was elevated compared with that of SW-PBS mice (Figure 5D,5E). The MSCs-culture medium (CM) treatment decreased the alveolar epithelial cell injury Snail protein level and increased the Slug protein level after seawater stimulation (Figure 5F), indicating that the epithelial cell Snail pathway was inhibited after MSCs treatment. Immunofluorescence detection revealed that TNFα (red) was elevated and SP-C (yellow) was decreased in the SWD-ALI group, which was also accompanied by increase in Snail (cyan) and decrease in Slug (green). However, after MSCs treatment, the opposite effects were observed (Figure 5G). Therefore, it was concluded that MSCs inhibit the TNFα/Snail/EMT pathway and play a therapeutic role in SWD-ALI.

MSCs nebulized inhalation can effectively improve SWD-ALI and chronic pulmonary fibrosis

We investigated the mechanism of MSCs treatment in SWD-ALI through transcriptome sequencing analysis. PCA of transcriptome data indicated significant grouping differences among the groups (i.e., the CON, SW-PBS, and SW-MSC groups) (Figure 6A). The Wayne plot showed 846 common genes in the CON/SW-PBS and SW-PBS/SW-MSC intersections. These genes were primarily inflammatory response genes (Figure 6B). The volcano plot provided additional evidence of the substantial changes in gene expression between the SW-MSC group and SW-PBS group (Figure 6C). The clustering heat map and trend analysis (Figure 6D) showed that MSCs treatment restored extracellular membrane-associated protein, ciliary protein, and viral infection gene expression. It suggested that MSCs improved SWD-ALI at the transcriptome expression level. The first 10 pathways analyzed by KEGG enrichment analysis suggested that MSCs treatment ameliorated SWD-ALI through signaling pathways such as TNFα (Figure 6E).

Figure 6 MSCs therapy inhibited TNFα pathways to treat SWD-ALI, and MSCs reduced pulmonary fibrosis. (A) PCA (n=3). (B) Venn diagram showed the number of DEGs in the intersection based on comparison of the CON and SW-PBS groups, and the SW-PBS and SW-MSC groups. Total of 846 DEGs were selected. (C) Volcano plots showed the up- and down-regulated genes in the SW-PBS and SW-MSC groups. (D) Genes with a meaningful log₂fold change absolute value ≥5 in each group were analyzed by cluster heatmaps and divided into six groups according to the clustering. (E) Top 10 KEGG enriched pathways analysis among the groups. (F) Pearson correlation analysis of the network diagram of the TNFα, Snail and EMT pathways, and the major genes of pulmonary fibrosis among the groups (positive correlation in red; negative correlation in green). (G) Masson staining was used to analyze the degree of peribronchial and par alveolar pulmonary fibrosis after 28 days in the three groups of mice, the box in the figure was the enlarged area below; scale bars: 50 and 20 μm. PCA, principal component analysis; TNFα, tumor necrosis factor alpha; SWD-ALI, seawater drowning induced acute lung injury; DEGs, differential expression genes; CON, control group; SW-PBS, seawater drowning + phosphate buffer solution group; SW-MSC, seawater drowning + mesenchymal stem cells group; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Subsequently, a correlation network diagram (Figure 6F) analysis was performed, which revealed that the major genes of TNFα, Snail, and EMT were correlated with some fibrosis genes. To observe the chronic changes of mouse lung tissue caused by SWD, we performed Masson staining of mouse lung tissue slides after 28 days (Figure 6G), and found that SWD could indeed cause lung tissue fibrosis in mice, and MSCs treatment could alleviate lung interstitial fibrosis in SWD mice.

Thus, these results indicated that MSCs nebulized inhalation can alleviate ALI and chronic pulmonary fibrosis in SWD mice.

Discussion

SWD is a serious medical condition with high morbidity and mortality rates, however, the scientific understanding of the pathogenesis of SWD is limited, as is the development of effective treatment methods. In our study, SWD induced severe acute and chronic lung injury through the activation of the EMT pathway mediated by the TNFα inflammatory response. The inhalation of MSCs via nebulization significantly attenuated ALI and alleviated long-term pulmonary fibrosis in the SWD mice (Figure 7).

Figure 7 Model diagram of the SWD-ALI injury mechanism and MSCs therapeutic effect. MSC, mesenchymal stem cell; TNFα, tumor necrosis factor alpha; AEC, alveolar epithelial cell; EMT, epithelial-mesenchymal transition; SWD-ALI, seawater drowning induced acute lung injury.

First, we constructed a SWD-ALI mouse model of 4 mL/kg seawater by LEI method for 6 h. After modeling, we successfully validated the model according to the evaluation criteria for ALI models in experimental animals proposed by the ATS Official Symposium (37). The criteria include histological evidence of tissue injury, alteration of the alveolar-capillary barrier, the presence of the inflammatory response, and physiological dysfunction. In terms of the functional evaluation, we innovatively applied the non-invasive pulmonary function test to the mice. The non-invasive pulmonary function test is not commonly used to evaluate ALI animal models (38). However, this method allowed for greater visualization of the activity and respiratory performance of the awake mice. The test data provided insights into mouse respiratory function, including the respiratory curve, tidal volume, and respiratory frequency. In addition, it consistently kept track of lung damage alterations in the mice. More importantly, the SWD-ALI model also revealed that mice lungs developed long-term pulmonary fibrosis after modeling. This suggests that we should pay attention to the longer-term complications of SWD.

Our study showed that the inflammatory response played an important role in model construction, similar to other causes of ALI, such as lipopolysaccharides (LPS), viruses (39-42). The genomics results suggested that the TNFα/Snail/EMT pathway plays an important role in the inflammatory response. The changes in the cell surface and basement membrane suggested the possibility of EMT (43-45), and that EMT may also occur in the acute phase, which is consistent with previous report (17). The question of whether its role is related to the abnormal repair of acute injury requires further study. At the same time, it has been suggested that TNFα drives the stabilization of the Snail protein and suppresses its degradation (19), while the zinc-finger transcription factor Snail protein can bind to E-cadherin and promote EMT (46), which may cause long-term pulmonary fibrosis. Interestingly, elevated Slug has been shown to promote EMT in most studies (47,48). However, Slug expression was decreased in our experiments. The exact relationship between our findings and alveolar collapse, fracture, or specific repair after alveolar cell injury remains unclear and requires further investigation.

Many studies have shown that MSCs can effectively treat ALI caused by LPS and viral infection (24-26,49). To examine whether MSCs were effective in treating SWD-ALI, we investigated the efficacy of administering MSCs via intratracheal nebulized inhalation. The route of administration can help circumvent the pulmonary embolism risks associated with intravenous stem cell administration. MSCs may also play several roles via direct cell-to-cell contact mechanisms and indirectly through the secretion of several anti-inflammatory factors to ALI. Further, we found that MSCs limited the occurrence of late lung fibrosis.

Conclusions

Taken together, early intervention in SWD-ALI events is crucial to reduce the severity of SWD-ALI and the risk of late complications. Our results suggested that MSCs treatment for SWD induced could alleviate ALI and delay later fibrosis. Future research could focus on developing spray devices to deliver highly effective genetically modified MSCs after clearing the airways of SWD patients. To further enhance the therapeutic efficacy of treatments and reduce complications.

Acknowledgments

The authors would like to thank Beijing Institute of Radiation Medicine and Chinese PLA General Hospital for their technical support.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1471/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1471/dss

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1471/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All the experimental procedures were approved by the Experimental Animal Welfare Ethics Committee of Chinese PLA General Hospital (No. 2024-X20-69), and adhered to the Guide for the Care and Use of Laboratory Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)