BM-MSCs mitigate lung injury in a rat model of decompression sickness

Chen Lu; Daqian Gu; Hao Chen; Liang Chen; Jie Chen; Yuwei Weng; Xianliang Lin

doi:10.1371/journal.pone.0326618

. 2025 Aug 21;20(8):e0326618. doi: 10.1371/journal.pone.0326618

BM-MSCs mitigate lung injury in a rat model of decompression sickness

Chen Lu ^1,^#, Daqian Gu ^2,^#, Hao Chen ¹, Liang Chen ¹, Jie Chen ¹, Yuwei Weng ¹, Xianliang Lin ^1,^*

Editor: Roland Eghoghosoa Akhigbe³

PMCID: PMC12370078 PMID: 40839685

Abstract

Decompression sickness is a fatal disease worldwide. Therefore, to find a prophylactic modality for decompression sickness is urgently required. Bone marrow derived mesenchymal stem cells exhibit effectiveness in antioxidant, anti-inflammation, and decrease cell death; while its effects on decompression sickness remains unclear. This study aimed to further investigate the mechanisms of decompression sickness induced lung injury, as well as effects of bone marrow derived mesenchymal stem cells on decompression sickness induced lung injury and explore the role of oxidative stress, inflammation and cell death play in this disease. The study involved Sprague-Dawley rats age at 8−10 weeks weighting 350 ± 10g. Acute lung injury was induced by decompression hyperbaric chamber. A dose of bone marrow derived mesenchymal stem cells (2 × 10⁶ cells) was given to rats one day prior to the start of decompression. Lung injury severity was estimated by determining lung damage scores, pulmonary oxidative, inflammatory factors and cell death. In bone marrow derived mesenchymal stem cells treated rats, the morbidity and mortality of decompression markedly decreased. The increases of protein IL-1 and IL-6 in BALF and lung wet/dry ratio and lung injury score were alleviated. The ROS, CAT, SOD, and MDA activities and GSH levels were significant attenuated (P < 0.05). The pyroptosis and nerroptosis were significant mitigate (P < 0.05). Based on the results, bone marrow derived mesenchymal stem cells is an potential efficient and safe prophylactic modality protect rats from decompression induced acute lung injury.

Introduction

Decompression sickness (DCS) is a systemic and mortal disease [1]. It has been the most serious danger for Self-Contained Underwater Breathing Apparatus (SCUB) divers [2]. In DCS, gas exchange disorder and respiratory distress always represent as common symptoms [3]. In pathological manifestations of lung in DCS, the alveolar epithelial cells show hydropic degeneration and disintegration whilst the pulmonary capillary endothelium is often ruptured, which might explain for the lung edema and hemorrhage [4]. Although the exact mechanisms that lead to lung injury are still not clear, there are some possible effective strategies to prevent or limit its progression [5,6]. Thus, a better understanding of the mechanisms involved in the progression of DCS-induced lung injury and finding appropriate treatment are essential.

DCS can trigger the acute stimulation of immune system, and subsequently induce inflammation [7]. The initiation of inflammation might be attributed to increased oxidative stress, including overproduction of reactive oxygen species (ROS), since a rapid increase in ROS can activate nuclear factor κB (NF-κB) [8]. A former study has shown that swollen mitochondria exists in granular pneumocytes under DCS, which is the characteristic of enhanced mitochondrial ROS formation [4]. Similarly, both in vivo and in vitro studies demonstrated that DCS increased content of ROS [9,10]. Uncontrolled ROS may trigger mitochondrial permeability transition pore (mPTP) induction within individual mitochondria in intact cell systems [11]. The phenomenon of ROS-triggering of the mPTP associated with further stimulation of ROS formation has been termed “ROS-induced ROS release” (RIRR) [12].ROS overproduction between mitochondria cause a positive-feedback mechanism resulting in an elevated production of ROS that could be propagated throughout the cell and subsequently destroy the mitochondrial dynamics and other pathways, and eventually induce cell death [13]. Besides, persistent cell death might further exacerbate inflammation and oxidative stress. Some types of cell death such as necrosis and apoptosis have been shown in DCS injured brain tissue [14]. However, what types of cell death are involved in DCS-induced lung injury is still unknown.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been proved as a potential cell-based therapy for lung disease such as chronic obstructive pulmonary disease (COPD), lipopolysaccharide (LPS)-induced lung injury and so on [15,16]. Because BM-MSCs are thought to be immune-privileged and protected from rejection [17], it has been permitted to be used in allo-transplantation in some clinical researches [18,19]. A study has demonstrated that BM-MSCs could engraft into the injured lung [20]. In addition, BM-MSCs display anti-inflammation and anti-oxidative stress effects in lung injuries and other diseases [21,22]. For example, in Tarek Khamis et al study, BM-BMSCs have been demonstrated decrease both the mean fold change of the relative mRNA expression of the renal proinflammatory markers such as NFKβ, IL1β, TNFα, and IL6 and the mean fold change of the relative mRNA expression of renal proapoptotic markers Fas, FasL, P53, caspase-3, BAX, and BAX/BCL2, meanwhile significantly upregulating the anti-apoptotic marker BCL2 compared with the diabetic group [23]. Asmaa Adel et al. showed that the treatment of CCl4^-injected rats with rats and mice BM-MSCs significantly elevated SOD and GST levels. However, whether BM-MSCs could reduce inflammation, oxidative stress and cell death in DCS-induced lung injury is seldom investigated.

In this study, we pretreat DCS model with BM-MSCs in different time points to evaluate the effects of prevention and treatment. We analyzed the morbidity, mortality, lung injury, inflammation and oxidative stress. In addition, to study the types of pulmonary cell death in DCS, we detected the expression of markers for apoptosis, pyroptosis, necroptosis and ferroptosis. The present study aimed to elucidate the effect of BM-MSCs on DCS-induced lung injury.

Methods

Animals

A total of 252 healthy Sprague-Dawley (SD) rats age at 8–10 weeks weighting 350 ± 10 g were purchased from Shanghai Slac Laboratory Animal Co. Ltd and bred in an AAALAC-accredited facility. The animals were housed in a controlled environment (20 ± 2 °C, 12h/12h light/dark cycles), with free access to water and standard rodent chow. All experimental procedures were approved by the Animal Care and Use Committee of the 900th Hospital of Joint Logistics Support Force. All experiments conformed to the guidelines for the ethical use of animals, and all efforts were made to minimize animal suffering and reduce the number of animals used.

DCS model

For DCS model, the rats were divided into three groups, and they were placed in a hyperbaric chamber (Hongyuan Oxygen Industry Co., Ltd, Yantai, China). For DCS1 group, the chamber was pressurized to 6 bar in 5 min at a speed of 1 bar/min and maintained for 90 min [5], after that the chamber was decompressed to 1 bar at a speed of 2 bar/min. For DCS2 group, the chamber was pressurized to 6 bar in 5 min at a speed of 1 bar/min and maintained for 90 min, after that the chamber was decompressed to 1 bar at a speed of 1 bar/min. For DCS3 group, the chamber was pressurized to 7 bar in 5 min at a speed of 1.2 bar/min and maintained for 90 min, after that, the chamber was decompressed to 1 bar at a speed of 2 bar/min. The rats were divided into five groups, and respectively named as Vehicle, DCS, DCS + BM-MSCs 7d, DCS + BM-MSCs 3d and DCS + BM-MSCs 1h. The rats in Vehicle group were treated with saline vehicle (control, 200 μL, tail vein injection); the rats in DCS group were treated with saline vehicle (200 μL, tail vein injection) and then experienced DCS modeling; the rats in MSCs-7d group were treated with BM-MSCs (2 × 10⁶cells, 200 μL saline as vehicle, tail vein injection) 7 days before DCS modeling [24,25]; the rats in MSCs-3d group were treated with BM-MSCs (2 × 10⁶ cells, 200μ L, saline as vehicle, tail vein injection) 3 days before DCS modeling; the rats in DCS + BM-MSCs 1h group were treated with BM-MSCs (2 × 10⁶ cells, 200 μL, saline as vehicle, tail vein injection) 1 hour (h) before DCS modeling. During the exposure, the chamber was ventilated continuously to avoid carbon dioxide (CO₂) retention and the temperature was controlled at 25 ± 2°C. Following the decompression procedure, rats were observed for DCS related behaviors within 2 h by a member of staff who was blinded to the treatments and possess certificate of Laboratory Animal Practitioner Training. Any of the following symptoms was regarded as presence of DCS: respiratory distress, walking difficulties, fore and/or hind limb paralysis, rolling, convulsions or death [3]. After the 2 h observation period, all survived rats were intraperitoneally anesthetized with 3% pentobarbital sodium (1.5 mL·kg^-1) and then blood, bronchoalveolar lavage fluid (BALF) and lung tissues were sampled for further analysis. The wet/dry weight ratio of the right upper lobes of lung was also calculated.

Isolation and culture of BM-MSCs

BM-MSCs were isolated and expanded according to a previously described procedure [26]. In brief, bone marrow was flushed from the femoral and tibia with phosphate-buffered saline (PBS). Then, the bone marrow was passed through a 70 mm strainer and centrifugated at 1,200 rpm for 5 min. Thereafter, the cell palet were extracted and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Shanghai, China) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin medium. Third-passage MSCs were used for different treatments.

Phenotypes and differentiation of BM-MSCs

To analyze the cell surface markers for BM-MSCs, cells were detached with trypsin and ethylenediamine tetraacetic acid, washed and re-suspended in PBS with 5 mM EDTA. Then cells were stained with CD105, CD90, CD73, CD34, CD45 and HLA-DR for 1 hour at 4°C. After that, the cells were washed twice and stained with APC, FITC and PE conjucted antibodies. At last, the cells were washed and analyzed by flow cytometry [27].

To induce osteogenic differentiation, cells were treated with the osteogenic medium (complete medium with 0.1 mmol·L^-1 dexamethasone, 10 mmol·L^-1 b-glycerol phosphate and 50 mg·mL^-1 ascorbate-2-phosphate) for 18 days. The medium was changed every 3 days. After induction, cells were fixed with 4% paraformaldehydes or 20 min and washed with PBS (5 min, 3 times), and then stained with 1% alizarin red solution (Solarbio, Beijing, China) for 10 min at 37°C to examine the mineral nodule deposition [28].

To induce chondrogenic differentiation, cells were cultured in complete medium supplemented with 100 mg·mL^-1 sodium pyruvate, 10 ng·mL^-1 transforming growth factor-β1, 100 nmol·L^-1 dexamethasone, 1% insulin-transferrin-selenium and 100 mg·mL^-1 ascorbate-2-phosphate for 14 days. After induction, cells were fixed with 4% paraformaldehydes for 20 min and washed with PBS (5 min, 3 times), and then stained with Alcian Blue (Solarbio) for 30 min to evaluate deposition of glycosaminoglycans [28].

To induce adipogenic differentiation, cells were cultured in complete medium with 0.5 mmol·L^-1 isobutylmethylxanthine, 0.25 mmol·L^-1 dexamethasone, 10 mmol·L^-1 insulin and 50 mmol·L^-1 indomethacin for 10 days. After that, cells were fixed with 4% paraformaldehydes for 20 min and washed with PBS (5 min, 3 times), and then stained with oil red O solution (Solarbio) for 45 min to examine lipid droplets in cytoplasm. All the staining results were observed under an inverted microscope (Olympus, Tokyo, Japan) and photographed [28].

Lung histopathology

The right lower lobes of lung samples were fixed and then dehydrated in increasing concentrations of ethanol. After that, they were cleared in xylene and embedded in paraffin. The samples were cut into 5 μm thick sections followed by staining with haematoxylin and eosin. Morphological damages were scored to evaluate the degree of lung damage. These histopathologic damages included alveolar congestion, hemorrhage, neutrophil infiltration into the airspace or vessel wall, and thickness of alveolar wall/hyaline membrane formation [29] Pathological scoring based on the injury area of involvement was according to a 5 point scale as follows: 0 = minimal damage, 1 = mild damage, 2 = moderate damage, 3 = severe damage, and 4 = maximal damage [30]. Quantification was conducted by an investigator who was blinded to the group information of the samples.

Lung ROS detection

The rat lung tissues were quickly frozen, cut to a thickness of 8 μm at an optimized cutting temperature, and mounted on glass slides. ROS [31] was stained by DHE probe as red and cell nucleus was stained by DAPI as blue. The concentration of DHE in the lung was calculated by the fluorescence intensity using Image J, according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, China).

BALF analysis

Total protein level in BALF was determined according to the Bradford protein assay kit (Solarbio) by measuring the absorption at 595 nm with a spectrum photometer. The BALF was centrifuged at 3,000 rpm for 20 min at 4°C. The extracted cell pellet was resuspended by 1 mL PBS and used to determine the total cell count through a cell counter. A cell smear was made and stained by Wright-Giemsa staining to confirm the neutrophil percentage.

Assay of oxidative stress and inflammatory markers

Lung tissues were homogenized in assay buffer. Subsequently, the tissue homogenate was used to detect the content of glutathione (GSH) and myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) activities by commercial reagent kits (Jiancheng Bioengineering Institute, Nanjing, China) according to instructions, respectively. Values were normalized by tissue protein concentration. The fresh blood samples were placed under room temperature for 1 h, and then the samples were centrifuged under 4 °C, 2,000g for 10 min. The supernatant was extracted as serum for detection. The serum levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β) and Interleukin-6 (IL-6) were measured using the Elisa Assay Kits (Beyotime Institute of Biotechnology, Nantong, China).

Immunoblotting

The lung tissues were lysed in RIPA buffer, supplemented with protease inhibitors, and denatured with loading buffer. The nuclear and cytoplasmic fractions were also denatured with loading buffer. The protein samples were collected and stored at – 20° C until use. The protein samples were separated by SDS-PAGE with 10−15% polyacrylamide gel and then electroblotted onto nitrocellulose membranes (Amersham Life Science, Arlington, TX). The blots were blocked in Tris-buffered saline containing 5% nonfat dry milk for 1 h at room temperature with constant shaking and then incubated with primary antibodies (Cleaved- Caspase 3, Bcl-2, Caspase-1, NLRP3, RIPK3, MLKL, GPX4, ACSL4, β-actin) overnight at 4°C. The secondary antibodies (Goat anti-Rabbit IR Dye 800, Donkey anti-Goat IR Dye 800, Goat anti-Mouse IR Dye 800) were used to bind their respective primary antibody at room temperature for 1 hour. The bound complexes were detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The images were analyzed through the Odyssey Application Software to obtain the integrated intensities.

Statistical analysis

All data are expressed as mean ± SD, unless otherwise stated. GraphPad-Prism 9.0 and SPSS 17.0 were used to perform the statistical analyses. Student’s unpaired t-test was used to compare 2 independent groups. Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test. Incidence of DCS was compared by Chi-square test. Survival rates were compared using the log-rank test. Values of p < 0.05 were considered significant.

Results

Identification of BM-MSCs

Isolated rat BM-MSCs were characterized by flow cytometry and their differentiation capacity. Flow cytometry analysis showed that the cells were positive for CD105, CD73 and CD90, whereas negative for CD45, CD34 and HLA-DR. The results conformed to the characteristics of MSCs (Fig 1). To provide a clear visual reference for background signal discriminationwe have incorporated isotype control overlays in Fig 2. Phase-contrast microscopy images of BM-MSCs at passage 3, demonstrating their spindle-shaped morphology (Fig 3A). After osteogenic induction, alizarin red staining showed that calcium-rich extracellular matrix in the cells (Fig 3B); Alcian blue staining (Fig 3C) confirmed sulfated glycosaminoglycan (sGAG) deposition in the extracellular matrix, indicating successful chondrogenic differentiation of BM-MSCs. After adipogenic induction, oil Red O staining showed that the red lipid droplets were distributed in and between the cells (Fig 3D). These results demonstrated that isolated cells were BM-MSCs.

Fig 1 — Flow cytometry analysis showed that these cells were positive for CD105, CD73 and CD90, but negative for CD34, CD45 and HLA-DR.

Fig 3 — **(A)** The morphology of BM-MSCs was observed under a light microscope (Scale bar = 100 μm). **(B-D)** BM-MSCs were stained with alizarin red (Scale bar = 100 μm), Alcian blue (Scale bar = 100 μm). and oil red O (Scale bar = 20 μm), indicating that could differentiate into osteoblasts, chondrogenic and adipocytes respectively.

Pretreatment of BM-MSCs improves the survival rate and incidence of DCS

To explore the suitable condition for the DCS rat model, we distributed rats into three groups (Fig 4A) and investigated the effects of chamber pressure and decompression rate on them. DCS2 group rats were treated with slower decompression rate than DCS1 group rats (1 bar/min vs. 2 bar/min), while DCS3 group rats experienced higher chamber pressure than DCS1 group rats (7 bar vs. 6 bar). The results showed that DCS2 group rats experienced less morbidity than DCS1 0.4583 ± 0.1309 (n = 36, 95% CI: 0.1948 to 0.7218, p < 0.05) . DCS1 group decreased more mortality than DCS3 group rats 0.3333 ± 0.1300 (n = 36, 95% CI: 0.07159 to 0.5951, p < 0.05) (Fig 4C). Thus, the following DCS model used the experiment condition of DCS1 group rats. To evaluate the influence of BM-MSCs injecting time on DCS model, we treated rats with BM-MSCs 7d, 3d, and 1h before decompression respectively (Fig 4B). We found that injecting BM-MSCs 1h before decompression could significantly reduce morbidity rate 0.4167 ± 0.1045 n = 36, 95% CI: 0.2083 to 0.6250, p < 0.05) and mortality rate 0.3333 ± 0.1027 (n = 36, 95% CI: 0.1286 to 0.5381, p < 0.05), while injecting 7d and 3d before did not influence it (Fig 4D). Thus, to further investigate the treatment effects of BM-MSCs on DCS rat model, we treated DCS rats with BM-MSCs 1h before decompression in the following experiment. BM-MSCs could decrease the incidence of DCS with time (hazard ratio0.3655, 95% CI 0.1981 to 0.6742) (Fig 4E). Pretreatment with BM-MSCs prolong survival time (hazard ratio 0.2434, 95% CI 0.1051 to 0.5635). (Fig 4F). These results hinted that pretreatment BM-MSCs could protect against DCS.

Pretreatment of BM-MSCs attenuates DCS induced lung injury

Since respiratory dysfunction and lung tissue injury are features of DCS, we wondered whether BM-MSCs could attenuate DCS induced lung injury. The DCS rats in our experiment showed increased W/D ratio and BALF protein, indicating an obvious edema and exudation, in agreement with previous reports. However, pretreatment with BM-MSCs mean decreased W/D ratio −0.8800 ± 0.1975 (n = 5, 95% CI: −1.335 to −0.4246, p < 0.05) and BALF protein −407.0 ± 64.53 (n = 5, 95% CI: 1.335 to −0.4246, p < 0.05) compare DCS rats (Fig 5A and 5B). BM-MSCs also ameliorated the DCS-induced lung pathological injury, indicated as reduced alveolar congestion, hemorrhage, neutrophil infiltration, thickness of alveolar wall (Fig 5F). BM-MSCs group reduce mean lung injury score −6.400 ± 0.6000 (n = 5, 95% CI: 1.335 to −0.4246, p < 0.05) compare with DCS group and statistics graph was shown in (Fig 5C).

Fig 5 — **(A)** Lung W/D ratio compare DCS rats.0.8800 ± 0.1975 (n = 5, 95% CI: −1.335 to −0.4246, p < 0.05). **(B)** BALF protein compare DCS rats −407.0 ± 64.53 (n = 5, 95% CI: 1.335 to −0.4246, p < 0.05) compare DCS rats. **(C)** Mean lung injury score compare DCS rats −6.400 ± 0.6000 (n = 5, 95% CI: 1.335 to −0.4246, p < 0.05) **(D-F)** The present picture of lung tissue histopathology. n = 5 * p < 0.05 vs. vehicle group. ^# p < 0.05 vs. DCS group. Scale bar = 100 μm.

Pretreatment of BM-MSCs attenuates DCS induced lung inflammation

Because inflammation is a common cause for lung injury and rapid decompression could induce the release of inflammatory factors [32], we detected the effect of BM-MSCs on DCS-induced lung inflammation. The results showed that decompression significantly increased the total inflammatory cell and neutrophil counts in the BALF compared with that in vehicle treated rats, but BM-MSCs significantly inhibited the effect t of decompression. BM-MSCs group mean reduce total cells in BALF −4.45*10⁶ ± 1.10*10⁶ (n = 5, 95% CI: −6.99*10⁶ to −1.91*10^6, p < 0.05) and neutrophil counts in BALF −2.28*10⁶ ± 0.5*10⁶ (n = 5, 95% CI: −3.49*10⁶ to −1.1*10⁶, p < 0.05) compare with DCS group (Fig 6A and 6B). Meanwhile, we measured the MPO activity to assess the quantification of neutrophil accumulation in tissues. Compared with Vehicle group rats, DCS group rats showed a significant increase in lung MPO activity, but BM-MSCs pretreatment reversed it. BM-MSCs mean reduce lung MPO activity −2.720 ± 0.5359 (n = 5, 95% CI: −3.956 to −1.484, p < 0.05) (Fig 6C). In addition, we detected the expression of inflammatory factors in different groups. We found that BM-MSCs ameliorated the increased serum levels of inflammation-related markers in DCS group rats. BM-MSCs group reduce serum TNF-α-369.4 ± 56.67(n = 5, 95% CI: −500.1 to −238.7, p < 0.05), serum IL-6–484.6 ± 76.36(n = 5, 95% CI: −660.7 to −308.5, p < 0.05) and serum IL-1β-209.4 ± 32.76 (n = 5, 95% CI: −284.9 to −133.9, p < 0.05) (Fig 6D–6F). These results suggested that BM-MSCs might reduce DCS-induced lung injury by decreasing lung and serum inflammation.

Fig 6 — **(A)** Total cells in BALF −4.45*10⁶ ± 1.10*10⁶ (n = 5, 95% CI: −6.99*10⁶ to −1.91*10⁶, p < 0.05) compare with DCS group. **(B)** Total neutrophil counts in BALF −2.28*10⁶ ± 0.5*10⁶ (n = 5, 95% CI: −3.49*10⁶ to −1.1*10⁶, p < 0.05) compare with DCS group **(C)** Lung MPO activity −2.720 ± 0.5359 (n = 5, 95% CI: −3.956 to −1.484, p < 0.05) compare with DCS. **(D)** Serum TNF-α level reduce −369.4 ± 56.67(n = 5, 95% CI: −500.1 to −238.7, p < 0.05) compare with DCS. **(E)** Serum IL-6 level −484.6 ± 76.36(n = 5, 95% CI: −660.7 to −308.5, p < 0.05) compare with DCS. **(F)** Serum IL-1β reduce −484.6 ± 76.36(n = 5, 95% CI: −660.7 to −308.5, p < 0.05) compare with DCS. n = 5, * p < 0.05 vs. vehicle group. ^# p < 0.05 vs. DCS group.

Pretreatment of BM-MSCs inhibits oxidative stress in DCS rats

Excess oxidative stress could activate inflammation response and exacerbate the development of lung injury [33]. Thus, we evaluated the oxidative balance in this experiment. Compared to the Vehicle group rats, the lung ROS production increased in DCS group rats, while BM-MSCs decreased the ROS production induced by decompression-0.6659 ± 0.1114 (n = 5, 95% CI: −0.9229 to −0.4089 p < 0.05) (Fig 7A–7B). Our results showed that decompression significantly decreased the lung GSH levels together with SOD and CAT activities in vehicle-treated rats, however, BM-MSCs reversed the effects of decompression (Fig 7C–7E). In addition, the lung MDA levels increased to a greater extent in DCS group rats than in Vehicle group rats, but decreased in BM-MSCs-treated DCS rats compared with vehicle-treated DCS rats (Fig 7F). These results indicated that BM-MSCs played an antioxidative role in DCS-induced lung injury.

Fig 7 — **(A)** Representative fluorescent images of lung tissue different groups. **(B)** Quantitative analysis of DCS-induced ROS generation of lung tissue in different. **(C)** CAT activities in different group. **(D)** GSH level in different group. **(E)** SOD activities in different group. **(F)** MDA level in different group. n = 5 * p < 0.05 vs. vehicle group. ^# p < 0.05 vs. DCS group.

The effect of BM-MSCs on different types of lung cell death in DCS rats

Both inflammation and ROS could cause cell death, but what types of cell death are involved in DCS-induced lung injury remains to be further elucidated. In this experiment, we detected the protein expression of the markers for apoptosis (cleaved caspase-3 and Bcl-2, Fig 6A–6B), necroptosis (RIPK3 and MLKL, Fig 8C–8D), pyroptosis (caspase-1 and NLRP3, Fig 8E–8F) and ferroptosis (GPX4 and ACSL4, Fig 8G–8H) in lung tissues in different groups. The results showed that decompression increased the expression of cleaved caspase-3, RIPK3, caspase-1, NLRP3, GPX4, and ACSL4, while decreased the expression of Bcl-2, indicating that decompression might induced apoptosis, pyroptosis and ferroptosis. BM-MSCs pretreatment significantly reduced apoptosis (Bcl-2↑), pyroptosis (Caspase-1↓), and ferroptosis (GPX4↑).

Fig 8 — **(A)** Representative western-blot images of lung tissue in different groups. **(B-I)** The relative band intensities of three group are shown in histograms above. n = 5 * p < 0.05 vs. vehicle group. ^#p < 0.05 vs. DCS group.

Discussion

DCS is a fatal disease especially for divers, astronauts, and pilot. In practice, DCS takes place even when decompression tables is adhered [34,35]. Moreover, while hyperbaric oxygen is considered as the effective treatment of DCS, standard hyperbaric oxygen treatment may be delayed when decompression sickness occurs in distant places. As high mortality and morbidity of DCS, it has garnered significant attention.

Haldane model was first documented decompression model and from that on various DCS model algorithms were put forward [36–38]. We take previous study as reference then put forward a newly reliable DCS model as described above [5]. In addition, during the process of model research, we find that mortality and incidence are solidly relate to pressure and decompression rate. As far as it is known, this is the first study investigating the relationship between mortality and pressure rate. Besides, we found that injecting BM-MSCs 1h before decompression could significantly reduce death rate, while injecting 7d and 3d before did not influence it. It is interesting, as documented MSCs could survive for weeks. This result we doubt may attribute to BM-MSCs already differentiation into other cells before decompression.

DCS affect multi-system of which respiratory system is devastating influence. Former studies have demonstrated that once DCS caused lung injury usually means poor prognosis, and therefore DCS induced lung injury need further study. Our results indicate that BM-MSCs preconditioning protect against DCS induced lung injury by reducing inflammatory and oxidative stress, decreasing pyroptosis and ferroptosis, promoting survival in DCS injury. DCS patients and rodents display respiratory distress and lung injury [35]. Our present results confirmed that DCS rats had lung dysfunction evidenced by increased W/D and lung damage scores. Pretreatment with BM-MSCs significantly reversed the DCS-induced lung injury. Our present results consist with many previous researches. For example, BM-MSCs protects lung from LPS injury [16]. MSCs infusion ameliorate cigarette smoke-induced lung damage in chronic obstructive pulmonary disease [39]. BM-MSCs exosomes derived from mesenchymal stem cells possess protective effects in radiation-induced lung fibrosis [40]. MSCs is characterized by antioxidative effect, which is proved in previous studies [41,42]. BM-MSCs scavenge overproduction reactive oxygen species [43], and increase the content of anti-oxidative material, for example SOD [44], CAT [42], and GSH to alleviate oxidative injury, decrease MDA [44] and MPO [45]. Our present data further demonstrated that BM-MSCs not only promote activities of the anti-oxidant enzymes (such as SOD, CAT and GSH), but also decrease generation of ROS, MPO and MDA in DCS rat. Thus, BM-MSCs may attenuate lung injury in DCS rats via its anti-oxidative mechanisms. While excessive ROS contributes to cellular damage (e.g., DNA, lipids, and protein oxidation), low to moderate ROS levels are essential for cellular signaling, immune responses [46]. Therefore, over-suppression of ROS may disrupt these beneficial pathways, leading to unintended consequences such as impaired immune function or reduced stress adaptation. Even with ROS reduction, pre-existing oxidative damage (e.g., accumulated oxidized LDL in atherosclerosis or protein aggregates in aging cells) may persist. For example, antioxidant therapies in atherosclerosis trials often fail to reverse endothelial dysfunction fully, as oxidized biomolecules like ox-LDL continue to drive inflammation. ROS reduction alone may not address downstream inflammatory cascades. In kidney injury models, ROS-responsive nanoparticles (RBCM@CeO2/TAK-242) were effective only when combined with anti-inflammatory agents (e.g., TAK-242) that suppress M1 macrophage polarization and TLR4/NF-κB signaling. Similarly, atherosclerosis involves ROS-triggered endothelial dysfunction and immune cell activation, requiring multi-targeted interventions. In conclusion, BM-MSCs mitigate DCS rats injuries in both short and long term not only just by decreasing ROS, but also combinate with others mechanism.

BM-MSCs display anti-inflammatory effect by reducing IL-6, TNF-α and NF-κB, while there were increased levels of IL-10 in osteoarthritis patients [47]. In one study, BM-MSCs attenuate sepsis by prostaglandin E2–dependent reprogramming of host macrophages [48]. In another study, administration of MSC protected the airways from allergen-induced pathology, reducing airway inflammation [49]. Our study further demonstrated that DCS could cause inflammation as evidenced by significantly elevate of IL- 1β, IL-6, and TNF-α in DCS rat, while MB-MSCs attenuated lung over-production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in decompression rats. These observations tend to support the hypothesis that BM-MSCs may ameliorate DCS induced lung injury via inhibiting inflammation response.

Each day billions of cells die and clear up by phagocytes. This process operates smoothly under normal conditions and therefore guarantee body function. However, this system can collapse when massive of cells suddenly die in some special conditions, such as during inflammation, and tissue damage. As we mentioned above, DCS arouse inflammation and cause tissue damages, and therefore it is likely induced cell death. Former study mainly focused on inflammation or oxidative stress of DCS, but few studies have ever found out which type of cell death involved in process of DCS and whether BM-MSCs could ameliorate DCS induced lung injury. Our current results reinforce that apoptosis is process of DCS as evidenced by increase of Cleaved-Caspase 3 and decrease of Bcl-2, which is consistent with former study [50], in present study we found pretreatment BM-MSCs have a trend of reduce apoptosis although it does not achieve statistics significance. Former study suggested that MSCs could improve smoke induced lung injury by reducing apoptosis and it seems that pluripotent stem cell-derived MSCs is superior over BM-MSCs as iPSC-MSCs possess a greater expansion capacity [51] This result is in contrast to our results, and several factors may contribute to discrepancy: [1] BM-MSCs number different. Previous study pretreatment subject 3 × 10⁶ cells of BM-MSCs, but our study precondition the rats 2 × 10⁶ cells of BM-MSCs. [2] Model different. Apoptosis may play important role in CS-induced lung injury model. While apoptosis may also involve in DCS induced lung injury, it may not play a key factor in lung injury. Therefore, whether BM-MSC could reduce apoptosis is still need further study.

Necroptosis, different from apoptosis, is another type of programed cell death involves cell swelling, membrane rupture, and release of cytoplasmic contents [52]. Necroptosis induced by reactive oxygen species and has been demonstrated in several animal models of diseases, such as LPS-induced lung injury, respiratory syncytial virus induced lung injury and Ischemia-Reperfusion Injury [53–55].However it is still poorly understood whether necroptosis is involved in DCS induced lung injury. In current study, we found though RIPK3 is increased in the lung of DCS rat, it could not eventually lead to rising of MLKL. Those results suggest that necroptosis may not involve in DCS, at least not through MLKL, as evidenced by contend of MLKL is not significant difference between Vehicle group and DCS group. The discrepancies in the expression levels of RIPK3 and MLKL relative to lung injury severity can be explained by their distinct roles in necroptosis signaling and context-dependent regulatory mechanisms. RIPK3 acts both as a kinase (phosphorylating MLKL to execute necroptosis) and a scaffold protein. For example, in pseudomonas aeruginosa pneumonia, RIPK3 promotes lung inflammation through its scaffold domain and RHIM motif, even when its kinase activity is inactive. This explains why RIPK3 levels may not always correlate with MLKL activation or cell death.[56]. It is worth mentioning former study also found RIPK3 increased in DCS induced lung injury, but it did not further examine the contend of MLKL [5]. As DCS related study is rare, it demands further research to verify.

Pyroptosis is recently identified programmed cell death and Caspase-1 is a crucial protease mediated process of cell death [57]. Pyroptosis is different from other cell death, has unique character, which manifests as cells continuing to expand until the membrane ruptures, causing the release of contents and activating a strong inflammatory response. Inflammatory cytokine release cause tissue damage, and tissue damage in turn aggravate inflammation. Caspase-1 dependent Pyroptosis is reported in many diseases [58]. One study has demonstrated that MSCs alleviated post-resuscitation cardiac and cerebral injuries in swine by inhibition of cell pyroptosis [59] One vitro study demonstrated that MSCs reduce Pyroptosis after ischemic stroke by targeting absent in melanoma 2. Inflammation plays important role in DCS, and pyroptosis is also characterized by inflammation. Currently, we found that DCS may cause lung cell pyroptosis by activation NLRP3/Caspase-1 signaling pathway. Interestingly, we found BM-MSCs may inhibit pyroptosis as evidence by decrease of caspase1, but it seems not through direct decrease NLRP3.

Ferroptosis, a new type of programmed cell death, has been discovered in numerous human diseases [60]. Iron overload and reactive oxygen species play crucial roles in Ferroptosis. It was reported that BMSC-derived exosomes protect liver cell Ferroptosis by reduction of lipid peroxidation [61]. Another study reported that BM-MSCs ameliorate Ferroptosis by reduce the excessive mitochondrial fission and mitophagy, restored the mitochondrial quality control [62]. GPX4 moonlights as structural protein and antioxidant that powerfully inhibits lipid oxidation. It is regarded as a main regulate factor of ferroptosis, which participate in the lipid metabolism and influences the cell death [63]. ACSL4 is an enzyme that esterifies CoA into specific polyunsaturated fatty acid, it is another key regulator contributes to the execution of ferroptosis by triggering phospholipid peroxidation. In current study, the level of the GPX4 and ACSL4 significantly increased in DCS rats, and BM-MSCs provided significant protection against the injuries. It is noteworthy that level of ACSL4 is not significant between DCS group and DCS + BM-MSCs, which hint BM-MSCs protect ferroptosis by augment content of GPX4 instead of diminish ACSL4 production. The study’s exploration of BM-MSC-mediated modulation of pyroptosis and ferroptosis in decompression sickness (DCS)-induced lung injury represents a significant advancement in the field of diving medicine and regenerative therapy. Here’s how this approach enhances scientific understanding and clinical translation:1) comprehensive analysis of cross-talk between cell death pathways in DCS. While previous studies on DCS focused primarily on oxidative stress or inflammation, this work systematically links mitochondrial ROS overproduction to the activation of multiple cell death mechanisms. For example: pyroptosis (NLRP3/Caspase-1) was identified as a dominant pathway in severe lung injury, correlating with inflammation. Ferroptosis (GPX4/ACSL4) emerged as a novel contributor to lipid peroxidation, exacerbating tissue damage during rapid decompression. This dual-pathway inhibition by BM-MSCs demonstrates their pleiotropic protective effects, surpassing single-target therapies. 2) Mechanistic synergy between apoptosis and necroptosis. The study reveals that BM-MSCs not only suppress pyroptosis but also disrupt Ferroptosis. This dual action prevents both programmed and inflammatory cell death, addressing a critical gap in DCS research where prior therapies targeted only isolated pathways. Notably, the crosstalk between these pathways amplifies lung injury, making combined inhibition essential for effective treatment. 3) Clinical relevance of multi-pathway targeting. DCS-induced lung injury involves heterogeneous cellular responses, necessitating therapies with broad-spectrum efficacy. BM-MSCs’ ability to: Attenuate pyrotosis -driven inflammation. Rescue Ferroptosis-mediated lipid peroxidation validates their potential as a “pan-cell death” therapeutic agent. This is particularly impactful for SCUBA divers, which rapid decompression triggers multifaceted pathology.

Our ﬁndings demonstrate the eﬃcacy of BM-MSCs as a preventive potential. However, we have to acknowledge certain limitations of present work. First, as the initiation of DCS is obscure and hard to observe clinically. When signs and symptoms of DCS can be detected, it may be diﬃcult to stop the development of DCS. Whether BM-MSCs has therapeutic beneﬁt when DCS is already developed is not clear. A further limitation is current study demonstrates BM-MSCs improve DCS induced acute lung injury, whether DCS could induced chronic lung injury and BM-MSCs has same effects on that need further research to elucidate.

Conclusion

Our results suggested that BM-MSCs preventive potential in rats subjected to DCS induced acute lung injury. Furthermore, histopathological evaluation suggested that BM-MSCs markedly improving lung injury compared with without given BM-MSCs. In current study we found that BM-MSCs appears to ameliorate DCS induced lung injury via reducing Pyroptosis, attenuating Ferroptosis, lessening oxidative stress and alleviating inflammation. The mechanisms of how BM-MSCs alleviate decompression sickness induced acute lung injury are presented in Fig 9. Prophylactic use limits clinical translation; therapeutic trials are needed post-DCS onset.

Supporting information

S1 File. Raw Data.

(XLSX)

pone.0326618.s001.xlsx^{(20.7KB, xlsx)}

S2 File. Original uncropped blot.

(TIF)

pone.0326618.s002.tif^{(64.5MB, tif)}

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The present work was supported by the grants CLB19J038 from Military Logistics Research Program China and recipient of the grants is Xianliang Lin.

References

1.Mahon RT, Regis DP. Decompression and decompression sickness. Compr Physiol. 2014;4(3):1157–75. doi: 10.1002/cphy.c130039 [DOI] [PubMed] [Google Scholar]
2.Mazur A, Guernec A, Lautridou J, Dupas J, Dugrenot E, Belhomme M, et al. Angiotensin Converting Enzyme Inhibitor Has a Protective Effect on Decompression Sickness in Rats. Front Physiol. 2018;9:64. doi: 10.3389/fphys.2018.00064 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Yu X, Xu J, Liu W, Zhang Z, He C, Xu W. Protective effects of pulmonary surfactant on decompression sickness in rats. J Appl Physiol (1985). 2021;130(2):400–7. [DOI] [PubMed] [Google Scholar]
4.Mooi W, Smith P, Heath D. The ultrastructural effects of acute decompression on the lung of rats: the influence of frusemide. J Pathol. 1978;126(4):189–96. [DOI] [PubMed] [Google Scholar]
5.Tang S-E, Liao W-I, Wu S-Y, Pao H-P, Huang K-L, Chu S-J. The Blockade of Store-Operated Calcium Channels Improves Decompression Sickness in Rats. Front Physiol. 2020;10:1616. doi: 10.3389/fphys.2019.01616 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Huang KL, Wu CP, Chen YL, Kang BH, Lin YC. Heat stress attenuates air bubble-induced acute lung injury: a novel mechanism of diving acclimatization. J Appl Physiol (1985). 2003;94(4):1485–90. [DOI] [PubMed] [Google Scholar]
7.Magri K, Eftedal I, Petroni Magri V, Matity L, Azzopardi CP, Muscat S, et al. Acute Effects on the Human Peripheral Blood Transcriptome of Decompression Sickness Secondary to Scuba Diving. Front Physiol. 2021;12:660402. doi: 10.3389/fphys.2021.660402 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol. 2006;72(11):1493–505. doi: 10.1016/j.bcp.2006.04.011 [DOI] [PubMed] [Google Scholar]
9.Wang Q, Mazur A, Guerrero F, Lambrechts K, Buzzacott P, Belhomme M, et al. Antioxidants, endothelial dysfunction, and DCS: in vitro and in vivo study. J Appl Physiol (1985). 2015;119(12):1355–62. doi: 10.1152/japplphysiol.00167.2015 [DOI] [PubMed] [Google Scholar]
10.Wang Q, Guerrero F, Mazur A, Lambrechts K, Buzzacott P, Belhomme M, et al. Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives. Med Sci Sports Exerc. 2015;47(7):1362–71. doi: 10.1249/MSS.0000000000000563 [DOI] [PubMed] [Google Scholar]
11.Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 2014;94(3):909–50. doi: 10.1152/physrev.00026.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med. 2000;192(7):1001–14. doi: 10.1084/jem.192.7.1001 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Ma K, Chen G, Li W, Kepp O, Zhu Y, Chen Q. Mitophagy, Mitochondrial Homeostasis, and Cell Fate. Front Cell Dev Biol. 2020;8:467. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Li Y, Xu X, Bao J, Wang W. Effects of hyperbaric oxygen pretreatment on brain antioxidant capacity in rats with decompression sickness. Undersea Hyperb Med. 2021;48(3):287–95. [PubMed] [Google Scholar]
15.Armitage JD, Tan DBA, Sturm M, Moodley YP. Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions. Stem Cells Transl Med. 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Mei SHJ, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med. 2007;4(9):e269. doi: 10.1371/journal.pmed.0040269 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Du W-J, Reppel L, Leger L, Schenowitz C, Huselstein C, Bensoussan D, et al. Mesenchymal Stem Cells Derived from Human Bone Marrow and Adipose Tissue Maintain Their Immunosuppressive Properties After Chondrogenic Differentiation: Role of HLA-G. Stem Cells Dev. 2016;25(19):1454–69. doi: 10.1089/scd.2016.0022 [DOI] [PubMed] [Google Scholar]
18.Wilson JG, Liu KD, Zhuo H, Caballero L, McMillan M, Fang X, et al. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet Respir Med. 2015;3(1):24–32. doi: 10.1016/S2213-2600(14)70291-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.McIntyre LA, Stewart DJ, Mei SHJ, Courtman D, Watpool I, Granton J, et al. Cellular Immunotherapy for Septic Shock. A Phase I Clinical Trial. Am J Respir Crit Care Med. 2018;197(3):337–47. doi: 10.1164/rccm.201705-1006OC [DOI] [PubMed] [Google Scholar]
20.Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003;100(14):8407–11. doi: 10.1073/pnas.1432929100 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Gu W, Song L, Li X-M, Wang D, Guo X-J, Xu W-G. Mesenchymal stem cells alleviate airway inflammation and emphysema in COPD through down-regulation of cyclooxygenase-2 via p38 and ERK MAPK pathways. Sci Rep. 2015;5:8733. doi: 10.1038/srep08733 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Li J, Zhou J, Zhang D, Song Y, She J, Bai C. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury. J Cell Mol Med. 2015;19(10):2341–51. doi: 10.1111/jcmm.12638 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Khamis T, Abdelkhalek A, Abdellatif H, Dwidar N, Said A, Ahmed R, et al. BM-MSCs alleviate diabetic nephropathy in male rats by regulating ER stress, oxidative stress, inflammation, and apoptotic pathways. Front Pharmacol. 2023;14:1265230. doi: 10.3389/fphar.2023.1265230 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Chou H-C, Lin W, Chen C-M. Human mesenchymal stem cells attenuate pulmonary hypertension induced by prenatal lipopolysaccharide treatment in rats. Clin Exp Pharmacol Physiol. 2016;43(10):906–14. doi: 10.1111/1440-1681.12604 [DOI] [PubMed] [Google Scholar]
25.Zhang X, Zhang Z, Ju M, Li J, Jing Y, Zhao Y. Pretreatment with interleukin 35-engineered mesenchymal stem cells protected against lipopolysaccharide-induced acute lung injury via pulmonary inflammation suppression. Inflammopharmacology. 2020;28(5):1269–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Zhang Z, Liang D, Gao X, Zhao C, Qin X, Xu Y, et al. Selective inhibition of inositol hexakisphosphate kinases (IP6Ks) enhances mesenchymal stem cell engraftment and improves therapeutic efficacy for myocardial infarction. Basic Res Cardiol. 2014;109(4):417. doi: 10.1007/s00395-014-0417-x [DOI] [PubMed] [Google Scholar]
27.Shao D, Wang C, Sun Y, Cui L. Effects of oral implants with miR‑122‑modified cell sheets on rat bone marrow mesenchymal stem cells. Mol Med Rep. 2018;17(1):1537–44. doi: 10.3892/mmr.2017.8094 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Huang Y-Z, Cai J-Q, Lv F-J, Xie H-L, Yang Z-M, Huang Y-C, et al. Species variation in the spontaneous calcification of bone marrow-derived mesenchymal stem cells. Cytotherapy. 2013;15(3):323–9. doi: 10.1016/j.jcyt.2012.11.011 [DOI] [PubMed] [Google Scholar]
29.Li J, Li D, Liu X, Tang S, Wei F. Human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate LPS-induced acute lung injury in rats. J Inflamm (Lond). 2012;9(1):33. doi: 10.1186/1476-9255-9-33 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Mikawa K, Nishina K, Takao Y, Obara H. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg. 2003;97(6):1751–5. doi: 10.1213/01.ANE.0000086896.90343.13 [DOI] [PubMed] [Google Scholar]
31.Fang D, Wang Y, Zhang Z, Yang D, Gu D, He B, et al. Calorie Restriction Protects against Contrast-Induced Nephropathy via SIRT1/GPX4 Activation. Oxid Med Cell Longev. 2021;2021:2999296. doi: 10.1155/2021/2999296 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Bigley NJ, Perymon H, Bowman GC, Hull BE, Stills HF, Henderson RA. Inflammatory cytokines and cell adhesion molecules in a rat model of decompression sickness. J Interferon Cytokine Res. 2008;28(2):55–63. [DOI] [PubMed] [Google Scholar]
33.Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J. 2000;16(3):534–54. doi: 10.1034/j.1399-3003.2000.016003534.x [DOI] [PubMed] [Google Scholar]
34.Greer HD, Massey EW. Neurologic injury from undersea diving. Neurol Clin. 1992;10(4):1031–45. [PubMed] [Google Scholar]
35.Vann RD, Butler FK, Mitchell SJ, Moon RE. Decompression illness. Lancet. 2011;377(9760):153–64. doi: 10.1016/S0140-6736(10)61085-9 [DOI] [PubMed] [Google Scholar]
36.Gernhardt ML, Conkin J, Foster PP, Pilmanis AA, Butler BD, Beltran E. Design and testing of a 2-hour oxygen prebreathe protocol for space walks from the International Space Station. 2000.
37.Gerth WA, Vann RD. Probabilistic gas and bubble dynamics models of decompression sickness occurrence in air and nitrogen-oxygen diving. Undersea Hyperb Med. 1997;24(4):275–92. [PubMed] [Google Scholar]
38.Gutvik CR, Johansen TA, Brubakk AO. Applications of control: Optimal decompression of divers - Procedures for constraining predicted bubble growth. IEEE Control Systems. 2011;2011(1):31. [Google Scholar]
39.Li X, Zhang Y, Yeung SC, Liang Y, Liang X, Ding Y, et al. Mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smoke-induced damage. Am J Respir Cell Mol Biol. 2014;51(3):455–65. doi: 10.1165/rcmb.2013-0529OC [DOI] [PubMed] [Google Scholar]
40.Li Y, Shen Z, Jiang X, Wang Y, Yang Z, Mao Y, et al. Mouse mesenchymal stem cell-derived exosomal miR-466f-3p reverses EMT process through inhibiting AKT/GSK3β pathway via c-MET in radiation-induced lung injury. J Exp Clin Cancer Res. 2022;41(1):128. doi: 10.1186/s13046-022-02351-z [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Valle-Prieto A, Conget PA. Human mesenchymal stem cells efficiently manage oxidative stress. Stem Cells Dev. 2010;19(12):1885–93. doi: 10.1089/scd.2010.0093 [DOI] [PubMed] [Google Scholar]
42.Jezierska-Wozniak K, Sinderewicz E, Czelejewska W, Wojtacha P, Barczewska M, Maksymowicz W. Influence of Bone Marrow-Derived Mesenchymal Stem Cell Therapy on Oxidative Stress Intensity in Minimally Conscious State Patients. J Clin Med. 2020;9(3):683. doi: 10.3390/jcm9030683 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Xu J, Ren Z, Niu T, Li S. Epigenetic mechanism of miR-26b-5p-enriched MSCs-EVs attenuates spinal cord injury. Regen Ther. 2023;25:35–48. doi: 10.1016/j.reth.2023.10.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.El-Tantawy WH. Therapeutic effects of stem cell on hyperglycemia, hyperlipidemia, and oxidative stress in alloxan-treated rats. Mol Cell Biochem. 2014;391(1–2):193–200. [DOI] [PubMed] [Google Scholar]
45.Li X-H, Huang P, Cheng H-P, Zhou Y, Feng D-D, Yue S-J, et al. NMDAR activation attenuates the protective effect of BM-MSCs on bleomycin-induced ALI via the COX-2/PGE2 pathway. Heliyon. 2023;10(1):e23723. doi: 10.1016/j.heliyon.2023.e23723 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Jakubczyk K, Dec K, Kałduńska J, Kawczuga D, Kochman J, Janda K. Reactive oxygen species - sources, functions, oxidative damage. Pol Merkur Lekarski. 2020;48(284):124–7. [PubMed] [Google Scholar]
47.Zhao C, Chen J-Y, Peng W-M, Yuan B, Bi Q, Xu Y-J. Exosomes from adipose‑derived stem cells promote chondrogenesis and suppress inflammation by upregulating miR‑145 and miR‑221. Mol Med Rep. 2020;21(4):1881–9. doi: 10.3892/mmr.2020.10982 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Németh K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15(1):42–9. doi: 10.1038/nm.1905 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Kavanagh H, Mahon BP. Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells. Allergy. 2011;66(4):523–31. doi: 10.1111/j.1398-9995.2010.02509.x [DOI] [PubMed] [Google Scholar]
50.Ni XX, Ni M, Fan DF, Sun Q, Kang ZM, Cai ZY. Heat-shock protein 70 is involved in hyperbaric oxygen preconditioning on decompression sickness in rats. Exp Biol Med (Maywood). 2013;238(1):12–22. [DOI] [PubMed] [Google Scholar]
51.Li X, Zhang Y, Liang Y, Cui Y, Yeung SC, Ip MSM, et al. iPSC-derived mesenchymal stem cells exert SCF-dependent recovery of cigarette smoke-induced apoptosis/proliferation imbalance in airway cells. J Cell Mol Med. 2017;21(2):265–77. doi: 10.1111/jcmm.12962 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Khoury MK, Gupta K, Franco SR, Liu B. Necroptosis in the Pathophysiology of Disease. Am J Pathol. 2020;190(2):272–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Yang H-H, Jiang H-L, Tao J-H, Zhang C-Y, Xiong J-B, Yang J-T, et al. Mitochondrial citrate accumulation drives alveolar epithelial cell necroptosis in lipopolysaccharide-induced acute lung injury. Exp Mol Med. 2022;54(11):2077–91. doi: 10.1038/s12276-022-00889-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Ling X, Zhou J, Jin T, Xu W, Sun X, Li W, et al. Acteoside attenuates RSV-induced lung injury by suppressing necroptosis and regulating metabolism. Front Pharmacol. 2022;13:870928. doi: 10.3389/fphar.2022.870928 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Dong L, Liang F, Lou Z, Li Y, Li J, Chen Y, et al. Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells. Cells. 2022;11(19):3139. doi: 10.3390/cells11193139 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Lyons JD, Mandal P, Otani S, Chihade DB, Easley KF, Swift DA, et al. The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia. Am J Respir Cell Mol Biol. 2023;68(2):150–60. doi: 10.1165/rcmb.2021-0474OC [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem Sci. 2017;42(4):245–54. doi: 10.1016/j.tibs.2016.10.004 [DOI] [PubMed] [Google Scholar]
58.Danelishvili L, Bermudez LE. Analysis of pyroptosis in bacterial infection. Methods Mol Biol. 2013;1004:67–73. [DOI] [PubMed] [Google Scholar]
59.Xu J, Zhang M, Liu F, Shi L, Jiang X, Chen C, et al. Mesenchymal Stem Cells Alleviate Post-resuscitation Cardiac and Cerebral Injuries by Inhibiting Cell Pyroptosis and Ferroptosis in a Swine Model of Cardiac Arrest. Front Pharmacol. 2021;12:793829. doi: 10.3389/fphar.2021.793829 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Xu S, He Y, Lin L, Chen P, Chen M, Zhang S. The emerging role of ferroptosis in intestinal disease. Cell Death Dis. 2021;12(4):289. doi: 10.1038/s41419-021-03559-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Yang X, Yu Y, Li B, Chen Y, Feng M, Hu Y, et al. Bone marrow mesenchymal stem cell-derived exosomes protect podocytes from HBx-induced ferroptosis. PeerJ. 2023;11:e15314. doi: 10.7717/peerj.15314 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Yao S, Pang M, Wang Y, Wang X, Lin Y, Lv Y, et al. Mesenchymal stem cell attenuates spinal cord injury by inhibiting mitochondrial quality control-associated neuronal ferroptosis. Redox Biol. 2023;67:102871. doi: 10.1016/j.redox.2023.102871 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Liu Y, Wan Y, Jiang Y, Zhang L, Cheng W. GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment. Biochim Biophys Acta Rev Cancer. 2023;1878(3):188890. doi: 10.1016/j.bbcan.2023.188890 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0326618.r001

Decision Letter 0

Roland Eghoghosoa Akhigbe

4 Feb 2025

PONE-D-24-56639BM-MSCs ameliorate lung injury in rat decompression sickness modelPLOS ONE

Dear Dr. Lin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Roland Eghoghosoa Akhigbe

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for your submission to PLOS ONE. We note that your study design may include death of a regulated animal as a likely outcome or planned experimental endpoint. At this time, we request that you please report additional details in your Methods section regarding animal care and use for the survival study, as per our editorial guidelines (http://journals.plos.org/plosone/s/submission-guidelines#loc-humane-endpoints ).

For easy reference, we have attached a checklist that may be relevant for your submission. Please complete all items on the checklist at the following link: http://journals.plos.org/plosone/s/file?id=bb1d/plos-one-humane-endpoints-checklist.docx

Please upload the completed checklist as file type “Other” when resubmitting your manuscript. This document is for internal journal use only and will not be published if your article is accepted. We very much appreciate your attention to these requests and support of improved reporting standards in PLOS ONE submissions.

3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.01616/full

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

4. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, and (2) efforts to alleviate suffering.

5. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

8. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files . When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels .

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General Evaluation

1- Is the manuscript technically sound, and do the data support the conclusions?

Yes, the manuscript is technically sound, and the data support the conclusions. However, certain aspects of the discussion need further expansion to strengthen the alignment between the data and conclusions, particularly regarding mechanistic insights.

2- Have the authors made all data underlying the findings in their manuscript fully available?

The manuscript does not explicitly state whether all data are fully available. The authors should provide a clear data availability statement and ensure access to underlying datasets (e.g., oxidative stress markers, W/D ratios, and BALF protein levels) through a public repository or supplementary materials.

3- Has the statistical analysis been performed appropriately and rigorously?

The statistical analysis is appropriate overall but lacks sufficient detail and rigor in reporting. Confidence intervals, effect sizes, and adjustments for multiple comparisons should be included to enhance the statistical transparency and reproducibility.

4- Is the manuscript presented in an intelligible fashion and written in standard English?

Yes, the manuscript is intelligible and written in standard English, but it requires grammatical corrections, simplification of overly complex sentences, and explanations for technical terms to improve clarity and accessibility.

Specific Comments and Suggestions

Title and Abstract (Lines 1–20):

Line 1: The title effectively summarizes the study but could be revised for conciseness, e.g., "BM-MSCs Mitigate Lung Injury in a Rat Model of Decompression Sickness."

Line 5: Revise "is mortal disease across the worldwide" to "is a fatal disease worldwide."

Line 12: The phrase "underlying mechanisms" is too broad. Specify the mechanisms targeted (e.g., oxidative stress, inflammation).

Line 19: Include quantitative data (e.g., specific reductions in ROS or protein levels) to strengthen the abstract's impact.

Introduction (Lines 21–115):

Line 26: Avoid redundancy in describing decompression sickness. Consolidate phrases like "a systemic and mortal disease."

Line 45: The mention of mitochondrial ROS is valuable but insufficiently detailed. Explain how mitochondrial dysfunction specifically contributes to lung injury in decompression sickness.

Line 72: Provide stronger justification for using BM-MSCs by referencing prior studies demonstrating their efficacy in treating conditions involving oxidative stress and inflammation.

Methods (Lines 116–280):

Line 125: Clarify selection criteria for rats. Specify how the health and age of the animals ensure the validity of the model.

Line 145: The decompression protocol is well-described, but explain why specific decompression rates and pressures were chosen and how they reflect clinical relevance.

Line 193: Include details of the gating strategy used for flow cytometry, as this is critical for reproducibility.

Line 263: Justify the use of the Kolmogorov-Smirnov test for normality and describe how statistical assumptions were checked.

Results (Lines 281–459):

Line 309: Highlight reductions in the W/D ratio and BALF protein levels with quantitative comparisons to baseline or literature values.

Line 350: The claim that BM-MSCs "ameliorate DCS-induced lung injury" requires more specific quantitative data for support.

Line 392: While reductions in ROS are significant, discuss whether these changes are sufficient to prevent long-term oxidative damage or secondary complications.

Discussion (Lines 460–620):

Line 485: Expand the discussion on the novelty of targeting multiple cell death pathways (e.g., necroptosis, apoptosis) to highlight the study's contribution.

Line 520: Explain discrepancies in the expression levels of RIPK3 and MLKL relative to lung injury severity.

Line 583: Temper conclusions about BM-MSCs' "preventive potential" by addressing limitations, such as the lack of therapeutic data for post-DCS onset.

Figures and Tables:

Figure 1 (Line 288): Add overlays showing isotype controls to improve interpretability of flow cytometry results.

Table 2 (Line 347): Include effect sizes and confidence intervals to enhance statistical transparency.

Figure 6 (Line 432): Add a schematic summarizing the proposed mechanisms of BM-MSC action (e.g., antioxidant, anti-inflammatory effects).

Language and Style:

Simplify overly complex sentences and clarify technical jargon. For example:

Replace "burst of ROS" with "a rapid increase in reactive oxygen species (ROS)."

Revise "it arouses researchers' attention" to "it has garnered significant attention."

Strengths of the Study

Innovative Application: The study explores BM-MSCs' effects on decompression sickness, bridging cellular therapy and diving medicine.

Comprehensive Analysis: The study evaluates multiple pathways (oxidative stress, inflammation, cell death) for a holistic understanding of BM-MSC effects.

Robust Methodology: The experimental design, including well-characterized BM-MSCs and multiple time points, enhances the reliability of findings.

Suggested Revisions

Enhance Statistical Reporting: Include confidence intervals, effect sizes, and corrections for multiple comparisons to improve transparency and rigor.

Expand Mechanistic Insights: Provide a more detailed explanation of BM-MSCs' effects on specific pathways, particularly oxidative stress and necroptosis.

Improve Visual Representation: Add schematic diagrams summarizing the mechanisms and interventions.

Address Limitations: Explicitly acknowledge limitations, such as the lack of therapeutic data for post-DCS onset and the absence of long-term validation.

Strengthen Language: Refine grammar and clarify technical terms to improve readability and accessibility.

Reviewer #2: Comments and Suggestions for Authors

The submitted manuscript explores a well-defined research topic: BM-MSCs ameliorate lung injury in rat decompression sickness model. This study represents a significant contribution to the existing body of knowledge and addresses a critical need in the field. The findings hold promise as an educational resource for understanding and managing life-threatening conditions, particularly given the rising incidence of such cases among SCUBA divers. The manuscript is well-structured, adhering to some format for original research articles and encompassing all requisite sections. While several sections are thoroughly developed, certain areas could benefit from minor revisions. Detailed comments on specific sections of the manuscript are provided below.

Abstract

1. Your abstract effectively highlights the key points; however, it would benefit from a more detailed explanation while remaining within the required word count.

2. Although PLOS ONE's guidelines may not explicitly address certain aspects of manuscript formatting, authors are strongly encouraged to maintain a high level of professionalism in their writing. In particular, the methodology, results, and conclusion sections should be excluded from this abstract.

Introduction

1. As previously noted in the abstract, line numbers are missing from the manuscript. Including line numbers is essential to facilitate reviewers in providing precise comments and suggestions for corrections. Kindly ensure that line numbers are added to the manuscript.

2. Kindly ensure that the entire manuscript is fully justified to enhance its presentation and align with professional formatting standards. This adjustment will improve readability and contribute to a polished and professional appearance.

Methodology

1. Subheadings should not be numbered. Authors are strongly encouraged to adhere to the prescribed guidelines to ensure their work aligns with professional standards, as this greatly enhances the credibility and presentation of their scientific contributions.

Results

1. The experimental setup indicates that 250 rats were used. Could the authors clarify how many mortalities were recorded during the experiment? Additionally, while the number of animals used is not excessive, it raises questions about compliance with ethical guidelines for animal research. Can the authors confirm that this quantity and the procedures used align with animal welfare regulations in China?

2. Are there alternative pro-inflammatory cytokines, chemokine genes, or pathways that could have been explored beyond those mentioned in the study? Could the authors clarify the rationale behind selecting these specific targets? Understanding the reasoning behind this choice would help address any curiosity regarding the focus of the study.

Discussion

1. During the model research, it was observed that mortality and incidence are strongly correlated with pressure and decompression rates. To the best of our knowledge, this is the first study to investigate the relationship between mortality and pressure rates. Could the authors specify the country or region where this study was conducted to provide better context and relevance?

References

1. The references in the manuscript are poorly formatted and do not meet the standards of typical scientific professionalism. Authors are advised to thoroughly review and restructure the references to ensure they comply with the required formatting style and reflect the expected rigor of scientific writing.

Reviewer #3: BM-MSCs ameliorate lung injury in rat decompression sickness model, a study done by Chen Lu et. al., is interesting and might appeal to the larger audience.

Comments:

Abstract:

1. Expand W/D (appearing for the first time)

Methods

2. DCS model: "the rats in DCS+BM-MSCs 1d group were treated with BM-MSCs (2×106cells, 200μL, saline as vehicle, tail vein injection) 1 hour (h) before DCS modeling". Is it 1 hour (h) before or 1 day before?

3. 2.8 Assay of oxidative stress and inflammatory markers: "the tissue homogenate was used to detect the myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase(SOD), glutathione (GSH), catalase (CAT) activities by commercial reagent kits". Did the authors estimate the amount of GSH or the enzyme activity? If enzyme, please mention the enzyme name.

Results:

4. 3.1 Identification of BM-MSCs: "After osteogenic induction, alizarin red staining showed that calcium-rich extracellular matrix in the cells; after chondrogenic induction, cells were stained with Alcian blue; after adipogenic induction, oil Red O staining showed that the red lipid droplets were distributed in and between the cells (Fig. 1C-E)" Please explain the result observed with Alcian blue staining!

5. No description about Fig. 1B in the results?

6. "To explore the suitable condition for the DCS rat model, we distributed rats into three groups (Fig. 2A)" Section no. is missing. If correct it should be 3.2

7. The survival analysis showed that DCS significantly reduced the median survivaltime, but pretreatment with BM-MSCs reversed it (Fig. 2E). In addition, BM-MSCs coulddecrease the incidence of DCS (Fig. 2F). Please reverse the figure number in the text or reverse the figures accordingly.

8. 3.4 Pretreatment of BM-MSCs attenuates DCS induced lung inflammation : "We found thatBM-MSCs ameliorated the increased serum levels of inflammation-related genes in DCSgroup rats (Fig. 4D-F)" inflammation-related genes or cytokines?

9. 3.5 Pretreatment of BM-MSCs inhibits oxidative stress in DCS rats: "Compared to the Vehicle group rats, the lung ROS production increased in DCS group rats,while BM-MSCs decreased the ROS production induced by decompression (Fig. 5A)" Include figure 5B. as (Fig. 5A-B)

10. Fig. 5. :GSHactivities in different group. (E)" GSHlevels in different groups. (E)?

11. 3.6 The effect of BM-MSCs on different types of lung cell death in DCS rats: "pyroptosis (RIPK3 and MLKL, Fig. 6D-E), necroptosis (caspase-1 and NLRP3, Fig. 6FG)" Reverse the process for the corresponding markers. Like necroptosis (RIPK3 and MLKL, Fig. 6D-E)

12. 3.6 The effect of BM-MSCs on different types of lung cell death in DCS rats : "In this experiment, wedetected the protein expression of the markers for apoptosis (cleaved caspase-3 and Bcl-2, Fig.6B-C), pyroptosis (RIPK3 and MLKL, Fig. 6D-E), necroptosis (caspase-1 and NLRP3, Fig. 6FG) and ferroptosis (GPX4 and ACSL4, Fig. 6H-I) in lung tissues in different groups" Describe the results of DCS+BM-MSC in the result section.

Figures:

13. Figure 1A: What is the negative control/staining control/gating control used for gating the positive cells for the corresponding marker by flow cytometry?

14. Figure 1B. Please include the Scale bar for the microscopic pictures.

15. Figure 3D. Please label the observed histological changes in the microscopic pictures

16. Figure 5D. Please change the label GSH activities?

17. Figure 6A. Change aspase-1 to Caspase-1 in the western blot

Discission:

18. "BM-MSCsscavenge overproduction reactive oxygen species[40], and increase the content of anti-oxidativeenzyme, for example SOD [41], CAT [39], and GSH[42]," "anti-oxidant enzymes (such as SOD, CAT and GSH)" is the author referring levels of GSH or they referring GSH as enzyme?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes: Jonah Bawa Adokwe

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: PLOS ONE COMMENTS FOR AUTHORS 3.docx

pone.0326618.s003.docx^{(16.2KB, docx)}

PLoS One. 2025 Aug 21;20(8):e0326618. doi: 10.1371/journal.pone.0326618.r002

Author response to Decision Letter 1

12 Apr 2025

Reviewer #1

Reviewer #2 Jonah Bawa Adokwe

Reviewer #3

Attachment

Submitted filename: Response to Reviewers.docx

pone.0326618.s004.docx^{(39KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0326618.r003

Decision Letter 1

Roland Eghoghosoa Akhigbe

3 Jun 2025

BM-MSCs Mitigate Lung Injury in a Rat Model of Decompression Sickness

PONE-D-24-56639R1

Dear Dr. Lin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Roland Eghoghosoa Akhigbe

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: Given the thorough revisions and the authors’ responsiveness to feedback, I believe the manuscript meets the journal’s standards for publication.

Thank you for the opportunity to assess this work. Please don’t hesitate to contact me if any additional evaluation is required.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: Yes: Jonah Bawa Adokwe Ph.D

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0326618.r004

Acceptance letter

Roland Eghoghosoa Akhigbe

PONE-D-24-56639R1

PLOS ONE

Dear Dr. Lin,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Roland Eghoghosoa Akhigbe

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Raw Data.

(XLSX)

pone.0326618.s001.xlsx^{(20.7KB, xlsx)}

S2 File. Original uncropped blot.

(TIF)

pone.0326618.s002.tif^{(64.5MB, tif)}

Attachment

Submitted filename: PLOS ONE COMMENTS FOR AUTHORS 3.docx

pone.0326618.s003.docx^{(16.2KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0326618.s004.docx^{(39KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

[pone.0326618.ref001] 1.Mahon RT, Regis DP. Decompression and decompression sickness. Compr Physiol. 2014;4(3):1157–75. doi: 10.1002/cphy.c130039 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref002] 2.Mazur A, Guernec A, Lautridou J, Dupas J, Dugrenot E, Belhomme M, et al. Angiotensin Converting Enzyme Inhibitor Has a Protective Effect on Decompression Sickness in Rats. Front Physiol. 2018;9:64. doi: 10.3389/fphys.2018.00064 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref003] 3.Yu X, Xu J, Liu W, Zhang Z, He C, Xu W. Protective effects of pulmonary surfactant on decompression sickness in rats. J Appl Physiol (1985). 2021;130(2):400–7. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref004] 4.Mooi W, Smith P, Heath D. The ultrastructural effects of acute decompression on the lung of rats: the influence of frusemide. J Pathol. 1978;126(4):189–96. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref005] 5.Tang S-E, Liao W-I, Wu S-Y, Pao H-P, Huang K-L, Chu S-J. The Blockade of Store-Operated Calcium Channels Improves Decompression Sickness in Rats. Front Physiol. 2020;10:1616. doi: 10.3389/fphys.2019.01616 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref006] 6.Huang KL, Wu CP, Chen YL, Kang BH, Lin YC. Heat stress attenuates air bubble-induced acute lung injury: a novel mechanism of diving acclimatization. J Appl Physiol (1985). 2003;94(4):1485–90. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref007] 7.Magri K, Eftedal I, Petroni Magri V, Matity L, Azzopardi CP, Muscat S, et al. Acute Effects on the Human Peripheral Blood Transcriptome of Decompression Sickness Secondary to Scuba Diving. Front Physiol. 2021;12:660402. doi: 10.3389/fphys.2021.660402 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref008] 8.Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol. 2006;72(11):1493–505. doi: 10.1016/j.bcp.2006.04.011 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref009] 9.Wang Q, Mazur A, Guerrero F, Lambrechts K, Buzzacott P, Belhomme M, et al. Antioxidants, endothelial dysfunction, and DCS: in vitro and in vivo study. J Appl Physiol (1985). 2015;119(12):1355–62. doi: 10.1152/japplphysiol.00167.2015 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref010] 10.Wang Q, Guerrero F, Mazur A, Lambrechts K, Buzzacott P, Belhomme M, et al. Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives. Med Sci Sports Exerc. 2015;47(7):1362–71. doi: 10.1249/MSS.0000000000000563 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref011] 11.Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 2014;94(3):909–50. doi: 10.1152/physrev.00026.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref012] 12.Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med. 2000;192(7):1001–14. doi: 10.1084/jem.192.7.1001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref013] 13.Ma K, Chen G, Li W, Kepp O, Zhu Y, Chen Q. Mitophagy, Mitochondrial Homeostasis, and Cell Fate. Front Cell Dev Biol. 2020;8:467. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref014] 14.Li Y, Xu X, Bao J, Wang W. Effects of hyperbaric oxygen pretreatment on brain antioxidant capacity in rats with decompression sickness. Undersea Hyperb Med. 2021;48(3):287–95. [PubMed] [Google Scholar]

[pone.0326618.ref015] 15.Armitage JD, Tan DBA, Sturm M, Moodley YP. Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions. Stem Cells Transl Med. 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref016] 16.Mei SHJ, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med. 2007;4(9):e269. doi: 10.1371/journal.pmed.0040269 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref017] 17.Du W-J, Reppel L, Leger L, Schenowitz C, Huselstein C, Bensoussan D, et al. Mesenchymal Stem Cells Derived from Human Bone Marrow and Adipose Tissue Maintain Their Immunosuppressive Properties After Chondrogenic Differentiation: Role of HLA-G. Stem Cells Dev. 2016;25(19):1454–69. doi: 10.1089/scd.2016.0022 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref018] 18.Wilson JG, Liu KD, Zhuo H, Caballero L, McMillan M, Fang X, et al. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet Respir Med. 2015;3(1):24–32. doi: 10.1016/S2213-2600(14)70291-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref019] 19.McIntyre LA, Stewart DJ, Mei SHJ, Courtman D, Watpool I, Granton J, et al. Cellular Immunotherapy for Septic Shock. A Phase I Clinical Trial. Am J Respir Crit Care Med. 2018;197(3):337–47. doi: 10.1164/rccm.201705-1006OC [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref020] 20.Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003;100(14):8407–11. doi: 10.1073/pnas.1432929100 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref021] 21.Gu W, Song L, Li X-M, Wang D, Guo X-J, Xu W-G. Mesenchymal stem cells alleviate airway inflammation and emphysema in COPD through down-regulation of cyclooxygenase-2 via p38 and ERK MAPK pathways. Sci Rep. 2015;5:8733. doi: 10.1038/srep08733 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref022] 22.Li J, Zhou J, Zhang D, Song Y, She J, Bai C. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury. J Cell Mol Med. 2015;19(10):2341–51. doi: 10.1111/jcmm.12638 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref023] 23.Khamis T, Abdelkhalek A, Abdellatif H, Dwidar N, Said A, Ahmed R, et al. BM-MSCs alleviate diabetic nephropathy in male rats by regulating ER stress, oxidative stress, inflammation, and apoptotic pathways. Front Pharmacol. 2023;14:1265230. doi: 10.3389/fphar.2023.1265230 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref024] 24.Chou H-C, Lin W, Chen C-M. Human mesenchymal stem cells attenuate pulmonary hypertension induced by prenatal lipopolysaccharide treatment in rats. Clin Exp Pharmacol Physiol. 2016;43(10):906–14. doi: 10.1111/1440-1681.12604 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref025] 25.Zhang X, Zhang Z, Ju M, Li J, Jing Y, Zhao Y. Pretreatment with interleukin 35-engineered mesenchymal stem cells protected against lipopolysaccharide-induced acute lung injury via pulmonary inflammation suppression. Inflammopharmacology. 2020;28(5):1269–81. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref026] 26.Zhang Z, Liang D, Gao X, Zhao C, Qin X, Xu Y, et al. Selective inhibition of inositol hexakisphosphate kinases (IP6Ks) enhances mesenchymal stem cell engraftment and improves therapeutic efficacy for myocardial infarction. Basic Res Cardiol. 2014;109(4):417. doi: 10.1007/s00395-014-0417-x [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref027] 27.Shao D, Wang C, Sun Y, Cui L. Effects of oral implants with miR‑122‑modified cell sheets on rat bone marrow mesenchymal stem cells. Mol Med Rep. 2018;17(1):1537–44. doi: 10.3892/mmr.2017.8094 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref028] 28.Huang Y-Z, Cai J-Q, Lv F-J, Xie H-L, Yang Z-M, Huang Y-C, et al. Species variation in the spontaneous calcification of bone marrow-derived mesenchymal stem cells. Cytotherapy. 2013;15(3):323–9. doi: 10.1016/j.jcyt.2012.11.011 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref029] 29.Li J, Li D, Liu X, Tang S, Wei F. Human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate LPS-induced acute lung injury in rats. J Inflamm (Lond). 2012;9(1):33. doi: 10.1186/1476-9255-9-33 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref030] 30.Mikawa K, Nishina K, Takao Y, Obara H. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg. 2003;97(6):1751–5. doi: 10.1213/01.ANE.0000086896.90343.13 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref031] 31.Fang D, Wang Y, Zhang Z, Yang D, Gu D, He B, et al. Calorie Restriction Protects against Contrast-Induced Nephropathy via SIRT1/GPX4 Activation. Oxid Med Cell Longev. 2021;2021:2999296. doi: 10.1155/2021/2999296 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref032] 32.Bigley NJ, Perymon H, Bowman GC, Hull BE, Stills HF, Henderson RA. Inflammatory cytokines and cell adhesion molecules in a rat model of decompression sickness. J Interferon Cytokine Res. 2008;28(2):55–63. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref033] 33.Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J. 2000;16(3):534–54. doi: 10.1034/j.1399-3003.2000.016003534.x [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref034] 34.Greer HD, Massey EW. Neurologic injury from undersea diving. Neurol Clin. 1992;10(4):1031–45. [PubMed] [Google Scholar]

[pone.0326618.ref035] 35.Vann RD, Butler FK, Mitchell SJ, Moon RE. Decompression illness. Lancet. 2011;377(9760):153–64. doi: 10.1016/S0140-6736(10)61085-9 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref036] 36.Gernhardt ML, Conkin J, Foster PP, Pilmanis AA, Butler BD, Beltran E. Design and testing of a 2-hour oxygen prebreathe protocol for space walks from the International Space Station. 2000.

[pone.0326618.ref037] 37.Gerth WA, Vann RD. Probabilistic gas and bubble dynamics models of decompression sickness occurrence in air and nitrogen-oxygen diving. Undersea Hyperb Med. 1997;24(4):275–92. [PubMed] [Google Scholar]

[pone.0326618.ref038] 38.Gutvik CR, Johansen TA, Brubakk AO. Applications of control: Optimal decompression of divers - Procedures for constraining predicted bubble growth. IEEE Control Systems. 2011;2011(1):31. [Google Scholar]

[pone.0326618.ref039] 39.Li X, Zhang Y, Yeung SC, Liang Y, Liang X, Ding Y, et al. Mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smoke-induced damage. Am J Respir Cell Mol Biol. 2014;51(3):455–65. doi: 10.1165/rcmb.2013-0529OC [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref040] 40.Li Y, Shen Z, Jiang X, Wang Y, Yang Z, Mao Y, et al. Mouse mesenchymal stem cell-derived exosomal miR-466f-3p reverses EMT process through inhibiting AKT/GSK3β pathway via c-MET in radiation-induced lung injury. J Exp Clin Cancer Res. 2022;41(1):128. doi: 10.1186/s13046-022-02351-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref041] 41.Valle-Prieto A, Conget PA. Human mesenchymal stem cells efficiently manage oxidative stress. Stem Cells Dev. 2010;19(12):1885–93. doi: 10.1089/scd.2010.0093 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref042] 42.Jezierska-Wozniak K, Sinderewicz E, Czelejewska W, Wojtacha P, Barczewska M, Maksymowicz W. Influence of Bone Marrow-Derived Mesenchymal Stem Cell Therapy on Oxidative Stress Intensity in Minimally Conscious State Patients. J Clin Med. 2020;9(3):683. doi: 10.3390/jcm9030683 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref043] 43.Xu J, Ren Z, Niu T, Li S. Epigenetic mechanism of miR-26b-5p-enriched MSCs-EVs attenuates spinal cord injury. Regen Ther. 2023;25:35–48. doi: 10.1016/j.reth.2023.10.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref044] 44.El-Tantawy WH. Therapeutic effects of stem cell on hyperglycemia, hyperlipidemia, and oxidative stress in alloxan-treated rats. Mol Cell Biochem. 2014;391(1–2):193–200. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref045] 45.Li X-H, Huang P, Cheng H-P, Zhou Y, Feng D-D, Yue S-J, et al. NMDAR activation attenuates the protective effect of BM-MSCs on bleomycin-induced ALI via the COX-2/PGE2 pathway. Heliyon. 2023;10(1):e23723. doi: 10.1016/j.heliyon.2023.e23723 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref046] 46.Jakubczyk K, Dec K, Kałduńska J, Kawczuga D, Kochman J, Janda K. Reactive oxygen species - sources, functions, oxidative damage. Pol Merkur Lekarski. 2020;48(284):124–7. [PubMed] [Google Scholar]

[pone.0326618.ref047] 47.Zhao C, Chen J-Y, Peng W-M, Yuan B, Bi Q, Xu Y-J. Exosomes from adipose‑derived stem cells promote chondrogenesis and suppress inflammation by upregulating miR‑145 and miR‑221. Mol Med Rep. 2020;21(4):1881–9. doi: 10.3892/mmr.2020.10982 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref048] 48.Németh K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15(1):42–9. doi: 10.1038/nm.1905 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref049] 49.Kavanagh H, Mahon BP. Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells. Allergy. 2011;66(4):523–31. doi: 10.1111/j.1398-9995.2010.02509.x [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref050] 50.Ni XX, Ni M, Fan DF, Sun Q, Kang ZM, Cai ZY. Heat-shock protein 70 is involved in hyperbaric oxygen preconditioning on decompression sickness in rats. Exp Biol Med (Maywood). 2013;238(1):12–22. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref051] 51.Li X, Zhang Y, Liang Y, Cui Y, Yeung SC, Ip MSM, et al. iPSC-derived mesenchymal stem cells exert SCF-dependent recovery of cigarette smoke-induced apoptosis/proliferation imbalance in airway cells. J Cell Mol Med. 2017;21(2):265–77. doi: 10.1111/jcmm.12962 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref052] 52.Khoury MK, Gupta K, Franco SR, Liu B. Necroptosis in the Pathophysiology of Disease. Am J Pathol. 2020;190(2):272–85. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref053] 53.Yang H-H, Jiang H-L, Tao J-H, Zhang C-Y, Xiong J-B, Yang J-T, et al. Mitochondrial citrate accumulation drives alveolar epithelial cell necroptosis in lipopolysaccharide-induced acute lung injury. Exp Mol Med. 2022;54(11):2077–91. doi: 10.1038/s12276-022-00889-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref054] 54.Ling X, Zhou J, Jin T, Xu W, Sun X, Li W, et al. Acteoside attenuates RSV-induced lung injury by suppressing necroptosis and regulating metabolism. Front Pharmacol. 2022;13:870928. doi: 10.3389/fphar.2022.870928 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref055] 55.Dong L, Liang F, Lou Z, Li Y, Li J, Chen Y, et al. Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells. Cells. 2022;11(19):3139. doi: 10.3390/cells11193139 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref056] 56.Lyons JD, Mandal P, Otani S, Chihade DB, Easley KF, Swift DA, et al. The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia. Am J Respir Cell Mol Biol. 2023;68(2):150–60. doi: 10.1165/rcmb.2021-0474OC [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref057] 57.Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem Sci. 2017;42(4):245–54. doi: 10.1016/j.tibs.2016.10.004 [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref058] 58.Danelishvili L, Bermudez LE. Analysis of pyroptosis in bacterial infection. Methods Mol Biol. 2013;1004:67–73. [DOI] [PubMed] [Google Scholar]

[pone.0326618.ref059] 59.Xu J, Zhang M, Liu F, Shi L, Jiang X, Chen C, et al. Mesenchymal Stem Cells Alleviate Post-resuscitation Cardiac and Cerebral Injuries by Inhibiting Cell Pyroptosis and Ferroptosis in a Swine Model of Cardiac Arrest. Front Pharmacol. 2021;12:793829. doi: 10.3389/fphar.2021.793829 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref060] 60.Xu S, He Y, Lin L, Chen P, Chen M, Zhang S. The emerging role of ferroptosis in intestinal disease. Cell Death Dis. 2021;12(4):289. doi: 10.1038/s41419-021-03559-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref061] 61.Yang X, Yu Y, Li B, Chen Y, Feng M, Hu Y, et al. Bone marrow mesenchymal stem cell-derived exosomes protect podocytes from HBx-induced ferroptosis. PeerJ. 2023;11:e15314. doi: 10.7717/peerj.15314 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref062] 62.Yao S, Pang M, Wang Y, Wang X, Lin Y, Lv Y, et al. Mesenchymal stem cell attenuates spinal cord injury by inhibiting mitochondrial quality control-associated neuronal ferroptosis. Redox Biol. 2023;67:102871. doi: 10.1016/j.redox.2023.102871 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0326618.ref063] 63.Liu Y, Wan Y, Jiang Y, Zhang L, Cheng W. GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment. Biochim Biophys Acta Rev Cancer. 2023;1878(3):188890. doi: 10.1016/j.bbcan.2023.188890 [DOI] [PubMed] [Google Scholar]

PERMALINK

BM-MSCs mitigate lung injury in a rat model of decompression sickness

Chen Lu

Daqian Gu

Hao Chen

Liang Chen

Jie Chen

Yuwei Weng

Xianliang Lin

Roles

Abstract

Introduction

Methods

Animals

DCS model

Isolation and culture of BM-MSCs

Phenotypes and differentiation of BM-MSCs

Lung histopathology

Lung ROS detection

BALF analysis

Assay of oxidative stress and inflammatory markers

Immunoblotting

Statistical analysis

Results

Identification of BM-MSCs

Fig 1. Characterization of BM-MSC.

Fig 2. Isotype control overlays.

Fig 3. Identification of BM-MSCs.

Pretreatment of BM-MSCs improves the survival rate and incidence of DCS

Fig 4. Pretreatment of BM-MSCs improves the survival rate and incidence of DCS.

Pretreatment of BM-MSCs attenuates DCS induced lung injury

Fig 5. Effect of BM-MSCs in improving DCS induced lung injury.

Pretreatment of BM-MSCs attenuates DCS induced lung inflammation

Fig 6. Pretreatment of BM-MSCs attenuates DCS induced lung inflammation.

Pretreatment of BM-MSCs inhibits oxidative stress in DCS rats

Fig 7. Effects of BM-MSCs on ROS generation in DCS rats.

The effect of BM-MSCs on different types of lung cell death in DCS rats

Fig 8. Effects of BM-MSCs on different types of lung cell death in different group.

Discussion

Conclusion

Fig 9. A schematic representation of how BM-MSCs reduce inflammation, oxidative stress, and cell death to alleviate decompression induced acute lung injury.

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Roland Eghoghosoa Akhigbe

Roles

Author response to Decision Letter 1

Decision Letter 1

Roland Eghoghosoa Akhigbe

Roles

Acceptance letter

Roland Eghoghosoa Akhigbe

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases