Human decidual mesenchymal stem cells obtained from early pregnancy attenuate bleomycin-induced lung fibrosis by inhibiting inflammation and apoptosis

doi:10.21203/rs.3.rs-3867170/v1

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Human decidual mesenchymal stem cells obtained from early pregnancy attenuate bleomycin-induced lung fibrosis by inhibiting inflammation and apoptosis

https://doi.org/10.21203/rs.3.rs-3867170/v1

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Background

Decidual mesenchymal stem cells (DMSCs) are easily obtained and exhibit strong anti-inflammatory and anti-apoptotic effects. Compared with bone marrow mesenchymal stem cells (BMSCs), their role in cell transplantation after idiopathic pulmonary fibrosis remains unclear.

Methods

BMSCs and DMSCs were derived from healthy donors. The anti-inflammatory and anti-apoptotic effects on both cell types were evaluated in vitro. The function of DMSCs in MLE-12 cells and mouse lung fibroblasts was examined using additional transwell coculture experiments in vitro. We investigated whether the transplantation of BMSCs and DMSCs could alleviate pulmonary inflammation and fibrosis in a bleomycin-induced mouse model of pulmonary fibrosis. Twenty-one days after MSC transplantation, we examined the inflammatory factors in the serum and bronchoalveolar lavage fluid, collagen content, pathology, fibrotic area, lung function, and micro-computed tomography of the lung tissue.

Results

DMSCs exhibited better anti-inflammatory and anti-apoptotic effects than BMSCs on MLE-12 cells in vitro. In addition, DMSCs inhibited tumor growth factor β-dependent epithelial-mesenchymal transition in MLE-12 cells and attenuated mouse lung fibroblasts fibrosis. Furthermore, transplantation of DMSCs in the mouse idiopathic pulmonary fibrosis model significantly attenuated pulmonary inflammation and lung fibrosis compared with BMSCs transplantation.

Conclusions

DMSCs exhibited better efficacy in improving pulmonary inflammation and lung fibrosis than BMSCs. Thus, DMSCs are a potential therapeutic target for pulmonary fibrosis.

Human decidual mesenchymal stem cells

lung fibrosis

inflammation

Pulmonary fibrosis is a progressive, usually fatal, form of interstitial lung disease of unknown etiology, characterized by the proliferation of fibroblasts and the accumulation of a large amount of extracellular matrix, accompanied by inflammatory damage and tissue structure destruction[1, 2]. The most common disease type is idiopathic pulmonary fibrosis (IPF), a severe inflammatory interstitial lung disease with poor prognosis, high mortality, and increasing incidence and prevalence[3]. Several drugs for IPF have been tested; however, their efficacy continues to be unsatisfactory. Lung transplantation is the best available treatment option for improving the survival of patients with IPF. However, this approach is constrained by the lack of donors[4]. Thus, there is an urgent need to identify new therapeutic approaches for pulmonary fibrosis. With the rapid development of cell therapies, stem cell therapy has emerged as a promising strategy for treating several diseases[5].

Bone marrow mesenchymal stem cells (BMSCs) are feasible candidates for cell transplantation in animal models of fibrosis because of their repair and protective effects[6, 7]. However, BMSCs have limited proliferative capacity, an age-dependent decrease in cell availability, and an insufficient supply[8, 9]. Decidual mesenchymal stem cells (DMSCs) from early pregnancy tissues significantly ameliorate endothelial cell proliferation, inflammation, and oxidative stress in a cell culture model of preeclampsia[10]. DMSCs are an essential part of the decidual tissue that originates from normal pregnancy and are easy to obtain[8]. In the field of regenerative medicine, DMSCs are considered promising therapeutic candidates for the treatment of IPF. Furthermore, our group has reported the therapeutic effects of DMSCs in myocardial infarction animal models, which can suppress cardiac fibrosis and improve ventricular function[8]. Based on our previous research, we explored the impact of DMSCs on bleomycin (BLM)-induced pulmonary fibrosis and found that DMSCs may have better anti-inflammatory and anti-apoptotic therapeutic potency than BMSCs.

In this study, we evaluated the therapeutic effects of DMSCs on BLM-induced lung fibrosis in mice. We used an in vitro coculture system to elucidate the underlying mechanism of DMSC activity. This study contributes to understanding the function of DMSCs in pulmonary fibrosis and suggests that DMSCs have better anti-inflammatory and anti-apoptotic properties than BMSCs.

1. Isolation and culture of human DMSCs and BMSCs

Human decidual samples were collected from healthy women aged 20–35 years old who underwent selective vaginal surgery to terminate early pregnancy. Human decidual tissue was washed with saline, and blood clots were removed. All samples were stored in phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin, cut into small pieces (1–2 mm), digested with 0.25% trypsin and 0.2% collagenase IV (Yeasen, Shanghai, China) for 1 h. Cell digests were collected every 15 min and passed through a nylon mesh (200 µm) to remove large particulate matter. Then, complete medium containing 10% fetal bovine serum (FBS; Sciencell, Sandiego, CA, USA) was added to terminate digestion. After centrifugation, DMSCs were cultured overnight in DMEM/F12 (Hyclone, South Logan, Utah, USA) containing 10% FBS. BMSCs were derived from female patients (20–35 years old) who underwent median sternotomy during open-heart surgery. Equal volumes of the lymphocyte separation solution and bone marrow were mixed and centrifuged, and the mononuclear cell layer was collected and washed twice with PBS. BMSCs were seeded in culture flasks with DMEM/F12 (HyClone) containing 10% FBS (ScienCell). A follow-up experiment was performed using the third-generation DMSCs/BMSCs. All the participants provided informed consent. All procedures performed in the experiments involving animals complied with the ethical standards of the First Affiliated Hospital of Anhui Medical University.

2. Morphology and identification of MSCs

The morphology and number of cells were observed under a light microscope (Leica, Germany) to determine the growth status of MSCs. To identify the MSC phenotypes, the cell suspension was mixed with CD73-PerCP, CD90-FITC, CD105-APC, CD34-FITC, and CD45-PerCP antibodies and incubated for 30 min in the dark. Stained cells were analyzed using flow cytometry (BD, USA).

3. Comparison of MSC proliferation capacity

DMSCs and BMSCs were cultured in a medium containing 10% serum at a density of 1 × 10³ in 96-well plates, followed by incubation for 12, 24, 48, and 72 h. The rate of cell proliferation was determined using a Cell Counting Kit-8 (Dojindo, China). DMSCs and BMSCs were seeded at densities of 250 and 500 cells/well, respectively, in six-well plates for two weeks. Cells were fixed with 4% paraformaldehyde for 15 min, rinsed with PBS, stained with crystal violet for 10 min, and rinsed again. Colonies containing > 20 cells were manually counted as clonal colonies.

4. MLE-12 and mouse lung fibroblast (MLF) isolation and culture

The murine lung epithelial type II cell line MLE-12 was purchased from the American Type Culture Collection. Upon thawing, the cells were cultured in DMEM/F12 (HyClone) containing 10% FBS (ScienCell). Primary MLFs were extracted from the lung tissues of 4-week-old C57BL/6J mice. Briefly, mice were sacrificed after being anesthetized with an intraperitoneal injection of tribromoethanol (500 mg/kg) to collect lung tissues. Lung tissues were washed with PBS and placed on a clean dish. Under aseptic conditions, lung tissues were trimmed into blocks approximately 1 mm in diameter. The tissue blocks were transferred to DMEM/F12 (HyClone) containing 10% FBS (ScienCell) for 72 h. MLFs were purified from macrophages by passage, and cells at passage five were used for the experiments.

5. Coculture experiments

MLE-12 cells were seeded into the lower chamber, MLE-12 cells were treated with BLM (5 ng/mL; Selleck, China) in DMEM/F12 medium for 24 h, and DMSCs or BMSCs (1:1 ratio) were added to the upper chamber of a 6-well transwell apparatus (Corning Costar). Four groups were established as follows: control group, MLE-12 cells; BLM group, MLE-12 cells treated with BLM; BMSC group, MLE-12 cells treated with BLM and cocultured with BMSC; and DMSC group, MLE-12 cells treated with BLM and cocultured with DMSC. Similarly, MLFs were seeded into the lower chamber, MLFs were treated with tumor growth factor (TGF)-β1 (3 ng/mL; Thermofisher, China) in DMEM/F12 medium for 24 h, and DMSCs or BMSCs (1:1 ratio) were added into the upper chamber of a 6-well transwell apparatus. Four groups were distributed as follows: the control group, MLFs; the TGF-β1 group, MLFs treated with TGF-β1; BMSC group, MLFs treated with TGF-β1 and cocultured with BMSC; and DMSC group, MLFs treated with TGF-β1 and cocultured with DMSC.

6. Apoptosis detection

The viability of BLM-treated MLE-12 cells was evaluated using live/dead staining. A live/dead cell staining kit (Solarbio, Beijing, China) was used according to the manufacturer’s instructions. The Calcein-AM was added to the plate and incubated for 20 min, 5 µL of propidium iodide (PI) was added, and incubation continued for 5 min. After washing with PBS, photographs were taken under an inverted fluorescence microscope, and live (green fluorescence, 488 nm) and dead (red fluorescence, 550 nm) cells were simultaneously detected.

Apoptosis in BLM-treated MLE-12 cells and TGF-β1-treated MLFs were determined using flow cytometry with annexin V/PI double staining (Beyotime, Shanghai, China). MLE-12 cells were cocultured with MSCs for 24 h, digested with pancreatin, and incubated with annexin V and PI. After washing with PBS, the cells were suspended in PBS, and flow cytometry was performed.

7. Clonogenic, scratch, and transwell invasion assays

MLE-12 cells were seeded in 6-well plates at a density of 1000 cells/well in four different groups and cultured for four days. Cells were fixed with 4% paraformaldehyde for 15 min, rinsed with PBS, stained with crystal violet for 10 min, and rinsed again. Colonies containing > 20 cells were manually counted as clonal colonies.

The coculture medium was used to observe healing in the scratched areas. For this assay, MLE-12 cells were seeded into 6-well plates in coculture medium and grown to 80–90% confluency. Then, a wound track was introduced by scraping the cell monolayer with a 100-µL yellow pipette tip. The monolayer wound areas were measured at 0 and 48 h after treatment.

The cell invasion assay was performed using 24-well transwell plates (Costar) coated with Matrigel (BD BioSciences) according to the manufacturer's instructions. The MLE12 cells were suspended in a serum-free medium and plated into the upper chamber of the transwell system with a pore size of 8 µm. The bottom chamber was filled with the coculture medium. After incubation for 12 h, the migrating cells in the bottom chambers were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope.

8. Enzyme-linked immunosorbent assay (ELISA)

Using a transwell chamber coculture experiment system, a coculture experiment was performed in the chambers of 24-well plates (Corning, USA). DMSCs (1 × 10⁴) and BMSCs were inoculated into the upper chamber, and MLFs (1 × 10⁴) were cocultured in the lower chamber for 24 h. MLFs without hMSCs treatment were used as the controls. The secretion levels of interleukin (IL)-10, IL-1β, and IL-6 were measured using ELISA kits (Cusabio, China) according to the manufacturer’s instructions.

9. Immunofluorescence staining

MLFs were cocultured with DMSCs or BMSCs for 24 h using a transwell chamber coculture experimental system. MLFs were rinsed with PBS, fixed with 4% paraformaldehyde at room temperature, permeabilized with Triton X-100 for 5 min, and blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. Then, the MLFs were incubated with anti-α-smooth muscle actin (SMA) (Abcam, China) at 4°C overnight, followed by incubations with Alexa Fluo-635-conjugated goat anti-mouse antibodies for 1 h. The coverslips were washed once with PBS and mounted with a DAPI solution. Images were obtained under a fluorescence microscope (Leika, Germany) and quantified using ImageJ software.

10. BLM-induced pulmonary fibrosis and MSC transplantation

To induce pulmonary fibrosis, the C57BL/6J mice were anesthetized with intraperitoneal tribromoethanol (500 mg/kg). Then, mice were administered intratracheally once with BLM (3 mg/kg) at day 0 and subsequently received 5 × 10⁵ MSCs in 500 µl of PBS via tail vein injection in two and seven days after BLM treatment; this group was referred to as the BMSC or DMSC group. Mice injected with an equal volume of PBS were referred to as the BLM group, and normal C57BL/6J mice served as blank controls.

11. Bronchoalveolar lavage fluid and blood analyses

After anesthesia, blood was drawn from the hearts of the mice into heparinized tubes. Serum samples were separated through immediate centrifugation at 1500 rpm for 10 min. The serum levels of IL-10, IL-1β, IL-6, and TGF-β1 were measured using ELISA kits (Cusabio, China) according to the manufacturer’s instructions. After blood collection, the chest cavity of the mice was opened, the right lung was ligatured, the left lung was lavaged three times through the bronchus with 0.4 mL of PBS, and the liquid was pulled away slowly. The bronchoalveolar lavage fluid (BALF) levels of IL-10, IL-1β, IL-6, and TGF-β1weres measured using ELISA kits. BALF cytospins were prepared, slides were fixed in acetone, and then Wright–Giemsa staining was performed. Finally, an optical microscope was used to count the total number of leukocytes at high magnification.

12. Immunohistochemistry and Masson’s trichrome staining

Lung tissues were isolated and fixed with 4% paraformaldehyde (Biosharp, Shangh ai,China) for 48 h before being embedded in paraffin to prepare 5-mm tissue sections. The tissue sections were deparaffinized and rehydrated using graded levels of xylene and ethanol, placed in a pressure cooker with sodium citrate, boiled, and cooled gradually to room temperature to complete antigen repair. Endogenous peroxidases were blocked with 3% H₂O₂ for 10 min and incubated with 3% bovine serum albumin in PBS for 1 h at room temperature. The sections were treated with TGF-β1, α-SMA, Col1a1, and fibronectin antibodies overnight at 4℃, following by incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotim e,Shangh ai,China) for 60 min followed by diaminobenzidine, and counterstained with hematoxylin. Immunohistochemical staining was performed using a standard light microscope. Masson’s trichrome and hematoxylin and eosin staining were performed according to the manufacturer’s instructions (Solarbio, Beijing, China), and pathological changes in the lung tissue were assessed using an optical microscope.

13. Determination of hydroxyproline

The collagen and hydroxyproline contents of the lung tissue were determined using the hydroxyproline method, and approximately 45 mg of the sample was weighed into test tubes according to the procedures described in the instructions of the hydroxyproline assay kit (Jiancheng, Nanjing, China). The supernatant was collected and added to a 96-well plate, and the absorbance of the supernatant was measured at 550 nm.

14. Measurements of lung function

Invasive pulmonary function tests were performed using the FlexiVent system (SCIREQ, Montreal, QC, Canada). Mice were anesthetized, their tracheas were cannulated under direct vision, and attached to a computer-controlled ventilator for forced oscillation measurements, s set at 150 beats/min with a tidal volume of 10 mL/kg. Each mouse was measured three times (coefficient of determination > 0.95), and the mean was used as the measured value for each mouse.

15. Micro-computed tomography (micro-CT) measurement validation

CT scanning was used to measure lung tissue density and degree of pulmonary fibrosis in each group of mice. A plain CT scan was performed as follows: mice were anesthetized with an intraperitoneal injection of tribromoethanol, placed in the supine position on the bed of the CT scanner, and the living lung tissue of the mice was scanned after setting the scanning parameters. The images were processed using a post-processing system workstation, all CT images were input into the software in the form of raw data, and the analysis results were obtained. To evaluate the severity of pulmonary fibrosis, both structural and morphological changes in lung tissue were observed.

16. Western blotting

Total protein was extracted from MLE12 cells, MLFs, BALF, and lungs using RIPA lysis buffer (Beyotime, Beijing, China) containing protease and phosphatase inhibitors. The protein concentration of the lysates was determined using the BCA assay (Beyotime, China).

Proteins were separated using sodium dodecylsulfate-polyacrylamide gel electrophoresis (10% gels) and transferred onto 0.22 µm polyvinylidene fluoride membranes (Millipore, China). Membranes were incubated with the primary antibody and then rocked gently overnight at 4°C, with anti-E-cadherin (Abcam, Shanghai, China), anti-N-cadherin (Abcam, Shanghai, China), α-SMA (Abcam, Shanghai, China), Col1a1 (Abcam, Shanghai, China), fibronectin (Abcam, Shanghai, China), surfactant protein C (SPC; Abcam, Shanghai, China), Smad3 (Abcam, Shanghai, China), BAX (Abcam, Shanghai, China), Bcl-2 (Abcam, Shanghai, China), cleaved casepase 9 (Affinity, Jiangsu, China), and cleaved casepase 3 (Affinity, Jiangsu, China). After incubation with horseradish peroxidase-labeled secondary antibodies (Abcam, Shanghai, China) at 37°C for 1 h, the protein bands were visualized using a Chemiluminescence Imaging System (Tanon, China).

17. Statistical analysis

All statistical analyses were performed using GraphPad Prism 5.0 software, and data are expressed as mean ± SD. A t-test was performed to compare two groups, anda one-way analysis of variance (ANOVA) was performed to compare multiple groups. P < 0.05 was considered statistically significant, and the experiments were repeated at least three times.

1.Identification and proliferation of MSCs

Stem cells from human DMSCs and BMSCs exhibited a typical fibroblast-like morphology and were slender and fusiform; however, the DMSCs group displayed a more plump and full morphology (Fig. 1A). Moreover, MSCs expressed the surface markers CD73, CD,90 and CD105, but did not express CD34 and CD45 (Fig. 1B). Cell Counting Kit-8 and cell cloning experiments were used to determin MSCs proliferation. DMSCs showed a higher proliferation rate than BMSCs (Fig. 1C and D).

2.DMSCs inhibit apoptosis and SPC activity of BLM‑induced MLE‑12 cells

We studied the effect of DMSCs on cell viability induced by BLM in MLE-12 cells using live–dead staining (green stain for live cells and red for dead cells). Our results indicate that DMSCs can better protect MLE‑12 cells from BLM‑induced injury than BMSCs (Fig. 2A). Additionally, flow cytometry results confirmed that coculture with DMSCs significantly reduced the rate of MLE-12 cell apoptosis compared with that in the BMSCs and BLM groups (Fig. 2B). Simultaneously, the clone formation experiment suggests that DMSCs can better promote the clone formation ability of MLE‑12 cells than other groups (Fig. 2C). We also investigated the expression of SPC. Western blotting demonstrated that after MLE-12 cells were cocultured with DMSCs, SPC increased significantly compared with the TGF-β1 + BMSCs and TGF-β1 groups (Fig. 2D). These results suggest that DMSCs exert anti-apoptotic effects on BLM-induced MLE-12 cells.

3.DMSCs inhibit epithelial cell interstitial transformation of BLM‑induced MLE‑12 cells

We assessed whether DMSC transplantation affected BLM-induced epithelial-mesenchymal transition (EMT). The wound healing and cell invasion assays showed that DMSCs significantly suppressed cell migration and invasion (Fig. 3A and B). We also investigated the expression of EMT-related proteins, Western blotting demonstrated that after MLE-12 cells were cocultured with DMSCs, E-cadherin levels increased significantly, while N-cadherin protein levels decreased (Fig. 3C). These results indicate that DMSCs can inhibit BLM-induced EMT in MLE-12 cells.

4.DMSCs inhibit the TGF-β1-induced apoptosis, inflammation-related factors, and fibroblast phenotype of MLFs

Pulmonary fibrosis is characterized by the accumulation of myofibroblasts and excessive deposition of the extracellular matrix (ECM). To assess the impact of DMSCs on TGF-β1-treated MLFs, we examined the apoptosis ratio in MLFs after being cocultured with DMSCs for 72 h using the annexin V-FITC/PI Apoptosis Detection kit. The present cell apoptotic rate of the DMSC group was significantly lower than that in the TGF-β1 and TGF-β1 + BMSCs group (Fig. 4A). Simultaneously, we determined the expression of inflammatory factors of the supernatant in the coculture system. The expression of IL-10, IL-1β, and IL-6 inflammatory factors in the TGF-β1 group were significantly increased, whereas the levels of inflammatory factors in the TGF-β1 + BMSCs and TGF-β1 + DMSCs groups were significantly decreased (Fig. 4B). We measured α-SMA,collagen1,and fibronectin levels in MLFs after being cocultured with DMSCs for 72 h using real-time PCR, and the results showed that collagen 1, α-SMA, and fibronectin expression in MLFs was decreased after being cocultured with DMSCs. In addition, when comparing the TGF-β1 + BMSCs group, the TGF-β1 + DMSCs exhibited lower mRNA expression levels of collagen 1, α-SMA, and fibronectin (Fig. 4C). Using immunofluorescence analyses, we showed that the expression of α-SMA in the TGF-β1 + DMSCs group was significantly decreased compared with the TGF-β1 + BMSCs and TGF-β1 groups. The TGF-β1 + BMSCs group exhibited lower fluorescence intensity compared with the TGF-β1 groups (Fig. 4D).

5.DMSCs ameliorate the inflammation of BLM-induced lung fibrosis

The effect of DMSCs on the number of leukocytes in the BALF of BLM-treated mice was determined. The results of Wright–Giemsa staining showed that the number of leukocytes in the BLM + DMSCs group was significantly lower than that in the BLM + BMSCs and BLM groups, and the BLM + BMSCs group exhibited a lower number of leukocytes than the BLM groups (Fig. 5A and B). Total BALF protein increased in the BLM group but decreased in the DMSCs group compared with that in the control group (Fig. 5C). In addition, DMSC treatment can reduce the inflammatory factors IL-10, IL-1β, IL-6, and tumor necrosis factor (TNF)-α levels in the serum (Fig. 5D). The levels of the BALF-associated inflammatory cytokines showed similar trends. Following DMSCs treatment, the expression of inflammatory cytokines IL-10, IL-1β, IL-6, and TNF-α was markedly reduced (Fig. 5E). As shown using immunohistochemistry, the increased expression of TGF-β1 induced by BLM was significantly inhibited by DMSCs (Fig. 5F). These results illustrate that DMSCs play a protective role against BLM-induced lung inflammation.

6.DMSCs relieve the symptoms of BLM-induced lung fibrosis

Lung structure and collagen deposition were observed using H&E and Masson staining in the lung interstitium of the four groups. The pathological results showed that the inflammatory infiltration of alveolar and interstitial cells and the destruction of lung structure were significantly attenuated in the DMSCs group compared with those in the BLM group (Fig. 6A). Furthermore, we evaluated the inflammatory scores from the hematoxylin and eosin staining and found that the inflammatory scores of the DMSCs + BLM group were significantly improved compared with those of the BLM and BMSCs + BLM groups (Fig. 6B). In addition, the Ashcroft fibrosis score and fibrosis area were used to grade the fibrosis scale, and the DMSCs + BLM group demonstrated significantly lower Ashcroft scores in fibrosis than the BLM and BMSCs + BLM groups (Fig. 6C and D). Hydroxyproline content can be used to determine the level of collagen to assess the degree of fibrosis. In addition, compared with controls, the expression of collagen deposition was also markedly increased by BLM, while DMSC transplantation reduced the deposition of collagen deposition compared with the BLM and BMSCs + BLM groups (Fig. 6E), DMSC transplantation also increased the dry/wet radioratioung tissues compared with those in the BLM and BMSCs + BLM groups (Fig. 6F). The body weight of mice in the BLM group was significantly lower than that of the control mice; however, the body weight of mice in the DMSCs + BLM group was significantly higher than that of the BLM and BMSCs + BLM groups (Fig. 6G). These results suggest that DMSC transplantation ameliorates BLM-induced pulmonary fibrosis.

7.DMSCs relieve the cell apoptosis and antifibrotic phenotype of BLM-induced lung fibrosis

After the DMSCs treatment, the fibrosis-related proteins Smad3 levels were significantly reduced (Fig. 7A).The expression of apoptosis-related proteins was significantly altered in bleomycin-induced mouse lung fibrosis, and the levels of proapoptotic signals, such as BAX, cleaved caspase-9, and cleaved caspase-3, were significantly increased, whereas the expression of anti-apoptotic Bcl-2 was decreased in the lung tissue of mice in the BLM groups; these changes were considerably attenuated by DMSCs treatment (Fig. 7B). Moreover, western blotting showed that DMSC treatment inhibited the BLM-induced upregulation of fibrosis-related proteins α-SMA, fibronectin, and collagen I. However, the regulation of BMSC treatment was much weaker than that of DMSCs (Figure S1). Similar results were detected using immunohistochemistry; DMSC ransplantation mitigated ECM deposition, collagen formation, and myofibroblast activation in BLM-treated mice. Nevertheless, the effect of BMSC therapy was weaker than that of DMSC therapy in BLM-treated mice (Fig. 7C). These data show that DMSC therapy reverses lung fibrosis in mice through an-apoptosis and anti-fibroticts.

8.DMSCs improve lung function of BLM-induced lung fibrosis

Forced vital capacity, forced expiratory flow (FEF), inspiratory capacity (IC), FEV0.1, and flow-volume loops were also improved by DMSC therapy in BLM-treated mice, indicating that DMSCs therapy was closely related to lung function (Fig. 8A). Additionally, micro-CT images of the lungs were used to predict the occurrence of lung shadows in the mouse lung fibrosis models. In a bleomycin-induced pulmonary fibrosis model, CT revealed the presence of ecin in the lung lobe, which prevented air from entering the lungs. The entire lung appeared as multiple spots and hyperdense shadows. In contrast, DMSCs-treated BLM-induced lung fibrosis showed fewer spots and hyperdense shadowed areas. However,BMSCs were weaker than the ith DMSCs in hyperdense shadowed areas of BLM-treated mice (Fig. 8B). As demonstrated in Figure S2, normal lung volume was lower in BLM-induced lung fibrosis mice than in control mice. However, compared with that in the BLM induction group, the volume of the lungs was significantly restored in the DMSCs treatment group. These data show that DMSC therapy reverses lung function in mice.

Pulmonary fibrosis (PF) is a chronic, progressive, and fatal disease of unclear etiology with an increasing global mortality rate[11]. Currently, there are only two FDA-approved drugs (nintedanib and pirfenidone) for the treatment of pulmonary fibrosis; however, these drugs are only effective in slowing down, but nt halting disease progression[12, 13]. MSCs are useful in preclinical and clinical studies because of their immune regulatory, anti-inflammatory, antibacterial, and tissue regeneration functions [14]. Previous studies have shown that engrafted stem cells have beneficial effects on injured lung tissues[5], blood-borne stem cells may contribute to lung tissue in recipients of lung transplantation[16]and human menstrual blood-derived stem cells may ameliorate bleomycin-induced pulmonary fibrosis[2]. Although MSC transplantation is a promising therapeutic strategy for lung diseases, the effects of clinical translational treatments are not ideal.

BMSCs, which are multipotent cells, are widely used in the treatment of various diseases owing to their self-renewability, differentiation, and immunomodulatory properties and have achieved some clinical effects[17]. Some experimental studies have also suggested that transplantation of BMSCs attenuates pulmonary fibrosis, which can improve the respiratory function of rats[18, 19]. However, BMSC therapy has not produced satisfactory results for tissue repair in clinical trials, partly due to limited proliferative ability, insufficient supply, and aging susceptibility [20]. DMSCs from early pregnancy tissues have been found to have a strong proliferation and anti-inflammatory capacity [21, 22]. We also found that DMSCs proliferated better than BMSCs, suggesting that they may be a favorable potential target for the treatment of pulmonary fibrosis.

Excessive ECM accumulation resulting from an imbalance between ECM protein synthesis and degradation can lead to pathological profiles of pulmonary fibrosis, which eventually cause impairment of gas exchange and respiratory failure[23]. Upon activation, fibroblasts transform into myofibroblasts, which produce the pathological ECM [6]. In addition, pulmonary epithelial cells transdifferentiate into myofibroblasts via EMT, which releases ECM components and leads to fibrosis[25]. BLM may lead to lung epithelial cell apoptosis or EMT, which is an important cause of BLMinduced pulmonary fibrosis[26]. Our group has previously demonstrated that DMSC treatment accelerates proliferation and suppresses apoptosis in BLM-induced MLE-12 cells. Additionally, DMSCs significantly inhibited BLM-induced EMT by downregulating N-cadherin and upregulating E-cadherin expression in MLE-12 cells. Others have shown that MSCs promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine effects[27]. Our current study showed that DMSC treatment promoted SPC expression in BLM-induced MLE-12 cells. These results indicate that DMSCs have great potential for reducing apoptosis and EMT in vitro.

We used MLFs to investigate the potential role of DMSCs and found that they could inhibit the apoptosis, inflammation, and activation of lung fibroblasts. MSC administration markedly alleviated TGF-β1-induced lunfibroblastts apoptosis and inflammation[7], while DMSCs display better anti-apoptotic and anti-inflammatory properties than BMSCs. Moreover, the MSCs attenuate the phenotypic activation of fibroblasts[28, 29]. We showed that DMSCs and BMSCs attenuated fibrotic phenotype TGF-β1-induced lung fibroblast,s whereas DMSC treatment appeared to be attenuated better than BMSCs treatments. These results suggest that DMSCs reduce inflammation, attenuate fibrosis, and reduce apoptosis in TGF-β1-induced lung fibroblasts.

BMSC treatment significantly reduces lung injury in mice and is associated with reduced pulmonary inflammation[30]Other studies have also confirmed that adipose-derived stem cells inhibit pulmonary inflammation duin BLM-treated mice during the pulmonary inflammatory phase. MSC transplantation attenuates inflammation and lung injury in an in vivo model of acute lung injury[32]. Although total leukocytes and flammatory cytokines IL-10, IL-1β, IL-6, and TNF-α in BALF in moumicegnificantly declined after bleomycin-induced pulmonary fibrosis and BMSC transplantation, the degree of inflammation was relatively low in the DMSC transplantation group compared with BMSC transplantation group. In addition, hematoxylin and eosin and Masson staining allowed us to assess the degree of damage and fibrosis in mouse lung tissue and found that DMSCs and BMSCs attenuated the degree of damage and fibrosis of bleomycin-induced pulmonary mouse lung tissue, whereas DMSC treatment appeared to be attenuated better than BMSCs treatment. Furthermore, the DMSC-treated groups showed lower degrees of lung inflammation and fibrosis, which accompanied by a lower degree of lung collagen deposition, as assessed by lung hydroxyproline content. MSCs have the potential to improve pulmonary function and have been proposed as a therapy for lung fibrosis[33–35]Bone marrow, adipose, umbilical cord, and menstrual blood-derived MSC transplantations improve the symptoms of pulmonary fibrosis[2, 34–36]. MSC treatment decreased lung tissue cell apoptosis and fibrosis in bleomycin-induced pulmonary mice, whereas the expression of proteins related to apoptosis and fibrosis in the DMSC treatment groups was lower than that in the BMSC treatment group. The CT scan and pulmonary function test results also showed a reduced degree of pulmonary fibrosis in mice treated with DMSCs compared with those treated with BMSCs.

DMSCs are a potential choice for stem cell transplantation in the treatment of treatingis. Our study also confirmed that DMSC-treated groups showed better antifibrotic and anti-inflammatory properties than BMSC-treated groups, although the underlying mechanisms are still not fully understood. In future studies, the related molecular mechanisms of DMSC-based therapy for pulmonary fibrosis will be explored further, making the results more valuable.

These results suggest that DMSC transplantation improved pulmonary function and reduced pulmonary fibrosis. The effect of DMSC transplantation was stronger than that of BMSC transplantation in pulmonary fibrosis.

DMSCs attenuated BLM-induced pulmonary fibrosis in mice, indicating that DMSC transplantation exhibited a better therapeutic effect than BMSC transplantation, which could provide a therapeutic strategy for pulmonary fibrosis.

BALF

bronchoalveolar lavage fluid

BLM

bleomycin

BMSCs

bone marrow mesenchymal stem cells

DMSCs

decidual mesenchymal stem cells

ECM

extracellular matrix

ELISA

enzyme-linked immunosorbent assay

EMT

epithelial-mesenchymal transition

FBS

fetal bovine serum

interleukin

IPF

idiopathic pulmonary fibrosis

Micro-CT

micro-computed tomography

MLFs

mouse lung fibroblastos

PBS

phosphate-buffered saline

propidium iodide

SMA

smooth muscle actin

SPC

surfactant protein C

TGF

tumor growth factor

TNF

tumor necrosis factor.

Acknowledgements: None.

Competing interests: All authors declare no conflicts of interest.

Funding: This work was supported by the Initial funding for doctoral research of the First Affiliated Hospital of Anhui Medical University (bskf2023014), the natural science research key project of Anhui (2022AH051150), the China Postdoctoral Science Foundation (2023M740027).

Availability of data and materials: Data and study materials are available.

Authors’ contributions: G N, X G performed the study; K Z and Y D participated in data processing and statistical analyses; C L , F X ,P C and R S provided experimental technical support; G N wrote the manuscript; K C , R Z and N K jointly conceived the study, and drafted the manuscript, and all authors reviewed and approved the final version of the manuscript.

Consent for publication: All authors give their full consent to publish the present article.

Ethics approval and consent to participate: Procedures involving animals and their care were conducted in conformity with the revised guidelines (Animals Act 1986) and were approved by the Committee on Animal Care of Anhui Medical University (Title: The role and mechanism of exosomal derived miR-34c in inhibiting alveolar epithelial cell apoptosis and alleviating pulmonary fibrosis through SYT1-ROS/JNK/p-53 pathway in human decidual mesenchymal stem cells of early pregnancy; Reference number: LLSC20232180; Date: April 01, 2023).

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No competing interests reported.

FigureS1.tif
Figure S1. DMSCs relieve antifibrotic phenotype of BLM-induced lung fibrosis (A) Expression of fibrosis-associated proteins in the lung tissues investigated using western blotting. (n=5; ^***P < 0.001 versus Control group;^###P < 0.001 versus BLM group.)
FigureS2.tif
Figure S2. DMSCs improve lung volume of BLM-induced lung fibrosis (A and B)3D micro-CT images indicated a restored volume in the lungs of BLM-induced lung fibrosis mice when DMSC treatment was required. (n=5; ^***P < 0.001 versus Control group;^###P < 0.001 versus BLM group.)
GRAPHICALABSTRACT.tif
GRAPHICAL ABSTRACT： The DMSCs and BMSCs were acquired from the human decidual tissue and sternum marrow, respectively. MSCs were isolated, separated, and cultured in vitro，and the third generation of cells was taken and intravenously injected into pulmonary fibrosis mice via the tail vein.
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Human decidual mesenchymal stem cells obtained from early pregnancy attenuate bleomycin-induced lung fibrosis by inhibiting inflammation and apoptosis

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

1. Isolation and culture of human DMSCs and BMSCs

2. Morphology and identification of MSCs

3. Comparison of MSC proliferation capacity

4. MLE-12 and mouse lung fibroblast (MLF) isolation and culture

5. Coculture experiments

6. Apoptosis detection

7. Clonogenic, scratch, and transwell invasion assays

8. Enzyme-linked immunosorbent assay (ELISA)

9. Immunofluorescence staining

10. BLM-induced pulmonary fibrosis and MSC transplantation

11. Bronchoalveolar lavage fluid and blood analyses

12. Immunohistochemistry and Masson’s trichrome staining

13. Determination of hydroxyproline

14. Measurements of lung function

15. Micro-computed tomography (micro-CT) measurement validation

16. Western blotting

17. Statistical analysis

Results

1.Identification and proliferation of MSCs

2.DMSCs inhibit apoptosis and SPC activity of BLM‑induced MLE‑12 cells

3.DMSCs inhibit epithelial cell interstitial transformation of BLM‑induced MLE‑12 cells

4.DMSCs inhibit the TGF-β1-induced apoptosis, inflammation-related factors, and fibroblast phenotype of MLFs

5.DMSCs ameliorate the inflammation of BLM-induced lung fibrosis

6.DMSCs relieve the symptoms of BLM-induced lung fibrosis

7.DMSCs relieve the cell apoptosis and antifibrotic phenotype of BLM-induced lung fibrosis

8.DMSCs improve lung function of BLM-induced lung fibrosis

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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