Intratracheal administration of mesenchymal stem cells ameliorates hyperoxia-induced bronchopulmonary dysplasia by inhibiting NLRP3 inflammasome activation: the critical role of Aldh1a2

Abstract

Background

Bronchopulmonary dysplasia (BPD) is a chronic lung disease driven by inflammation and oxidative stress. Mesenchymal stem cells (MSCs) have shown protective effects against hyperoxic lung injury. However, few studies have thoroughly examined the significantly differentially expressed genes (DEGs) in the lungs before and after MSC treatment. In this study, we analyzed the significant DEGs in lung tissues during both in vivo and vitro umbilical cord-derived mesenchymal stem cells (UCMSCs)-mediated repair of hyperoxic lung injury and investigated their potential mechanisms of action.

Methods

Neonatal rats were exposed to hyperoxia and subsequently treated with UCMSCs. Inflammatory responses were quantified via ELISA and RT‒qPCR, while Western blotting (WB) and immunohistochemistry (IHC) were used to examine NLRP3 inflammasome and IL-1β expression. Transcriptomic analysis of UCMSC-mediated lung repair revealed 46 DEGs, which were validated by RT‒qPCR, and WB verified the significant differential expression of ALDH1A2. In RLE-6TN cells, Aldh1a2 expression was reduced during MSC-mediated repair of H₂O₂-induced oxidative stress injury. Functional evaluations were performed. WB further analyzed NLRP3 inflammasome and IL-1β expression in these processes. A recombinant adenoviral overexpression vector was intratracheally administered to hyperoxia-exposed neonatal rats. Arterial blood gas and RT‒qPCR were performed, and ELISA, WB, and IHC were used to evaluate the impact of Aldh1a2 overexpression on lung inflammation and oxidative stress, focusing on the NLRP3 inflammasome.

Results

UCMSCs ameliorated hyperoxia-induced alveolar simplification and microvessel loss, reduced inflammation and oxidative stress injury, and inhibited the expression of the NLRP3 inflammasome. RT‒qPCR and WB analyses revealed significant differential expression of Aldh1a2 in UCMSC-treated hyperoxia-induced lung injury. UCMSCs also mitigated H₂O₂-induced oxidative stress injury in RLE-6TN cells. Inhibition of Aldh1a2 expression exacerbated oxidative stress, upregulated NLRP3 inflammasome and IL-1β expression, and impaired the reparative effects of UCMSCs. Conversely, Aldh1a2 overexpression or UCMSC intervention ameliorated hyperoxia-induced alveolar simplification and microvascular abnormalities, suppressed inflammation, and enhanced lung ventilation and angiogenesis. These findings indicated that Aldh1a2 overexpression inhibits NLRP3 inflammasome activation and IL-1β release.

Conclusions

Aldh1a2 was significantly differentially expressed in hyperoxic lung injury, with hyperoxia suppressing its expression and UCMSC treatment promoting its upregulation. Aldh1a2 mitigates lung inflammation and oxidative stress by inhibiting NLRP3 inflammasome activation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04851-z.

Background

Bronchopulmonary dysplasia (BPD) is a chronic lung disease prevalent in premature infants driven by excessive inflammation and oxidative stress. It is a clinically heterogeneous condition primarily linked to oxygen supplementation [1, 2]. BPD is characterized by alveolar simplification, pulmonary fibrosis, and vascular dysplasia [1, 2]. Despite advancements in neonatal intensive care, the incidence of BPD has risen significantly [1]. Current management strategies, including gentle volume-assured ventilation and medication-assisted therapy, have shown limited efficacy and pose developmental risks [3, 4]. Thus, there is an urgent need to develop novel and effective treatments for BPD [5].

Mesenchymal stem cells (MSCs) are multipotent stromal cells with significant potential for organ repair in preclinical models, primarily due to their immunomodulatory and anti-inflammatory properties [6]. Studies have shown that MSCs alleviate acute lung injury by reducing inflammation, modulating inflammatory mediators, inhibiting neutrophil activation, and suppressing macrophage polarization [6, 7]. Additionally, MSCs can regulate neonatal lung development, alveolar epithelial cell apoptosis, the blood‒air barrier, and microvascular reconstruction by modulating vascular endothelial growth factor (VEGF) and the renin‒angiotensin system (RAS) [6]. Emerging evidence suggests that MSCs effectively treat BPD [6–8]. In neonatal rat models of hyperoxic lung injury, MSCs have been shown to suppress lung inflammation, promote microangiogenesis, and improve cardiac and renal function [8]. However, the precise molecular mechanisms underlying these effects require further investigation.

Our preliminary transcriptomic analysis [8] revealed that aldehyde dehydrogenase 1 family member A2 (Aldh1a2) is significantly differentially expressed in a rat model of MSC-mediated repair of hyperoxic lung injury. Aldh1a2 is a rate-limiting enzyme in cellular retinoic acid synthesis [9]. Research has shown that Aldh1a2 may be involved in immunomodulation and apoptosis by participating in the expression of DCs and Tregs, regulating macrophage polarization, and influencing lymphocyte recruitment [9–12]. This study aimed to investigate the regulatory mechanisms of Aldh1a2 in MSC-mediated lung injury repair.

Methods

Culture and identification of UCMSCs

Human umbilical cord-derived mesenchymal stem cells (UCMSCs) were cultured following previously established protocols. Flow cytometry was used to characterize UCMSC surface markers, which were assessed as described in earlier studies [8].

Animals and study design

The study adheres to the guidelines established by the ARRIVE 2.0 checklist (Supplementary material). Timed pregnant Sprague‒Dawley rats were obtained from the Peking University Health Science Center and housed in an SPF-grade environment at the Animal Facility of Shandong University. All experiments were conducted in accordance with protocols approved by the Animal Care and Use Committee of Qilu Hospital, Shandong University (Approval No. DWLL-2021-035, Jinan, China). To minimize bias, only the corresponding author was aware of the group assignments. All experimental procedures, including interventions, data collection, and outcome assessment, were performed by personnel blinded to the group allocation.

Construction of the BPD rat model and UCMSC treatment

Newborn rats were randomly divided into three groups: the normoxia group (n = 10), the hyperoxia group (n = 10; exposed to 80% O₂ and treated with 40 µL PBS intratracheally on postnatal days 7 (P7) and 10 (P10), and the hyperoxia + MSCs group (n = 10; exposed to 80% O₂ and treated with 1 × 10⁶ UCMSCs in 40 µL intratracheally on P7 and P10). On P21, rats were euthanized via intraperitoneal injection of pentobarbital sodium. Lungs were excised and fixed in 4% paraformaldehyde overnight for histological analysis or stored at −80 °C for RT‒qPCR, western blot, and cytokine analyses.

For intratracheal administration, neonatal rats were anesthetized via intraperitoneal injection of pentobarbital sodium and positioned on a rodent intubation stand (CG-02 M, Yuyan, Shanghai, China) with neck hyperflexion. A small animal laryngoscope (SR310-M, Yuyan) was used to expose the inverted V-shaped vocal folds. The drug was delivered into the bronchoalveolar system through the vocal folds via a liquid pulmonary delivery nebulizer (Model Yan 30012, Yuyan). After administration, the rats were allowed to recover from anesthesia and were returned to their dams. No mortality was observed during the intratracheal administration procedure.

Aldh1a2 overexpression

The rat Aldh1a2 gene was cloned and inserted into a GFP-adenoviral vector to construct Ad-Aldh1a2 (Shanghai GeneChem Co., Ltd., Shanghai, China), while an empty adenoviral vector (Ad-NC) served as the negative control. To induce Aldh1a2 overexpression, hyperoxia-exposed rats were administered Ad-Aldh1a2 (3 × 10⁸ pfu/mL) via intratracheal injection through an endotracheal tube at P5, P8, and P11.

Fifty neonatal Sprague‒Dawley (SD) rats were used in this study, following the same experimental design as in the first animal model. The rats were randomly assigned to five groups: (1) the normoxia group (n = 10); (2) the hyperoxia group (n = 10, exposed to 80% O₂ and treated with PBS at P5, P8, and P11); (3) the hyperoxia + Ad-NC group (n = 10, exposed to 80% O₂ and treated with Ad-NC at P5, P8, and P11); (4) the hyperoxia + Ad-Aldh1a2 group (n = 10, exposed to 80% O₂ and treated with Ad-Aldh1a2 at P5, P8, and P11); and (5) the hyperoxia + MSC group (n = 10, exposed to 80% O₂ and treated with 1 × 10⁶ hUC-MSCs at P5, P8, and P11). All neonatal rats were sacrificed after 14 days of hyperoxia exposure.

Quantitative assessment of lung tissue morphology, inflammatory responses, and ventilatory function

Protein content and white blood cell count in BALF

BALF was collected prior to sacrificing the rats, following the methods described in a previous study [8]. Subsequently, protein concentrations and leukocyte counts in the BALF were analyzed.

Histological examination and immunohistochemistry

Lung tissue sections were subjected to hematoxylin‒eosin (H&E) and Masson staining following established protocols and the manufacturer’s instructions. Immunohistochemistry (IHC) was then performed on the same sections, as detailed in Supplementary material 1.

Measurement of malondialdehyde, myeloperoxidase and inflammatory cytokines in lung tissue

Myeloperoxidase (MPO) and malondialdehyde (MDA) levels in lung tissue were measured based on previous studies [8]. Additionally, the concentrations of interleukin-6 (IL-6), IL-10, IL-1β, and tumor necrosis factor-alpha (TNF-α) in the lung tissue homogenates were quantified via enzyme-linked immunosorbent assay (ELISA), as detailed in Supplementary material 1.

Arterial blood gas analysis

Arterial blood samples were collected from the rats in each group prior to sacrifice. The samples were analyzed via a GEM Premier 5000 blood gas analyzer (WALFEN, USA) to measure the pH, partial pressure of oxygen (PaO₂), and partial pressure of carbon dioxide (PaCO₂).

Analyzing the mechanisms using the RLE-6TN cell line

RLE-6TN cell culture and treatment

Primary rat type II alveolar epithelial cells (RLE-6TNs) and experimental groupings are detailed in Supplementary material 1. Oxidative stress was induced by exposing the cells to 0.2 mM H₂O₂ for 2 h. RLE-6TN cells were subsequently cocultured with UCMSCs to promote cellular repair.

Cell transfection and SiRNA treatment

To investigate the role of Aldh1a2 in oxidative stress injury, Aldh1a2 expression was silenced via siRNA (GenePharma, Shanghai, China). RLE-6TN cells were seeded in 12-well plates and cultured to 25–30% confluence prior to transfection. Following the manufacturer’s protocol, the cells were transfected with 40 nM siRNA via Lipofectamine 2000 (11668019, Invitrogen, USA), and the medium was replaced 6 h post-transfection. After 48 h and 72 h, the cells were analyzed by RT‒qPCR and WB, respectively, confirming that siRNA-1508 and siRNA-830 significantly reduced Aldh1a2 expression compared to the negative control (NC). These results validated successful siRNA transfection and target gene silencing.

For subsequent experiments, RLE-6TN cells were transfected for 36 h with Aldh1a2-siRNA1508, Aldh1a2-siRNA830, or NC, followed by exposure to 0.2 mM H₂O₂ for 2 h. The transfected cells were then co-cultured with UCMSCs in transwell chambers. After 48 h, apoptosis, proliferation, RT‒qPCR, and WB analyses were performed to assess the effects of Aldh1a2 downregulation.

Apoptosis and cell proliferation assays

The apoptosis and proliferation of RLE-6TN cells were assessed following standard protocols and the manufacturer’s instructions, as detailed in Supplementary material 1.

RT‒qPCR validation of differentially expressed genes

In earlier studies, animal experiments were conducted to investigate the reparative effects of UCMSCs on hyperoxic lung injury. Transcriptomic analysis via RNA sequencing [8] revealed 46 significant DEGs related to immune regulation. These genes were validated via RT‒qPCR. Furthermore, RT‒qPCR was employed to identify siRNAs with significant inhibitory effects, and functional studies on Aldh1a2 were performed both in vitro and in vivo. The detailed RT‒qPCR methods are provided in Supplementary material 1.

Western blotting (WB)

WB analysis of lung tissue and RLE-6TN cells was performed following the standard protocol outlined in Supplementary material 1.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism (v9.0) and SPSS (v26). Data from three independent experiments are presented as mean ± SEM. One-way or two-way ANOVA with Tukey’s multiple comparisons was used for statistical evaluation. Bonferroni tests were applied to group comparisons for significant ANOVA results. A P value < 0.05 was considered statistically significant.

Results

UCMSCs identification

The flow cytometry analysis of UCMSCs is provided in Supplementary material 2-Fig. 1.

UCMSCs increased the survival rate of neonatal rats with hyperoxia injury and promoted lung development

The survival curve was drawn from 7 days after birth. The survival rate of the hyperoxia group decreased, whereas that of the UCMSC group increased (Fig. 1A). Hyperoxic lung injury hindered weight gain, while UCMSC administration mitigated the growth retardation caused by hyperoxia (Fig. 1B).

Fig. 1.

UCMSCs increased the survival rate of neonatal rats and promoted lung development. (A) Kaplan-Meier survival curves of rat pups treated as indicated; (B) Body weight changes; (C) H&E staining of lung sections; magnification, 200×; scale bar = 100 μm. (D-G) Quantitative analysis of the MLI, MAD, RAC, and lung wet/dry weight ratio in different treatment groups (n = 5 for each group, 10 fields/animal). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

H&E staining revealed an intact lung structure, normal alveolar epithelium, and regular alveolar septa in the normoxia group (Fig. 1C). In contrast, the hyperoxia group exhibited simplified alveolar morphology, characterized by decreased radial alveolar count (RAC) and increased mean linear intercept (MLI) and mean alveolar diameter (MAD). UCMSC treatment significantly reduced hyperoxia-induced increases in MLI, MAD, and lung wet/dry weight ratio, while markedly improving the RAC (Fig. 1D-G).

UCMSCs attenuated hyperoxia-induced pulmonary artery remodeling and vascular loss

Alpha-SMA IHC staining and Masson’s trichrome staining were used to assess pulmonary vascular remodeling and interstitial hyperplasia (Fig. 2A). Compared with the normoxia group, the hyperoxia group presented an increased medial wall thickness index (MTI) and collagen fiber proliferation in the peripheral pulmonary vasculature. These changes were considerably alleviated by UCMSC treatment (Fig. 2BC).

Fig. 2.

UCMSCs attenuated hyperoxia-induced pulmonary artery remodeling and vascular loss. (A) Representative Masson, α-SMA, and vWF staining. Representative micrographs (scale bars = 100 μm) at 200× (Masson staining and α-SMA) and 400× (VWF). (B-D) Quantification of Maason staining, MTI, and MVD (n = 5 for each group, 5 fields/animal). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

To assess the impact of hyperoxia on peripheral pulmonary vessels, von Willebrand factor (vWF) staining was performed. Microvessel density (MVD) was significantly reduced in the hyperoxia group compared to the normoxia group, but UCMSC treatment effectively restored vascular density (Fig. 2D). Additionally, RT‒qPCR revealed decreased vascular endothelial growth factor (VEGF) mRNA expression in the hyperoxia group, which was partially restored by UCMSC intervention (Fig. 3).

Fig. 3.

Based on transcriptomic analysis of UCMSCs repairing hyperoxic lung injury, RT-qPCR identified 17 significant DEGs (n = 6). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

UCMSCs mitigated hyperoxia-induced lung inflammation and oxidative stress injury

To evaluate the effects of UCMSCs on hyperoxia-induced lung injury, inflammatory and oxidative stress markers were analyzed in BALF and lung tissue. Hyperoxia exposure significantly increased BALF protein concentrations, leukocyte counts, and lung tissue levels of MPO and MDA (Fig. 4A-D), which are indicators of neutrophil activity and oxidative stress, respectively. These changes were markedly reversed by UCMSC treatment. Cytokine analysis via ELISA revealed elevated levels of IL-1β, IL-6, and TNF-α in hyperoxia-exposed animals. In contrast, UCMSC intervention significantly reduced the levels of these proinflammatory cytokines and increased the levels of the anti-inflammatory cytokine IL-10 (Fig. 4E-H). IHC and WB analyses revealed increased caspase-3 expression under hyperoxia, which was considerably reduced by UCMSC treatment (Figs. 5 and 6). Moreover, IHC revealed a reduction in Ki67-positive cells in the hyperoxia group. In contrast, UCMSC administration restored Ki67 expression (Fig. 5).

Fig. 4.

UCMSCs mitigated hyperoxia-induced lung inflammation and oxidative stress injury. (A-B) MPO and MDA levels in lung tissues. (C-D) Leukocyte counts and protein concentrations in the BALF. (E-H) Levels of IL-10, TNF-α, IL-1β, and IL-6 in lung tissues. n = 5. * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

Fig. 5.

UCMSCs significantly suppressed CASPASE3 activation and concurrently upregulated Ki-67 expression in pulmonary tissues. (A) Representative immunohistochemistry image. Magnification 200×; scale bar = 100 μm. (B-C) Dual quantification of apoptotic and proliferative indices (n = 5). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

Fig. 6.

Hyperoxia exposure activated the NLRP3 inflammasome, triggered IL-1β release, and upregulated CASPASE3 expression, whereas UCMSC reversed these alterations. (A) Western blot analysis of key inflammasome components. Full-length blots/gels are presented in Supplemental Material 4. (B-F) Quantitative analysis of protein levels (normalized to GAPDH, n = 3). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

UCMSCs modulated the NLRP3 inflammasome signaling pathway in hyperoxia-induced lung injury

The expression levels of NLRP3, Caspase-1, ASC, and IL-1β in lung tissue were assessed by WB and IHC to investigate the effect of UCMSCs on NLRP3 inflammasome activity. Compared with those in the normoxia group, the NLRP3 levels were increased in the hyperoxia group, which could be partly reversed by UCMSC treatment. Similar changes were observed in Caspase-1, ASC, and IL-1β protein levels. We found that UCMSCs significantly inhibited the expression of the NLRP3 inflammasome signaling pathway in hyperoxia-induced lung injury (Fig. 6).

UCMSCs enhanced Aldh1a2 expression following hyperoxic lung injury

Previous transcriptome sequencing (CRA031576) of lung tissue during UCMSC-mediated repair [8] revealed 46 DEGs associated with immune regulation, macrophage differentiation, and the expression of channel proteins or neuroreceptors. The PCR primers used in the present study are detailed in Tables 2 and 4 of Supplementary Material 3. RT‒qPCR analysis further validated the significance of 17 DEGs during UCMSC-induced lung repair, including Tnfsf15, Gdf2, Aldh1a2, Krt14, Anxa3, Retnla, Gpnmb, Fcrl2, CD177, Sirpa, Igfbp6, Ascl, Nmur2, Vegf, Ccdc80, Chrm2, and Lamp3 (Fig. 3). These findings were consistent with transcriptome sequencing results, and all the differences were statistically significant. Functional analysis of these genes, focusing on immune microenvironment alterations during lung injury, identified Aldh1a2 (aldehyde dehydrogenase family 1, member A2) as a key target in immunomodulation for further investigation.

WB and RT‒qPCR assessed ALDH1A2 levels in lung tissue. Hyperoxia significantly reduced ALDH1A2 expression compared to normoxia, whereas UCMSC intervention markedly increased ALDH1A2 levels (Fig. 7BC). IHC analysis further confirmed these findings, showing that hyperoxia decreased ALDH1A2 expression, which was restored by UCMSC treatment (Fig. 7A, D). Additionally, IHC revealed that ALDH1A2 was predominantly expressed in alveolar epithelial cells.

Fig. 7.

UCMSCs upregulate ALDH1A2 expression in hyperoxic lung injury. (A, D) IHC staining and quantification. Magnification 200×; scale bar = 100 μm (n = 5). (B, C) Western blot analysis across lung groups. Full-length blots/gels are presented in Supplemental Material 4 (n = 3). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group

Aldh1a2 positively contributes to lung injury repair

In vitro validation of Aldh1a2 in UCMSC-mediated lung repair

To investigate the role of Aldh1a2 in UCMSC-mediated repair and its underlying mechanism, in vitro experiments were conducted using rat type II alveolar epithelial cells (RLE-6TNs). Oxidative stress injury was induced with H₂O₂, followed by UCMSC treatment. Apoptosis and proliferation assays confirmed that UCMSCs effectively repaired oxidative stress-induced injury, as detailed in Supplementary material 2-F3. PCR (Fig. 8) and WB (Fig. 14AB) analyses revealed a decrease in ALDH1A2 expression in the H₂O₂ group, which was restored upon UCMSC administration. These findings suggest that UCMSCs may protect against hyperoxic lung injury by regulating Aldh1a2 expression. To further validate this hypothesis, Aldh1a2 siRNAs were used in vitro. Among the tested siRNAs, siRNA1508 and siRNA830 effectively suppressed Aldh1a2 expression, with siRNA1508 exhibiting the most potent inhibitory effect, as detailed in Supplementary material 2-F4.

Fig. 8.

Suppression of Aldh1a2 exacerbated alveolar oxidative damage. (A) UCMSCs alleviated H₂O₂-induced alveolar oxidative stress injury. *P < 0.05 vs. Ctr group, ^P < 0.05 vs. H₂O₂ group. (B-C) Aldh1a2 downregulation aggravated oxidative stress injury and compromised the repair capacity of UCMSCs. n = 6. * P < 0.05 vs. Ctr-NC group; ^ P < 0.05 vs. H₂O₂-NC group; # P < 0.05 vs. H₂O₂ + MSCs-NC group

Fig. 14.

UCMSC or Aldh1a2 overexpression attenuated hyperoxia-induced NLRP3 inflammasome activation and pro-inflammatory signaling. Full-length blots/gels are presented in Supplemental Material 4 (n = 3). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

Cell apoptosis and proliferation assays were performed to evaluate the role of Aldh1a2 in oxidative stress-related lung injury. Annexin V/propidium iodide double staining revealed that siRNA transfection significantly increased apoptosis rates across all groups, with siRNA1508 inducing the highest level of apoptosis. However, UCMSC treatment effectively reversed these changes. Additionally, siRNA1508 reduced the proliferation index (Fig. 9).

Fig. 9.

Aldh1a2 inhibition attenuated UCMSC-mediated repair of oxidative stress injury in RLE-6TN cells. (A-B) Aldh1a2 suppression increased apoptosis. (C-D) Aldh1a2 downregulation impaired proliferation. n = 5. * P < 0.05 vs. Ctr-NC group; # P < 0.05 vs. H₂O₂-NC group; ^ P < 0.05 vs. H₂O₂ + MSCs-NC group

To examine the effects of UCMSCs on injured lung tissue, RT‒qPCR was used to assess genes involved in immune regulation, alveolar water balance, microangiogenesis, and pulmonary surfactant (PS) synthesis. The results revealed that oxidative stress injury increased lung inflammation, disrupted the alveolar water balance and PS synthesis, and impaired the production or secretion of microcirculatory factors in alveolar capillaries. UCMSC treatment considerably reversed or alleviated these changes. However, siRNA1508 transfection exacerbated H₂O₂ injury-induced disruption and diminished the reparative effects of UCMSCs (Fig. 8). These findings suggest that Aldh1a2 plays a crucial role in UCMSC-mediated repair of lung injury, with its down-regulation aggravating oxidative stress-induced damage to lung epithelial cells.

In vivo validation of the protective role of Aldh1a2 in lung injury

UCMSCs and Aldh1a2 overexpression enhanced Aldh1a2 expression (Supplementary material 2-F5)

Aldh1a2 mitigated hyperoxia-induced alveolar simplification and vascular damage

Survival analysis from postnatal day 5 revealed decreased survival rates in the hyperoxia group. In contrast, UCMSC and Ad-Aldh1a2 treatments significantly improved survival, with UCMSC showing a more pronounced effect (Fig. 10A). Additionally, hyperoxic lung injury impaired weight gain, but the administration of UCMSCs or Ad-Aldh1a2 reversed the growth retardation caused by hyperoxia (Fig. 10B).

Fig. 10.

UCMSCs or Ad-Aldh1a2 improved survival and lung development in neonatal rats with hyperoxia injury. (A) Kaplan-Meier survival curves. (B) Body weight alteration. (C) H&E staining of lung sections. Magnification 200×; scale bar = 100 μm. (D-F) Quantitative analysis of RAC, MLI, and MAD under different treatments (n = 6 for each group, 10 fields/animal). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

HE staining revealed intact lung tissue structure in the normoxia group, whereas the hyperoxia group exhibited simplified alveolar morphology (Fig. 10C). Both Aldh1a2 overexpression and UCMSC treatment significantly improved alveolar simplification (Fig. 10D-F).

Compared with those of the normoxia group, Masson staining and α-SMA immunohistochemistry revealed increased collagen fiber deposition and MTI in the peripheral pulmonary vasculature of the hyperoxia group. These changes were significantly reversed by UCMSC or Ad-Aldh1a2 treatment (Fig. 11A-D). Similarly, vWF staining revealed a marked reduction in the MVD in the hyperoxia group, which was considerably restored following UCMSC or Ad-Aldh1a2 intervention (Fig. 11EF). Furthermore, RT‒qPCR analysis demonstrated decreased VEGF mRNA expression in the hyperoxia group, which was increased after UCMSC or Ad-Aldh1a2 treatment (Fig. 12A).

Fig. 11.

UCMSCs or Ad-Aldh1a2 attenuated hyperoxia-induced pulmonary artery remodeling and vascular loss. Magnification 200×; scale bar = 100 μm. (A-B) Masson’s trichrome staining. (C-D) α-SMA IHC staining. (E-F) vWF staining. n = 6 for each group, 5 fields/animal. * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

Fig. 12.

UCMSC or Aldh1a2 overexpression attenuated hyperoxia-induced lung inflammation and improved pulmonary ventilation. (AB) RT‒qPCR analysis (n = 6). (C-E) Blood gas analysis (n = 5). (F-M) Inflammatory factor changes (n = 5). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

Aldh1a2 overexpression preserved alveolar function and improved pulmonary ventilation

RT‒qPCR analysis revealed that hyperoxia disrupted the alveolar water balance, impaired PS synthesis, and inhibited alveolar microangiogenesis. These changes were partially reversed or alleviated by UCMSC pretreatment or Aldh1a2 overexpression (Fig. 12AB). Compared with those in the normoxia group, arterial blood gas analysis showed significantly decreased PaO₂, increased PaCO₂, and reduced pH levels in the hyperoxia group. In the UCMSC and Ad-Aldh1a2 groups, the dysfunction of gas exchange induced by hyperoxia was partially reversed (Fig. 12C-E).

Aldh1a2 attenuated hyperoxia-induced lung inflammation and oxidative stress injury

Compared with those in the normoxia group, the BALF protein concentration and leukocyte count were significantly elevated in the hyperoxia group. Treatment with UCMSCs or Aldh1a2 overexpression reduced these increases (Fig. 12LM). Similarly, lung tissue levels of MPO and MDA followed the same trend (Fig. 12FG). The ELISA results revealed that hyperoxia exposure considerably upregulated pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while IL-10 levels were reduced. These inflammatory changes were notably reversed by UCMSC or Ad-Aldh1a2 intervention (Fig. 12H-K). Furthermore, IHC (Fig. 13A-D) and WB (Fig. 14) analyses revealed hyperoxia-induced upregulation of Caspase-3 and IL-1β, which was markedly mitigated after administration of UCMSCs or Ad-Aldh1a2. Conversely, Ki67 expression showed the opposite trend, indicating enhanced lung repair (Fig. 13EF). The RT‒qPCR results further indicated that hyperoxia promoted lung inflammation, and these effects were attenuated by UCMSC or Ad-Aldh1a2 treatment (Fig. 12AB).

Fig. 13.

IHC analysis of CASPASE3, IL-1β, and Ki67 expression in lung tissue. Magnification 200×; scale bar = 100 μm (n = 6). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

UCMSCs protected against lung injury by modulating the NLRP3 inflammasome pathway via Aldh1a2

In vitro (Fig. 15AB) and in vivo (Figs. 3 and 7) experiments demonstrated that the expression of Aldh1a2 decreased after lung injury while elevated after UCMSC administration. Hyperoxic lung injury significantly upregulated the expression of NLRP3 inflammasome components (NLRP3, ASC, and Caspase-1) and IL-1β in lung tissues, and these changes were reversed by UCMSC treatment (Fig. 6). Similarly, in RLE-6TN cells exposed to H₂O₂, injury-induced upregulation of the NLRP3 inflammasome and IL-1β was attenuated by UCMSC administration (Fig. 15C-H). Furthermore, siRNA1508 transfection significantly suppressed Aldh1a2 expression (Supplementary material 2-F4), leading to a marked elevation of inflammation-related proteins across all groups (Fig. 16). These findings suggest that Aldh1a2 may regulate the NLRP3 inflammasome pathway during lung injury and UCMSC-mediated repair. Notably, in a second animal experiment, Aldh1a2 overexpression considerably improved hyperoxia-induced lung injury. As shown in Figs. 14 and 17, hyperoxia increased the levels of the NLRP3 inflammasome and IL-1β, and these effects were mitigated by UCMSC treatment or Aldh1a2 overexpression.

Fig. 15.

UCMSCs upregulated ALDH1A2 expression and attenuated NLRP3 inflammasome-mediated inflammation in H₂O₂-induced injury. Full-length blots/gels are presented in Supplemental Material 4. (A-B) ALDH1A2 expression dynamics. (C-H) Inflammatory protein profiles. n = 3. * P < 0.05 vs. Control group; # P < 0.05 vs. H₂O₂ group

Fig. 16.

H₂O₂ exposure activated the NLRP3 inflammasome and upregulated the expression of inflammatory mediators, which were attenuated by UCMSC treatment. Aldh1a2 suppression exacerbated these H₂O₂-induced inflammatory responses. Full-length blots/gels are presented in Supplemental Material 4 (n = 3). * P < 0.05 vs. Control group; # P < 0.05 vs. H₂O₂ group; ^ P < 0.05 vs. H₂O₂ + MSC group; @ P < 0.05 vs. Control + siRNA group; & P < 0.05 vs. H₂O₂ + siRNA group. siRNA: 1508

Fig. 17.

Aldh1a2 overexpression suppresses NLRP3 inflammasome-associated protein expression. Magnification 200×; scale bar = 100 μm (n = 6). * P < 0.05 vs. Normoxia group; # P < 0.05 vs. Hyperoxia group; ^ P < 0.05 vs. Hyperoxia + MSC group; @ P < 0.05 vs. Hyperoxia + Ad-NC group

Discussion

To explore the molecular mechanisms underlying BPD, in vivo and in vitro lung injury models were established via neonatal SD rats exposed to hyperoxia and RLE-6TN cells exposed to H₂O₂. This study demonstrated that hyperoxia aggravated alveolar inflammation, resulting in alveolar simplification, impaired vascular development, and abnormal lung function. The application of UCMSCs in these models effectively attenuated inflammation, promoted alveolar structural repair and microangiogenesis, and significantly improved lung function. Lung transcriptomics analysis revealed DEGs, which were validated via RT‒qPCR and Western blotting, leading to the identification of the immunoregulatory gene Aldh1a2. Further investigation revealed that the upregulation of Aldh1a2 inhibited, while its downregulation promoted, the expression of the NLRP3 inflammasome, highlighting the critical role of this gene in modulating inflammatory responses during lung injury (Fig. 18).

Fig. 18.

Potential mechanisms of Aldh1a2-mediated mitigation of hyperoxia-induced lung injury in neonatal rats. Hyperoxia exposure activates the NF-κB pathway, leading to NLRP3 inflammasome activation and subsequent cellular damage accompanied by alveolar simplification. Aldh1a2 promotes AT2-to-AT1 differentiation, enhances VEGF expression, and suppresses the NF-κB pathway, thereby attenuating NLRP3 inflammasome activation and ultimately mitigating alveolar injury

MSCs exhibit diverse therapeutic functions, including immunomodulation, angiogenesis, inhibition of apoptosis, and tissue repair through differentiation and paracrine secretion [6, 7]. In the context of BPD, MSCs demonstrate anti-inflammatory and antioxidative effects, facilitate macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, promote angiogenesis, and enhance the differentiation of alveolar type II cells (AT2) into gas-exchanging alveolar type I cells (AT1) [6]– [7, 13–16]. Together, these processes contribute to restoring damaged lung tissue and normalizing alveolar gas exchange.

Inflammation-induced by oxidative stress injury is a key pathway involved in the development of the BPD phenotype [2, 3]. The NLRP3 inflammasome, a multiprotein complex comprising NLRP3, ASC (apoptosis-associated speck-like protein), and caspase-1, is upregulated during oxidative stress and systemic infections [17, 18]. As an essential component of innate immunity, the NLRP3 inflammasome detects soluble pathogens and triggers the release of the proinflammatory cytokines IL-1β and IL-18 [19]. It has been implicated in lung injury caused by hyperoxia, sepsis, mechanical ventilation, and ischemia‒reperfusion [20, 21]. Liao et al. highlighted its critical role in BPD pathogenesis [22]. Macrophages are the primary site of NLRP3 inflammasome activation, although they are also observed in lung epithelial cells [18]. This study demonstrated that UCMSCs effectively reversed hyperoxia-induced upregulation of the NLRP3 inflammasome and IL-1β. UCMSCs appear to mitigate inflammation by suppressing NLRP3 inflammasome activation, thereby facilitating lung tissue repair and functional recovery.

UCMSCs maintain their stemness and multilineage differentiation potential through epigenetic mechanisms, including histone modifications and DNA methylation. Through paracrine signaling, UCMSCs remodel the epigenetic landscape of adjacent cells, thereby promoting immunomodulation, tissue repair, and regeneration [23]. In a murine model of auricle regeneration, introduction of the rabbit-derived enhancer 1 (AE-1) restored Aldh1a2 expression and regenerative capacity. In AE-1 transgenic mice, ALDH1A2 protein localized to both epithelial cells and MSCs, with the highest expression detected in the epidermal layer. The Activator Protein 1 (AP-1) complex directly binds to the regulatory region of Aldh1a2 to initiate its transcription, which is further enhanced by retinoic acid signaling during regeneration [24]. UCMSCs significantly upregulated Aldh1a2 expression in alveolar epithelial cells, a process hypothesized to be mediated by UCMSC-derived paracrine factors that remodel the epigenetic state at Aldh1a2 regulatory elements AE1 and AE5. Specifically, UCMSCs increase active histone modifications (H3K4me3 and H3K27ac) while concurrently reducing local DNA methylation, constituting an epigenetic reprogramming event that stabilizes and robustly activates the Aldh1a2 transcriptional program. Additionally, UCMSCs secrete various bioactive factors, including exosomes, transforming growth factor-beta, and hepatocyte growth factor, which are proposed to activate the AP-1 transcription factor complex in epithelial cells through MAPK pathway modulation [6, 25], thereby facilitating AP-1 binding to Aldh1a2 regulatory elements and enhancing transcription.

Aldh1a2, encoding the rate-limiting enzyme for retinoic acid synthesis, plays a critical role in embryonic septum and cardiovascular formation and in the normal development of the diaphragm, cardiopulmonary, and urinary systems [26]. In addition, Aldh1a2 is a critical regulator of tissue regeneration in mammals [24]. ALDH1A2 is a cytosolic protein characterized by a highly restricted expression pattern, predominantly localized to specific epithelial cells, immune cells, and developing progenitor cells [24]. In the respiratory system, it is predominantly enriched in epithelial cells with regenerative capacity [27, 28]. Within the immune system, its expression is largely limited to dendritic cells and regulatory T cells, which directly modulate immune responses [9–12]. Aberrant expression of Aldh1a2 has been associated with congenital diaphragmatic lung injury [29]. Additionally, beyond its involvement in lymphocyte recruitment [9–12], Aldh1a2 modulates the immune microenvironment by driving macrophage polarization from the M1 to the M2 phenotype via the regulation of MMP-2 and MMP-9 activity [11, 30, 31]. In sepsis-associated lung injury repair, Aldh1a2 has been shown to facilitate AT2-to-AT1 cell differentiation, suppress inflammation, and enhance alveolar ventilation [32]. Consistent with these findings, we hypothesize that Aldh1a2 plays a pivotal role in immune regulation and AT2-to-AT1 differentiation during UCMSC-mediated lung repair.

To test this hypothesis, Aldh1a2 expression was downregulated in RLE-6TN cells. This downregulation exacerbated inflammation and NLRP3 inflammasome activation, increased apoptosis, inhibited cellular proliferation and microvascularization, disrupted surfactant synthesis and the water balance, and impaired the reparative effects of UCMSCs. Conversely, overexpression of Aldh1a2 via recombinant adenoviral vectors resulted in reparative effects similar to those of UCMSCs in hyperoxic lung injury. Aldh1a2 overexpression reduced tissue inflammation, promoted cell proliferation, inhibited apoptosis, ameliorated alveolar simplification and microvascular deficiency, maintained surfactant secretion and water balance homeostasis, and improved alveolar ventilation. These results suggest that Aldh1a2 is a key regulator of UCMSC-mediated repair and a promising therapeutic target for hyperoxic lung injury.

Studies have shown that Aldh1a2 is a key component of the RA-Hedgehog-Wnt gene regulatory network [33]. The Wnt signaling pathway is crucial for cardiopulmonary vascular development and is implicated in the progression of pulmonary hypertension (PPHN) [34]. We hypothesize that Aldh1a2 may regulate the Wnt signaling pathway during UCMSC-mediated lung injury repair, thereby inhibiting PPHN development. In hyperoxic lung-injured neonatal rats, UCMSCs mitigate peripheral pulmonary vascular loss, reduce the mean linear intercept (MLI), and decrease vascular smooth muscle thickness. PCR analysis revealed that hyperoxia downregulated Vegf expression, whereas UCMSC treatment restored its expression. Further investigations demonstrated that Aldh1a2 downregulation inhibited Vegf expression in RLE-6TN cells, whereas Aldh1a2 upregulation in hyperoxic rats enhanced microvascular formation, alleviated alveolar simplification, and increased Vegf expression. VEGF, a potent mitogen, plays a critical role in angiogenesis and alveolar development in endothelial cells [35]. These findings suggest that Aldh1a2 promotes UCMSC-mediated lung repair by ameliorating alveolar structural abnormalities, mitigating microvascular loss, and increasing Vegf-mediated angiogenesis (Fig. 18).

Downregulation of Aldh1a2 promotes NLRP3 inflammasome expression, whereas Aldh1a2 upregulation suppresses NLRP3 activation. The NLRP3 inflammasome contributes to the pathogenesis of lung injury by signaling pathways such as the NF-κB and MAPK/NF-κB pathways, which regulate macrophage activation, polarization, cell proliferation, and apoptosis [36–40]. Aldh1a2 has been shown to exert immunomodulatory effects by influencing macrophage polarization and dendritic cell activity [10, 11, 41, 42]. Cui et al. reported that somatostatin 4 inhibits oral squamous cell carcinoma progression through the NF-κB/DNMT1/ALDH1A2 axis [43], highlighting a potential link between Aldh1a2 and NF-κB signaling. Although research on the interplay between Aldh1a2 and NLRP3 inflammasome activation remains limited, both are implicated in macrophage activation, polarization, and the NF-κB signaling pathway. We propose that Aldh1a2 exerts anti-inflammatory effects by inhibiting NLRP3 inflammasome expression during lung injury and UCMSC-mediated repair (Fig. 18).

In UCMSC-mediated lung injury repair, Aldh1a2 exerts anti-inflammatory effects by suppressing NLRP3 inflammasome expression, inducing AT2-to-AT1 differentiation to enhance alveolar ventilation, promoting microangiogenesis, and supporting alveolar development by maintaining VEGF expression. Additionally, it inhibits the progression of PPHN by regulating the Wnt signaling pathway. This study revealed that Aldh1a2 facilitates the repair of hyperoxic lung injury through the inhibition of the NLRP3 inflammasome. Further exploration of this mechanism will be a key focus of future research.

Conclusions

This study revealed that UCMSCs mitigate lung injury by upregulating Aldh1a2, which suppresses NLRP3 inflammasome formation. Further research is needed to clarify the precise regulatory mechanisms of Aldh1a2 and the specific signaling pathways involved in its targeting of the NLRP3 inflammasome during UCMSC-mediated lung repair.

Abbreviations

BPD

Bronchopulmonary dysplasia

MSC

Mesenchymal stem cells

UCMSC

Umbilical cord-derived MSC

ALDH1A2

Aldehyde dehydrogenase 1 family member A2

NLRP3

Nod-like receptor protein 3

Ad-Aldh1a2

Recombinant adenovirus vector for Aldh1a2 overexpression

NC

Negative control

Ad-NC

Adenovirus-mediated negative control for Aldh1a2 overexpression

RLE-6TN

Rat type II alveolar epithelial cells

BALF

Bronchoalveolar lavage fluid

Author contributions

Xuejing Xu completed the experiment and analyzed the results statistically; Xiuli Ju and Dong Li proposed the design and conception of the experiment; Na Dong, Tianqing Xin and Qing Shi assisted with the collection of tissue and blood samples from animals; Linghong Liu supervised the animal protocols and integrated the materials; Xuejing Xu wrote the first manuscript of the study; Xiuli Ju and Dong Li revised it critically; and all authors have read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (No. ZR2023MH079); the Ji’Nan Science and Technology Bureau (No. JNKCJN202201); the Shandong Provincial Hospital for Maternal and Child Health, Ministry of Health Key Laboratory of Reproductive Regulation Technologies (No. 2023005), and the Natural Science Foundation of Shandong Province (ZR2024ZD23).

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Ethics Committee on Scientific Research of Qilu Hospital of Shandong University (The approved title is Umbilical Cord Mesenchymal Stem Cell Therapy Attenuates Hyperoxia-Induced Multisystem Organ Injury in Neonatal Rats (Approval No. DWLL-2021-035; Date of Approval: March 25, 2021). The hUC-MSCs used in this study were provided for research purposes by Shandong Provincial Umbilical Cord Blood Hematopoietic Stem Cell Bank. The supplier confirmed that original umbilical cord tissue collection was conducted following the protocol, “Umbilical Cord Mesenchymal Stem Cell Therapy in Preterm Infants with Bronchopulmonary Dysplasia: A Prospective Randomized Controlled Trial” approved by the Ethics Committee on Scientific Research of Qilu Hospital of Shandong University (Approval No. KYLL-202008-111; Date of Approval: August 31, 2020); in addition, they confirmed that informed consent was obtained from all donors prior to tissue collection. As this study was conducted on pre-collected, anonymized cells, and did not involve direct human participation, no additional ethics approval was required for their use.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.