Comparison of bone marrow stem cells and cell-derived exosomes on oleic acid-induced lung injury in rats

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RESEARCH ARTICLE

Hippokratia 2024, 28(3): 100-108

Önsöz E1, Ayar G1, Üstük HS2, Bahar L1, İnce B1, Köksel MO2, Maytalman E3, Büyükafşar K1,4
1Departments of Stem Cell and Regenerative Medicine, Health Science Institute, Mersin University
2Departments of Thoracic Surgery, School of Medicine, Mersin University
3Departments of Pharmacology, School of Medicine, Alaaddin Keykubat University
4Departments of Pharmacology, School of Medicine, Mersin University
Türkiye

Abstract

Background: Acute lung injury and its complication, acute respiratory distress syndrome, are significant clinical burdens that have a limited number of therapeutic approaches. Among them, stem cells seem to be a promising alternative therapy. In this study, we investigated for the first time and compared bone marrow stem cells (BMSCs) and their exosome fractions on lung injury induced by oleic acid in rats.

Methods: We administered oleic acid (60 mg/kg) by intravenous route to induce acute lung injury; thereafter, we intravenously applied rat BMSCs (1 x 106) and exosome fractions (obtained from 1 x 106 BMSCs by a commercially available kit) to the tail vein two hours after the oleic acid administration. Twenty-four hours later, the rats were sacrificed by deep anesthesia, and we obtained lung tissues. The examination of hematoxylin and eosin-stained samples was evaluated using the parameters of bleeding, leukocyte infiltration, edema, and hyperplasia. A flow cytometer verified the exosomes obtained from the stem cell culture medium.

Results: Administration of stem cells and exosome fractions after lung injury restored all those parameters; however, the regenerative capacity of BMSCs was better than its exosome fraction (p =0.004).

Conclusion: Stem cells and their exosome fractions can be regarded as therapeutic alternatives in treating oleic acid-induced lung injury in rats, being rather than exosomes their maternal source; the stem cells seem more potent. HIPPOKRATIA 2024, 28 (3):100-108. 

Keywords: Acute lung injury, oleic acid, bone marrow stem cells, bone marrow stem cell-derived exosome, acellular therapy

Corresponding author: Leyla Bahar (MD, PhD), Departments of Stem Cell and Regenerative Medicine, Health Science Institute, Mersin University, Mersin, Türkiye, tel:+903242410000 (21793), e-mail: drleylabahar33@gmail.com

Introduction

Acute respiratory distress syndrome (ARDS) is an important clinical entity that can be developed following a number of lung or systemic diseases due to lung inflammation, alveolar damage, and pulmonary edema1. At present, it is one of the fatal syndromes with no particular remedies. In experimental studies, several approaches are made to induce ARDS in animals, e.g., oleic acid, lipopolysaccharides (LPS), hyperoxia, sepsis, inhalation of harmful substances (phosgene, toluene, etc)2,3. Particularly, oleic acid is generally used in rat experimental models of ARDS. The fatty acid upregulated Rho-kinase (ROCK-1 and ROCK-2) and elevated the markers of oxidative and nitrosative stress, i.e., myeloperoxidase, malondialdehyde, nitrite/nitrate, and 3-nitro-l-tyrosine4. Furthermore, it increased interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, IL-8, and MIP-1α levels, triggering apoptotic signals, eventually resulting in the loss of lung epithelial cells, rendering the status a vicious cycle5,6.

As for the treatment of ARDS, according to the current algorithm, glucocorticoids and other anti-inflammatory drugs [e.g., non-steroidal anti-inflammatory drugs (NSAIDs)], as well as some adjunctive drugs, have been used. However, pharmacological trials have not provided a therapeutically effective strategy. The current research primarily focuses on mesenchymal stem cell therapy’s role in cell regeneration, reduction of death rate, and rapid healing of damaged tissue.

Many benefits of mesenchymal stem cells (MSCs), such as self-renewal, doubling capacity, in vitro proliferation, antiapoptotic, anti-fibrotic, anti-inflammatory, immunosuppressive, and immunomodulatory effects and paracrine nature, have been indicated in various pre-clinical studies and clinical evidence. Regarding cureless pathologies with medications, stem cells seem to be an interesting alternative due to their ability to repair tissue and organ degeneration. These cells are preferred as a therapeutic approach for a number of degenerative diseases, including respiratory system disorders. For example, the treatment of lung fibrosis can be achievable using stem cells7,8. On the other hand, MSCs (mainly bone marrow-derived) were experimentally tried in the treatment of ARDS. Accordingly, in the rat acute lung injury (ALI) model induced by LPS or bleomycin, bone marrow stem cells (BMSCs) healed lung degeneration by downregulating apoptosis-related protein expression (p-GSK-3β and Bax) and inflammatory and catastrophic cytokines such as IL-1β, IL-6, and TNF-α but upregulating of antiapoptotic protein, Bcl-2 and anti-inflammatory IL-4, and IL-10 levels9,10.

As a mode of action in healing degeneration, MSCs can induce progenitor cells and multiple trophic factors to stimulate the healing of damaged cells and induce matrix remodeling11. It has been reported that MSCs-mediated healing involves, at least in part, exosomes and some other microvesicles, the spherical vectors carrying several regenerating agents, i.e., miRNA, siRNA, ceramide, mRNA, DNA fragments and numerous factors, which take roles in healing. The use of exosomes in regenerative studies has recently been commented on more in the literature. For instance, in the ARDS model induced by LPS, MSC-derived exosomes decreased the levels of inflammatory cytokines (e.g., TNF-α, IL-1β)12. In another lung injury study induced by intestinal ischemia-reperfusion, BMSCs and their derivative exosomes were used to show regeneration in lung tissue and downregulation of the TLR4/NF-kB pathway13. It has been reported that the amount of miR-384-5p in the exosomes is critical because the microRNA seems crucial to alveolar lung regeneration. Moreover, MSCs-derived exosomes provided a protective effect against ALI induced by mustard gas in a dose-dependent manner12. The mechanism of regenerating action of MSCs has been attributed to the inhibition of proinflammatory factors (e.g., TNF-α, IL-6, and IL-8) and, on the contrary, induction of IL-10 level10,11,14.

However, to our knowledge, no studies have compared stem cells and their derivative exosomes in the rat oleic acid-induced ARDS model. Therefore, in the present study, we investigated the curative potential of BMSCs on lung injury induced by oleic acid in rats. Furthermore, we compared BMSC-derived exosomes and their maternal source, BMSCs itself, for the first time.

Materials and methods

Experimental Animals

In the study, we used 27 adult male Wistar Albino rats (220-270 g, 3-4 months old) obtained from the Mersin University Faculty of Medicine Experimental Animals Unit. The experimental protocol of the study was approved by the local ethical committee of Mersin University (2021/33), and we treated all rats humanely in accordance with the Guide for the Care and Use of Laboratory Animals. We calculated the sample size within the scope of the study using the GPower statistical package program, with Type I error, alpha set at 0.05, and the power of the test at 0.80. The effect size was 0.7, and the sample size was calculated as a minimum of six rats for each group. In order for BMSC isolation, three of the rats were sacrificed with a blow to the head (to rule out that anesthetic agents may cause physiological changes in the tissues, as confirmed by the ethical committee). The remaining 24 rats were divided into the following experimental groups.

Group 1, the Control group, consisted of six rats with alcohol and saline solution administered through the tail vein (40 µl 70 % ethyl alcohol + 260 µl saline solution).

Group 2, the Oleic acid group, consisted of six rats with an oleic acid solution administered through the tail vein [40 µl oleic acid (60 mg/kg) dissolved in 300 µl alcohol + saline solution].

Group 3, the BMSC group, consisted of six rats with BMSCs solution injected into each rat through the tail vein (1 x 106 BMSCs, dissolved in 500 µl Phosphate Buffered Saline (PBS) two hours after oleic acid [60 mg/kg, intravenous (i.v.)] administration.

Group 4, the Exosome group, consisted of six rats with exosomes obtained from 1 x 106 BMSCs, dissolved in 500 µl PBS injected into each rat through the tail vein two hours after oleic acid (60 mg/kg, i.v.) administration. 

Establishment of ARDS model with oleic acid in rats

We modeled ALI by i.v. administration of 60 mg/kg oleic acid (cis-9-octadecanoic acid). We dissolved the fatty acid in ethanol and then prepared it with saline at a final 60 mg/ml concentration4. 

Administration of stem cells and exosomes

Two hours after the application of oleic acid we administered BMSCs and its derivative exosomes in order for avoiding encounter of oleic acid with stem cells and its exosomes in the blood as oleic acid has the potential to destroy administered stem cells and exosomes. 

Obtaining stem cells from bone marrow

The femur and tibia bones of the rats sacrificed for BMSC production were removed in a sterile environment. Alpha MEM (Modification of Eagles Medium-Alpha) medium containing 10 % fetal bovine serum (FBS), penicillin, and streptomycin (100 U/mL and 0.1 mg/mL, respectively) was prepared to obtain bone marrow and to be used in cell culture. Mononuclear cells were isolated from bone marrow using the density gradient method. Mononuclear cells were seeded with one million/ml cells in each T25 flask. The primary cells passaged when cells reached approximately 80 % of the flask base and were allowed to proliferate for 6-8 days, and the passage was defined as 0 (P0). Thereafter, subsequent passages were done. Passage 3 (P3) was sufficient MSC for both direct cell and exosome administration. The P4 cells obtained after this stage were used directly in the experimental groups to which the cells were applied. Similar to the reports of Liu et al, stem cells were obtained from bone marrow in our study13.

Total exosome isolation from BMSC culture medium and exosome analysis

In passage 3, rat bone marrow-derived MSC cells were cultured with Alpha MEM containing 10 % exosome-depleted FBS, 1 % L-Glutamine, and 1 % penicillin-streptomycin15. Total exosomes were isolated from the obtained cell (1 x 106) culture medium with the Invitrogen Total Exosome Isolation kit (Catalog No: 4478359, Thermo Fisher Scientific, Waltham, Massachusetts, USA). CD9, CD63, and CD81 surface markers were labeled using MACSPlex Exosome kit (Catalog No: 130-122-209, Miltenyi Biotec, Bergisch Gladbach, Germany) and identified with flow cytometry16. 

Collecting blood and tissue samples

Twenty-four hours after administration of BMSCs or BMSC-derived exosomes with oleic acid, rats were administered ketamine/xylazine. Lungs were quickly removed and left in 10 % formaldehyde solution for histopathological examination.

Examination of lung tissue histology

After routine tissue tracing procedures of the tissues taken from the right lung of each rat, paraffin blocks were obtained and sections were taken (4-5 μm). Tissue samples were then stained with hematoxylin-eosin17. Lung tissue damage findings; It was evaluated by calculating the “Total Damage Score”, which includes the parameters i) Alveolar hyperemia-congestion, ii) mononuclear/neutrophilic infiltration, iii) perivascular-interstitial edema, and iv) cellular hyperplasia (alveolar wall thickening). Pathological findings adapted from the literature were graded in four stages: 0: normal, 1: mild, 2: moderate, and 3: severe. 

Measurement of Total Antioxidant Status (TAS) and Total Oxidant Status (TOS)

TOS in serum at the 24th hour after exosome and stem cell injection will be detected using the Erel method. We used a colorimetric test for TAS, and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) determined the antioxidant status of the samples. ABTS, used as a chromogenic agent, is a chemical compound useful for observing the reaction kinetics of specific enzymes. 

Statistical analysis

We express data as mean ± standard error of the mean; we used for statistical evaluation analysis of variance (ANOVA) and Dunn-Sidak multiple comparison tests (as a post hoc). A p-value of less than 0.05 was considered significant. 

Results

Characterization of bone marrow stem cell-derived exosomes

Characterization of bone marrow stem cell-derived exosomes was identified by flow cytometer analysis (Figure 1). As shown in Figure 1A, exosomes marked with CD 9, CD 63, and CD 81 markers in the exosome isolation kit gave signals in 101 Allophycocyanin (APC) areas. The APC 100 area was considered a negative control. Flow cytometer analysis indicates that the fraction contained ~74.5 % exosomes (Figure 1B). 

Figure 1: Images showing flow cytometry analysis of bone marrow stem cell-derived exosomes. A) Exosomes are grouped by positive and negative signal domains. B) Total exosomes show the area between cells (~74.5 % exosomes).

Effect of BMSCs and their derivatives exosomes on the lung injury by oleic acid

Applied 60 mg/kg oleic acid caused significant ALI in the rats compared to the control group. Massive bleeding, extensive infiltration, dramatic edema, and hyperplasia were all common in lung tissue sections in the oleic acid group. As for its score, oleic acid provoked three times more destruction in the lung than in vehicle-injected control sections (Figure 2A, Figure 2B). However, 1 x 106 BMSCs conspicuously restored all four parameters, i.e., bleeding, infiltration, edema, and hyperplasia (Figure 2C). In another group of experiments, exosomes derived from the BMSCs were tested, and we obtained substantial repairing effects again as with the stem cell itself. Histomorphological improvement findings were examined in the “Total Damage Score” values formed by the application of bone marrow stem cell-derived exosomes in lung tissue exposed to oleic acid. It has been observed that 1 x 106 BMSCs-derived exosomes provide significant improvement in oleic acid-induced ALI in rats (Figure 3). However, interestingly enough, the curative effects of exosomes were less pronounced compared with the maternal stem cells. A comparison of the BMSCs and their derivative exosomes is demonstrated in Figure 4. 

Figure 2: Histology images (A-C) showing histomorphological improvement findings in the “Total Damage Score” values resulting from applying bone marrow-derived mesenchymal stem cells in lung tissue exposed to oleic acid. A) Control group (Hematoxylin-Eosin stain, x 10), B) Oleic acid group (Hematoxylin-Eosin stain, x 20), and C) Oleic acid + bone marrow stem cells (Hematoxylin-Eosin stain, x 20). D) Histogram showing the therapeutic effect of bone marrow stem cells on oleic acidinduced acute lung injury.
BMSC: bone marrow stem cell, A: alveolus, AD: alveolar ductus, K: bleeding, NI: Neutrophil infiltration, ****: p<0.001, #: p<0.001. The straight line demonstrates terminal bronchiole, and the two-way arrow demonstrates edema.

Figure 3: Histology images (A-C) showing histomorphological improvement findings in the “Total Damage Score” values resulting from applying bone marrow stem cell-derived exosomes in lung tissue exposed to oleic acid. A) Control group (Hematoxylin-Eosin stain, x 10), B) Oleic acid group (Hematoxylin-Eosin stain, x 20), and C) Oleic acid + bone marrow stem cell-derived exosomes (Hematoxylin-Eosin stain, x 20). D) Histogram showing the therapeutic effect of bone marrow stem cell-derived exosomes on oleic acid-induced acute lung injury.
BMSC: bone marrow stem cell, ALI: acute lung injury, OA: oleic acid, A: alveolus, AD: alveolar ductus, K: bleeding, NI: Neutrophil infiltration, ****: p<0.001, ***: p<0.001. The straight line demonstrates terminal bronchiole, and the two-way arrow demonstrates edema.

Figure 4: Histology images (A-D) showing a comparison of histomorphological application of bone marrow stem cells and bone marrow stem cell-derived exosomes in lung tissue exposed to oleic acid. A) Control group (Hematoxylin-Eosin stain, x 10), B) Oleic acid group (Hematoxylin-Eosin stain, x 20), C) Oleic acid + bone marrow stem cells (Hematoxylin-Eosin stain, x 20), and D) Oleic acid + bone marrow stem cell-derived exosomes (Hematoxylin-Eosin stain, x 20). E) Histogram comparing the therapeutic effects of bone marrow stem cells and bone marrow stem cell-derived exosomes on oleic acid-induced acute lung injury. It was observed that 1 x 106 bone marrow stem cells administered and exosomes obtained from the same number of bone marrow stem cells significantly improved oleic acid-induced acute lung injury in rats.
BMSC: bone marrow stem cell, OA: oleic acid, A: alveolus, AD: alveolar ductus, K: bleeding, NI: Neutrophil infiltration, ***: p<0.001, &: p<0.001, a: p =0.004, #: p<0.001. The straight line demonstrates terminal bronchiole, the two-way arrow demonstrates edema, and the arrowhead demonstrates terminal bronchiole.

Data regarding TAS and TOS

We evaluated the effects of oleic acid and subsequent BMSCs application on serum TAS and TOS in rats. We found no change in the serum total oxidant stress parameter for statistical analysis (Figure 5A and Figure 5B). When we examined the effect of BMSC-derived exosomes on TAS and TOS in rats, oleic acid reduced TAS and TOS values compared to the control group. In contrast, BMSC-derived exosomes did not improve TAS suppressed by oleic acid. When we compared the effect of oleic acid and subsequent BMSC-derived exosomes in terms of TOS, we found no change between them (Figure 5C and Figure 5D). We applied ANOVA for statistical analysis and the Dunn-Sidak multiple comparison test for post-hoc testing.

Figure 5: Histograms demonstrating the effects A) of oleic acid and subsequent bone marrow stem cell application on total antioxidant status in rats and B) of oleic acid and subsequent bone marrow stem cell application on serum total oxidant status in rats. No change was found in the statistical analysis regarding the serum total oxidant stress parameter. Histograms demonstrating the effects C) of bone marrow stem cell-derived exosomes on total antioxidant status in rats and D) the effect of oleic acid and subsequent bone marrow stem cell-derived exosome application on total oxidant status in rats. No change was found
between groups.
TAS: total antioxidant status, TOS: total oxidant status, OA: oleic acid, BMSC: bone marrow stem cell, *: oleic acid reduced significantly the total antioxidant status and total oxidant status compared to the control group (p =0.02).

Statistical analysis

Flow cytometer analysis indicated that the fraction contained ~74.5 % exosomes characterizing BMSC-derived exosomes (Figure 1). In the Dunn-Sidak multiple comparison test statistical evaluation, oleic acid damage was found to be significant compared to the control group (p <0.001), and the oleic acid + BMSC group was found to be significant compared to the oleic acid group damage (p <0.001), (Figure 2). Oleic acid damage was found to be significant compared to the control group (p <0.001), and the BMSC-derived exosome significantly healed the tissue damage (p <0.001) (Figure 3). Furthermore, Figure 4 displays the comparison of the Oleic acid group to the control group (p <0.001), the BMSCs group to the Oleic acid group (p <0.001), the BMSC-derived exosome group to the Oleic acid group (p =0.002), the BMSCs group to the BMSCs-derived exosome group (p =0.004) regarding the induced ALI (Figure 4). When we evaluated the groups regarding TAS and TOS, we found no difference between BMSCs and BMSC-derived exosomes (Figure 5). 

Discussion

In this study, we aimed to compare the regenerative capacity of BMSCs and their derivative exosomes in the ALI induced by oleic acid in rats. Therefore, we applied bone-marrow-originated stem cells (1 x 106) and their derivative exosomes (harvested from 1 x 106 stem cells) after i.v. oleic acid injection to induce ALI in rats and lung specimens were evaluated using the hematoxylin-eosin examination. The exosomes were verified by flow cytometer analysis.

Mainly, MSCs are multipotent stem cells found in the umbilical cord, bone marrow, and adipose tissue. MSCs are generally preferred in regenerative medicine due to their low immunogenicity, multiple differentiation potential, high self-renewal capacity, and intense therapeutic potential18. MSCs have several distinguished properties, i.e., anti-inflammatory, less immunogenic, regenerative potentials, and easy to obtain, rendering the cells preferable in experimental and clinical research. The mode of action of stem cell-mediated regeneration is the subject of debate and is multifactorial. As mentioned above, trans-differentiation, anti-inflammatory, and immunomodulatory properties are the most possible mechanisms. However, it is also suggested that these cells have secretory properties, contributing to their regenerative capacity19. For example, MSCs migrate to the site of action during tissue repair in lung fibrosis, and the repair process is largely mediated by the extracellular vesicles they secrete. These vesicles are generally hypothesized to transfer nucleic acids (mRNAs and miRNAs), lipids, and intercellular communication proteins between cells to induce biological responses in recipient cells. BMSCs and their derivatives exosomes showed a high regenerative capacity. The mechanisms underlying stem cells and their exosomes are multifactorial, e.g., anti-inflammatory, immunomodulatory, and trans-differentiation of stem cells, as well as the secretome profile of the cells, especially via microvesicles and exosomes. MSCs exosome has a large cargo of several substances and different kinds of miRNAs (~150 miRNAs)20-23. These exosomes are rich in substances, including noncoding proteins, nucleic acids, and DNA. MSCs’ exosome significantly inhibits inflammatory factors, reduces oxidative stress, promotes normal lung cell proliferation, and leads to reduced apoptosis by delivering noncoding RNAs18,23. Previously, MSCs were tested on oleic acid-evoked lung injury in rats24,25.

In our study, however, we first compared maternal stem cells and their derivative exosomes in the healing phenomenon. We have demonstrated that both bone marrow-originated MSCs and their derivative exosomes have the capability to repair the oleic acid-induced ALI. Xu et al demonstrated that i.v. MSCs transplantation could preserve the pulmonary alveolar-capillary barrier’s integrity and modulate the inflammatory response to attenuate the experimental ALI/ARDS24, and our data support this report. However, in our study, we compared both MSCs and their derivative exosomes. Although both products significantly healed the lung injury, the MSCs induced a more pronounced effect, probably due to the more diverse and rich therapeutic components than exosomes. It is reasonable that whole cells (i.e., stem cells) contain exosomes and other potential curative ingredients, explaining why whole stem cells evoked better regenerative effects than exosomes.

The reason we administered BMSCs and their derivative exosomes two hours after the application of oleic acid is that oleic acid may destroy BMSCs and exosomes, exogenously administered (i.v. route in this study) when they come across in the blood since oleic acid may not only harmful to lung epithelial cells (to induce experimental ARDS) but also damage these therapeutic cells and their derivatives, exosomes.

On the other hand, MSCs’ regenerative pathways are not clearly understood. However, various studies focused on compatibility with macrophages, immune responses, well known pathways such as hedgehog, Wnt/β-katenin, transforming growth factors beta (TGF-βs), bone morphogenic proteins (BMPs), and fibroblast growth factor (FGF). Regeneration dedicates the transformation of niche areas and the arrangement of the microenvironment. Differentiation of mesenchymal stem cells (MSCs) is promoted with TGF-β1, TGF-β3, IGF-1, FGF-2, BMP-2 factors, and collagens in the niche. Among the mode of action of MSCs for therapeutic purposes, these cells increase the expressions of these factors, differentiation of macrophages for example, M1 macrophages to M2, suppression of inflammation responses, and regulation of hedgehog, Wnt/β-katenin, NF-kB, TGF-βs, BMPs and FGFs pathways9,10,14. Some of these beneficial effects of stem cells may result from their fractions, such as exosomes and other microvesicles. Exosome-specific surface markers are found in the cytoplasmic cell membrane. These markers are looked at to identify exosomes in experiments13,26.

MSC-derived exosomes (MSC-Exos) represent a cell-free therapy that seems to have tremendous potential for lung disease application as a safer and more stable option than traditional cell therapies. Compared to stem cell-based therapies, MSC-Exos can overpower challenges, such as host rejection, effectively and exhibit therapeutic effects on cell/tissue injury, including the nervous system, renal injury, cardiovascular system, skin scar, liver injury, osteoarthritis, autoimmune diseases, and lung injury18. Exosomes are released from cell types containing inner membranes, so a cytoplasmic cell membrane surrounds them. These cell-to-cell communication vehicles are rich in different substances, including nucleic acids, proteins, and DNA. Exosome studies focus on miRNA efficiency in regeneration besides proinflammatory factors. Delivery of non-coding RNAs (ncRNAs) enables MSC-Exos to communicate with target cells. Bone marrow MSC-exosomes induced axon branches and length in vitro study. In addition, an in vivo study with mice proved that human BMSCs and their exosomes caused functional healing after traumatic head-injured mice. The miR-17-92 found in BMSCs-exosomes induced Schwann cell’s axonal growth and neuronal network regeneration15,16,23,27. MSC-Exos dramatically suppress inflammatory factors, reduce oxidative stress, promote normal lung cell proliferation, and reduce apoptosis by delivering ncRNAs. These cargo particles can reduce oxidative stress and, thereby, inhibit inflammation. Also, they influence the apoptosis, proliferation, invasion, and metastasis of lung cancer cells. In a clinical sense, MSC-Exos have potential in lung diseases, mainly including lung injury, asthma, pulmonary fibrosis, ischemia/reperfusion injury, and lung cancer18,28. At the same time, MSC-Exos (10, 20, 40 μg) provided a protective effect against lung edema, and it was observed that the regeneration effect increased as the number of exosomes increased29

However, more decisive studies are needed regarding efficacy, safety, and suitability before clinical transition. Besides, different application routes, such as inhalation, also need to be tested, as Chu et al26 pointed out.

The limitation of the study is that antioxidants such as Catalase (CAT), Glutathione peroxidase (GPx), Superoxide dismutase (SOD), and oxidation parameters such as Hydrogen peroxide (H2O2) and Malonyldialdehyde (MDA) could not be measured due to the insufficient amount of blood taken from the rats after sacrificing.

Conclusion

This study compared stem cells and their derivative exosomes in the same degenerative lung model. For the first time, we found that maternal stem cells were more effective in repairing ALI caused by oleic acid in rats. This result may point to a new cell-free treatment alternative for lung injury. Moreover, more studies are needed to clarify the results and functions of exosomes in the future.

Conflict of interest

No conflicts of interest are declared by the authors.

Acknowledgments

This study was financially supported by the Mersin University Scientific Research Projects Unit (Project No: 2021-2-TP2-4522). The authors are indebted to Dr. Dilara Nemutlu Samur for her help in cell culture experiments. 

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