Deficiency of SPP1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice

doi:10.21203/rs.3.rs-4148717/v1

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Deficiency of SPP1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice

https://doi.org/10.21203/rs.3.rs-4148717/v1

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Bronchopulmonary dysplasia (BPD) is a common chronic lung disorder characterized by impaired proximal airway and bronchoalveolar development in premature births. SPP1 is involved in lung development and lung injury events, while its role was not explored in BPD. In the current study, the hyperoxiainduced lung injury animal models were generated by exposing neonatal mice to hyperoxia for 7 days after birth, and alveolar myofibroblasts (AMYFs) were treated with hyperoxia to establish the in vitro models of BPD. Based on the scRNA-seq analysis of lungs of mice housed under normoxia or hyperoxia conditions, mouse macrophages and fibroblasts were main different cell clusters between the two groups, and differentially expressed genes in fibroblasts were screened. Further GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly enriched in the pathways related to cell proliferation, apoptosis as well as the PI3K-AKT and ERK/MAPK pathways. SPP1 was found up-regulated in the lung tissues of hyperoxia mice. We also demonstrated the up-regulation of SPP1 in the BPD patients as well as hyperoxiainduced lung injury mouse models and cells. SPP1 deficiency was revealed to reduce the hyperoxia-induced apoptosis, oxidative stress and inflammation and increase the viability of AMYFs. In the hyperoxiainduced lung injury mouse models, SPP1 deficiency was demonstrated to reverse the hyperoxia-induced alveolar growth disruption, oxidative stress and inflammation. Overall, SPP1 exacerbates BPD progression in vitro and in vivo by regulating oxidative stress and inflammatory response via the PI3K-AKT and ERK/MAPK pathways, which might provide novel therapeutic target for BPD therapy.

SPP1

bronchopulmonary dysplasia

oxidative stress

preterm birth

The mechanical ventilation or supplementation with oxygen is often required by premature infants with respiratory disorders,and the hyperoxia can lead to inflammation and development of bronchopulmonary dysplasia (BPD) [1]. As the most prevalent chronic lung diseases in newborn infants, around 40–45% of extremely preterm infants are estimated to develop BPD, and the risk of BPD is thought to be negatively related to the birth weight and gestational age [2, 3]. The development of BPD is associated with longstanding consequences such as persistent pulmonary dysfunction in young adults, and is a systemic condition that affects the life quality of patients [4]. Currently, the BPD management mainly includes high-oxygen therapy and pharmacotherapy such as caffeine and vitamin A [5]. Whereas, the BPD incidence also shows an increasing trend in spite of/due to the progressed neonatal intensive care [6]. Thus, it is essential to understand the molecular basis and develop effective strategies for the prevention and treatment of BPD.

Exposure to hyperoxia is a primary risk factor for preterm infants, and as a result of the immature antioxidant defense system, oxidative stress can be induced and lead to oxidative damage in multiple organs, BPD included [7]. Oxidative stress damage involves the inflammatory injury and the evolved chronic and structural scarring related to BPD, and contributes to the increased morbidity [8]. Targeting the oxidative lung injury is suggested as a novel therapeutic strategy for BPD. Additionally, inflammation also plays an important role in the pathogenesis of BPD.Hyperoxia is revealed to increase the infiltration of inflammatory cells to the lungs and facilitate the secretion of chemotactic factors. Moreover, the excessive oxygen exposure also elevates the ROS production and stimulate the secretion of proinflammatory cytokines [9, 10]. Therefore, understanding the potential mechanism regulating oxidative stress and inflammation might benefit the targeted therapy of BPD.

Secreted phosphoprotein 1 (SPP1), also known as osteopontin (OPN), is an extracellular secreted glycol phosphoprotein [11]. Previously, it is reported to be expressed by osteoclasts, macrophages, and epithelial cells. Accumulating studies have also shown that SPP1 is involved in the progression of many cancers, including lung cancer [12, 13]. Additionally, SPP1 is reported to be dysregulated in pulmonary disorders and lung injury [14, 15], and the anti-inflammatory or pro-inflammatory role of SPP1 is reported in the context of the injury model. For example, SPP1 promotes the lung injury and fibrosis in the progression of acute respiratory distress syndrome by increasing Th17/Treg and M1/M2 ratios [16]. The administration of bone marrow stromal cell-conditioned media (BMSC-CM) show protective effects on BPD mice and reduces alveolar loss and accumulation of neutrophils and macrophages. SPP1 is up-regulated in BMSC-CM and is suggested to be associated with this immunomodulatory process [17]. However, the explicit function and mechanism of SPP1 in BPD progression still requires further investigation.

This study investigated the differentially expressed genes in the lung fibroblasts from normal oxygen or hyperoxia-induced neonatal mice, and SPP1 was found to be up-regulated by hyperoxia exposure. The effects of SPP1 deficiency on BPD progression in vitro and in vivo were explored, which might provide clues for the molecular basis underlying BPD development.

Neonatal tracheal aspirate samples

Totally 20 human neonates with BPD and 20 control counterparts who were intubated for primarily non-pulmonary diseases were recruited in this study.All BPD infants met the 2018 diagnostic criteria for BPD by the National Institute of Child Health and Human Development (NICHD)[18].The two groups of infants were matched in gender and gestational age.Tracheal aspirate (TA) samples were collected via in-line suctioning from intubated neonatal at the Intensive Care Unit (ICU).The study was approved by the Ethics Committee of the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University.

Animal experiment

The procedures of the animal study were performed under the approval of the Ethics Committee of our hospital. Neonatal mice were provided by Aniphe Biolaboratory Inc. (Jiangsu, China) and randomized into the Air,O₂,O₂ + sh-NC and O₂ + sh-SPP1 groups (n = 6 in each group). Mice were fed by the dams in sealed plexiglass chambers at22-27°C and 55% humidity. Mice in the Air group were exposed to room air (21% O₂) from postnatal day 1 to 7. Mice in the O₂ group were exposed to continuous oxygen at a flow rate of 5L/min to maintain FiO₂ > 95% from postnatal day 1 to 7. To explore the effects of SPP1 knockdown on hyperoxia-induced lung injury, mice from postnatal day 2 were intratracheally instilled with 1 µL normal saline containing adenovirus vector expressing sh-NC or sh-SPP1(GenePharma, Shanghai, China) at a titer of 1.58× 10⁹PFU/L for two times with an interval of 24 h.

Lung sample collection and histological analysis

Mice were anaesthetized by intraperitoneal injection of 10 mg/kg lidocaine on postnatal day 7, tracheas were cannulated, and the lungs were excised, followed by euthanasia with CO₂. The upper lungs were snap frozen in liquid nitrogen and stored at -80°C. The rest lung tissues were fixed with 4% paraformaldehyde (Beyotime, China)at 4°C overnight, embedded in paraffin, and sliced into 5-µm sections. Then the sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (Beyotime, China) following the manufacturer’s protocol. The histological changes in mouse lung tissues were observed using a light microscope(Nikon, Tokyo, Japan). The radial alveolar count (RAC) and mean linear intercept (MLI) was evaluated as previously described[19].

Immunohistochemistry(IHC)

The paraffin-embedded lung tissues were dewaxed, rehydrated and processed with 3% H₂O₂ for 10 min at room temperature. The samples were heated for 10 min at 105°C in citric acid buffer for antigen retrieval, and subsequently blocked with normal goat serum at 37°C. Then the samples were incubated with anti-α-SMA, anti-E-Cadherin,anti-p-PI3K, anti-p-AKT, anti-p-MEK1/2, and anti-p-ERK1/2 overnight at 4°C. Next, the samples were cultured with the HRP-conjugated secondary antibody for 40 min. After color development with diaminobenzidine peroxide solution and counterstaining with hematoxylin, the images were captured using a microscope.

Glutathione (GSH) and Malondialdehyde (MDA) measurement

The GSH levels and MDA levels in transfected cells and mouse lung tissues were determined to use a total GSH assay kit (BC1175, Solarbio, China) and an MDA Assay Kit (D799762, Sangon Biotech, Shanghai, China) respectively following the manufacturer’s protocol.

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of inflammatory cytokines (TNF-α, IL-1β) in mouse lung tissue homogenates and culture supernatant of AMYFs were measured using specific commercial ELISA kits obtained from Beyotime, in accordance with the manufacturer’s instructions.

Single-cell RNA sequencing (scRNA-seq) analysis

scRNA-seq analysis was conducted by the Metware Inc. (Wuhan, China). Single-cell suspension (1000 cells/µL) was prepared as previously described[20]. Mouse lungs were perfused with PBS, minced with scissors and digested at 37°C for 35 min. After filtration with a cell strainer, the red blood cells were lysed with ACK lysis, followed by resuspending the single cells in FACS buffer with 1% FBS (Gibco, USA) and 1 mM EDTA. Next, the 10X Genomics was applied to perform the scRNA-seq analysis following the manufacturer’s guide. Briefly, the single-cell suspension was loaded in 10x Chromium, and gel beads with barcode information were mixed with individual cells and enzymes in oil droplets based on microfluidic techniques to form the single-cell gel beads-in-emulsion (GEMs), followed by dissolving the beads and lysing the cells for Barcode sequence release, which was subsequently reverse transcribed into cDNAs and amplified. Next, the production of GEMs was mixed, and library was prepared based on the standard protocol. The Illumina platform (NovaSeq 6000) was applied for RNA sequencing. The Raw data were processed using Cell Ranger software (Version 7.1.0), and the gene expression matrix of all types of cells was obtained. Data dimension reduction, clustering, analysis and differential expression analyses were then performed using Seurat (Version 4.1.1). Single R and scCATCH software were applied for cell type identification and labelling. Gene Ontology (GO) and KEGG enrichment analyses were performed to analyze the signaling pathways affected by hyperoxia using the cluster Profiler package in R software (Version 4.1.2).

Cell culture and treatment

Mouse AMYFs were isolated from wild-type C57BL/6J mice as previously described [21]. Raw264.7 cells were purchased from IMMOCELL (Xiamen, China). Cells were cultured in DMEM containing 10%fetal bovine serum (FBS,Gibco, USA) and Penicillin-Streptomycin(1%, Sigma-Aldrich) at 37°C with 5% CO₂.To construct the in vitro model of BPD, cells were exposed to 95% O₂ and 5% CO₂ in sealed glass chambers. Cells exposed to normoxia (21% O₂ and 5% CO₂) were used as the control group.

Cell transfection

Short hairpin RNAs (shRNAs) targeting SPP1 (sh-SPP1) and scrambled control shRNAs (sh-NC) were designed and synthesized by GenePharma (Shanghai, China). Cells were transfected with the sh-SPP1 or sh-NC vectors using Lipofectamine 2000 (ThermoFisher Scientific) following the manufacturer’s protocol for 48 h.

Western blot

Total protein in lung tissues and fibroblasts was extracted using the RIPA lysis buffer (Beyotime, China). Next,the protein concentration was assessed using a bicinchoninic acid protein assay kit. After separation with the 8% SDS-PAGE gelsat 100 V, the samples were electro-transfer onto the PVDF membranes, followed by blocking with 5% skimmed milk. Subsequently, the membranes were incubate dover night at 4°C with the anti-SPP1, anti-Bax, anti-Bcl-2, anti-α-SMA, anti-E-Cadherin, anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2, anti-p-ERK1/2 with GAPDH as the internal control. Next, the membranes were washed with TBST and cultured with the HRP-conjugated secondary antibodies for 60 min at ambient temperature. All antibodies were obtained from Abcam (Shanghai, China). The target blots were developed with enhanced chemiluminescence reagents(Thermo Fisher Scientific) and quantified using the Image Lab 5.0 software (Bio-Rad).

Quantitative Real‑Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from cells, mouse lung tissues and neonatal tracheal aspirate samples using Trizol (Invitrogen, USA), and then reverse transcribed into cDNA using aVic qRT Super Kit (Vicmed, Xuzhou, China). Next, PCR was conducted with the qPCR SYBR® Green Master Mix(Vicmed) following the manufacturer’s instructions.Relative expression of RNAs was quantified with the 2^−ΔΔCt method, with GAPDH as the internal control.

Cell viability

Cells were grown into 96-well plates at 5000 cells/well. After incubation for 48 h, 10 µLCell Counting Kit‑8 (CCK-8) reagent (Beyotime, China) was added into each well and cultured for 3 h at 37°C, and the absorbance values at 450 nm was determined using a microplate reader (Bio-Rad, USA).

Flow cytometry analysis

Transfected cells were harvested and seeded into six-well plates at 3 × 10⁵ cells/well. The Annexin-V-FITC/PI apoptosis kit (Absin, Shanghai, China) was applied for determining the cell apoptosis rate following the manufacturer’s protocol. Briefly, cells were washed with cold PBS and resuspended in binding buffer. Subsequently,5 µL Annexin V-FITC and 10 µL propidium iodide (PI) were added and cultured for 10 min in dark at room temperature. Finally, the apoptotic rate was detected using flow cytometry (CytoFLEX, Beckman, USA) and analyzed with CytExpert software (Beckman).

Statistical analysis

Data analysis was conducted using GraphPad Prism9. Data are shown as mean ± standard deviation (SD). The t-test was used for comparing the statistical difference of two groups, and that of multiple groups was analyzed with one-Way ANOVA followed by Tukey’s post hoc test. P < 0.05 was regarded as statistical significance.

Hyperoxia induced alveolar growth arrest in hyperoxiainduced lung injury mouse models

For the establishment of lung injury mouse models, wild-type neonatal mice were maintained in hyperoxia conditions (95% O₂) for 7 days. There were no death in the model(O₂)and control (Air)groups. As shown in HE staining images of mouse lung tissues, those exposed to hyperoxia showed enlarged alveoli and decreased number of alveoli compared with mice exposed to room air (Fig. 1A). Meanwhile, the mean linear intercept (MLI) showed significant increase in the O₂ group relative to the normoxia, while the radial alveolar count (RAC) was reduced by hyperoxia exposure relative to room air (Fig. 1B-C). Overall, these results suggested that hyperoxia leads to alveologenesis disruption in lung injury mouse models.

Hyperoxia induces SPP1 up-regulation in mouse lungfibroblasts

To investigate the molecular alteration caused by hyperoxia in neonatal mouse lungs, scRNA-seq was used to profile the transcriptome data of mouse lungs in each group. The UMAP showed 30 distinct cell clusters were identified corresponding to 9 major cell lineages (Fig. 2A). We found that compare with the Air_lung group, 13 clusters showed significant difference in the O₂_lung group (Fig. 2B). Furthermore, as annotated in Fig. 2C, the fibroblasts were the major cell lineage differentially distributed between the Air_lung and O₂_lung groups (Fig. 2C). Since BPD is characterized by the alveologenesis disruption, and fibroblasts are reported to regulate the lung branching and alveogenesis [22], and macrophages are closely associated with the pulmonary fibrosis process [23]. We focused on the molecular changes in lung fibroblasts and macrophages. The differentially expressed genes were screened, and the volcano plot showed the significantly upregulated and downregulated RNAs in the lung fibroblasts and macrophages of hyperoxia and normoxia mice. SPP1 was confirmed as a highly-expressed gene in lung fibroblasts and macrophages of hyperoxia mice (Fig. 2D-G). Furthermore, the according to GO and KEGG enrichment analysis, we found that the hyperoxia significantly affected the signaling pathways related to cell proliferation, apoptosis, inflammation in fibroblasts and macrophages in GO analysis and the ERK/MAPK as well as the PI3K/Akt pathways in KEGG analysis (Fig. 2H-K). Overall, these data indicated that hyperoxia induces aberrant up-regulation of SPP1 in mouse fibroblasts and macrophages and may affect cell functions by regulating the related pathways.

SPP1 was up-regulated by hyperoxia and regulated the cell-cell interactions

As shown in Fig. 3A-B,in cell-cell communication, the interaction between different types of cells was explored. We found that the cell-cell interactions were enriched in the fibroblasts and macrophages with several different types of cells such as endothelial cells and epithelial cells. Moreover, we also identified that the SPP1-CD44 regulated the macrophage-fibroblast crosstalk (Fig. 3C). The percentage of SPP1⁺ fibroblasts/macrophages was significantly elevated in the hyperoxia mice relative to normaxia mice (Fig. 3D-E). Thus, we further focused on the role of SPP1 in BPD development. The expression of SPP1 in lung tracheal aspirate samples was detected, and SPP1 was revealed to be highly expressed in BPD neonates relative to the healthy controls (Fig. 3F). Similarly, SPP1 mRNA and protein expression also showed significant up-regulation in lung tissues of hyperoxia mice relative to normaxia mice (Fig. 3G-H).

SPP1 knockdown inhibited the cell fibrosis in vitro

As SPP1 was found differentially expressed in fibroblast and macrophages in hyperoxia mice, the function of SPP1 in vitro was further explored using hyperoxia-induced alveolar myofibroblasts (AMYFs) or RAW264.7 macrophages. We found that SPP1 expression was elevated in hyperoxia-stimulated RAW264.7 cells relative to control, and SPP1 was successfully knocked down in hyperoxia-induced RAW264.7 cells (Fig. 4A-B). The macrophage phenotype defined by FN1, Col1a1, and Acta2 expression was associated with fibrosis, and the expression of these fibrosis-related markers was detected after indicated treatment. The results revealed that the expression of FN1, Col1a1, and Acta2 were elevated in hyperoxia-induced RAW264.7 cells, and their levels were significantly reduced in response to SPP1 knockdown (Fig. 4C). Moreover, alveolar myofibroblasts (AMYFs) were cocultured with RAW264.7 macrophages exposed to hyperoxia. Similarly, we also confirmed that the levels of FN1, Col1a1, and Acta2 were significantly decreased by silencing SPP1 in AMYFs (Fig. 4D). These results indicated that SPP1 knockdown attenuated fibrotic potential of macrophages in vitro.

SPP1 knockdown inhibited hyperoxia-induced cell apoptosis, oxidative stress and inflammation response

The role of SPP1 in BPD development in vitro was further explored using the hyperoxia-induced alveolar myofibroblasts (AMYFs). We found that the viability of AMYFs was significantly reduced by exposure to hyperoxia, and SPP1 knockdown increased the viability of AMYFs relative to the hyperoxia + sh-NC group (Fig. 5A). Meanwhile, hyperoxia was also demonstrated to cause the elevation in AMYF apoptosis, which was restored by silencing SPP1 (Fig. 5B). The expression of apoptosis-related proteins was also detected, and the results identified the up-regulation of Bax and down-regulation of Bcl-2 in AMYFs exposed to hyperoxia, while SPP1 deficiency was revealed to reduce the Bax level and increase Bcl-2 expression in hyperoxia-induced AMYFs (Fig. 5C). The fibrosis marker α-SMA was shown to be elevated by hyperoxia stimulation while level of epithelial cell marker E-Cadherin was reduced in hyperoxia-treated AMYFs. However, SPP1 knockdown was revealed to reverse the effects of hyperoxia on the expression of α-SMA and E-Cadherin in AMYFs (Fig. 5D). As evidenced by the reduced GSH content and increased MDA concentration, hyperoxia was also revealed to induce oxidative stress in AMYFs, while SPP1 deficiency partially increased the GSH content and reduced the MDA concentration (Fig. 5E). Furthermore, the concentration of inflammatory cytokines (TNF-α, IL-1β) was elevated by hyperoxia and showed significant reduction by silencing SPP1 in hyperoxia-induced AMYFs (Fig. 5F). These data indicated that SPP1 deficiency increased the growth and reduced the apoptosis, inflammation as well as oxidative stress in hyperoxia-induced AMYFs.

SPP1 deficiency ameliorated the hyperoxia-induced BPD in neonatal mice

The effects of SPP1 knockdown on BPD development in vivo was further explored using neonatal mice exposed to hyperoxia. We found that the hyperoxia caused enlargement in mouse alveoli was significantly reduced by SPP1 deficiency, which suggested thatSPP1 deficiency promoted the alveologenesis in BPD mice (Fig. 6A). Moreover, according to the histological analysis, the hyperoxia-caused elevation in MLI and reduction in RAC was reversed by SPP1 knockdown in the neonatal mice (Fig. 6B-C). Meanwhile, IHC was adopted to evaluate the expression of α-SMA and E-Cadherin in the mouse lung tissues, and we found the α-SMA-positive cells were elevated while E-Cadherin-positive cells were reduced in the O₂ group, and SPP1 deficiency reversed the hyperoxia-induced changes in α-SMA and E-Cadherin expression, suggesting the inhibitory effects of SPP1 deficiency on lung fibrosis (Fig. 6D). Moreover, we also identified that hyperoxia induced a significant reduction in GSH and elevation in MDA content, and SPP1 deficiency attenuated the hyperoxia-caused oxidative stress in BPD mice by elevating GSH and reducing MDA production (Fig. 6F). Additionally, we discovered that the hyperoxia exposure increased the inflammation response in mouse lungs, as exhibited by the dramatic elevation in TNF-α as well as IL-1β production. However, after SPP1 knockdown, the TNF-α as well as IL-1β level showed significant decrease relative to the O2 + sh-NC group, suggesting the inhibitory effects of SPP1 silencing on the inflammatory response in the process of BPD (Fig. 6G).

SPP1 promoted the activation of the PI3K/AKT and ERK/MAPK signalings in Hyperoxiainduced lung injury mice and cell models

As KEGG analysis revealed that the PI3K/AKT and ERK/MAPK signaling are significantly affected by hyperoxia in mouse lung fibroblast, we further explored the potential effects of SPP1 deficiency on these pathways in BPD mice. As exhibited in Fig. 7A-B, p-PI3K and p-AKT protein increased in AMYFs exposed to hyperoxia and lung samples of hyperoxia mice, and SPP1 knockdown was demonstrated to inhibit the activation of the PI3K/AKT signaling by reducing the phosphorylation level of PI3K and AKT. Similarly, we also found that hyperoxia activated the ERK/MAPK pathway in AMYFs as well as BPD mouse lung tissues, as exhibited by elevated levels of p-MEK1/2 and p-ERK1/2, while SPP1 knockdown was revealed to reverse the hyperoxia-induced up-regulation of p-MEK1/2 and p-ERK1/2 both in vitro and in vivo (Fig. 7C-D). Overall, these data suggested that SPP1 deficiency suppressed the hyperoxia-induced activation of the PI3K/AKT and ERK/MAPK signalings in the process of BPD.

Multiple factors are involved in the pathogenesis of BPD, and exposure to hyperoxia remains a main risk factor of BPD in preterm infants [24, 25]. The better understanding of the molecular basis of BPD contributes to the improvement of targeted disease management [6]. In this work, SPP1 was aberrantly upregulated in the lungs of hyperoxia mice and hyperoxia-induced AMYFs. SPP1 knockdown was revealed to attenuate the hyperoxic lung injury in vitro and in vivo by regulating oxidative stress and inflammation.

Hyperoxia is revealed to hinder alveologenesis and induce respiratory dysfunction of normal lungs [26]. Studies have also shown that the hyperoxia exposure in neonatal animal models cause lung injury similar to human infants [27–29]. In this study, we established the hyperoxiainduced lung injury mouse models, and alveolar enlargement was observed in mice exposed to hyperoxia. The MLI was increased while the RAC was reduced by exposure to hyperoxia, which indicated the reduced respiratory function of mouse lungs [27]. The mouse models were successfully established with impaired lung development in neonatal mice.

Myofibroblasts are revealed to play a critical role in lung development and participate in the regulation of alveogenesis, the disruption of which is regarded as the hallmark of BPD [22, 30]. Macrophages as heterologous phagocytes are also considered to be involved in the lung injury and fibrosis [23]. In this study, we conducted the scRNA-seq analysis of mouse lungs in the hyperoxia and normoxia mice. We found that hyperoxia induced the cellular changes in the lungs of neonatal mice, and fibroblasts and macrophages are the main different cell cluster. The molecular profile in mouse lung fibroblasts and macrophages was further investigated, and the differentially expressed genes between the hyperoxia and normoxia groups were screened. SPP1 is found up-regulated in the lungs of hyperoxia mice, which was also validated by the results of western blot and qRT-PCR. Additionally, SPP1 was demonstrated to be highly expressed in the macrophages exposed to hyperoxia. In previous reports, SPP1 is excessively highly expressed in the alveolar macrophages of ARDS patients [31]. Similarly, SPP1 is demonstrated to be upregulated in ARDS mice, and SPP1 knockdown attenuates the lung injury and fibrosis in ARDS mice [16]. A previous study has also revealed that SPP1 from kidney tubule cells secreted by kidney tubule cells can lead to lung endothelial leakage, inflammatory response, and respiratory failure, and SPP1 can be used as a drug target for acute lung injury therapy [32]. These findings suggest that SPP1 plays a crucial role in the process of lung injury and fibrosis. In our work, we found that SPP1 knockdown inhibited the pro-fibrotic potential of Raw264.7 macrophages in vitro. The implication of SPP1 in the hyperoxiainduced lung injury development was also explored in animal and cell models. SPP1 deficiency protected from hyperoxic injury in neonatal mouse lungs by promoting alveologenesis, improving respiratory function, inhibiting oxidative stress and inflammation. SPP1 deficiency also protected lung AMYFs from hyperoxia-induced apoptosis, oxidative stress and inflammatory response and increased AMYF viability.

According to the enrichment analyses of DEGs in hyperoxia mouse lung tissues, the PI3K/AKT and ERK/MAPK signalings are two main pathways affected by hyperoxia in mouse fibroblasts. Previous studies have shown that the activation of the two pathways might protect alveolar epithelial cells from hyperoxic injury and apoptosis [33–35]. However, studies have also revealed that the activation of these pathways in fibroblasts is associated with the pulmonary fibrosis,inflammation and oxidative stress. As fibroblasts are required in the alveolarization and alveolar lung development [36], the effects of hyperoxia on these pathways in neonatal mice and fibroblasts were examined. We found that hyperoxia led to the activation of the PI3K/AKT and ERK/MAPK pathways, as shown by the increased phosphorylation levels of PI3K, AKT, MEK1/2 as well as ERK1/2. However, SPP1 deficiency counteracted the promoting effects of hyperoxia on the activation of these pathways in vivo and in vitro, suggesting that SPP1 promoted the activation of the two pathways under hyperoxia conditions, which further clarified that SPP1 not only activates these signaling in normoxia conditions [37, 38].

In conclusion, SPP1 was up-regulated in lungs of BPD mice and hyperoxia-induced lung fibroblasts. SPP1 knockdown improved the alveologenesis and respiratory function of BPD mice and protected fibroblasts from hyperoxia-caused apoptosis, oxidative stress and inflammation while inhibited the fibrotic potential of macrophages, which might deepen the understanding of molecular basis of BPD.

Funding

This study was supported by The affiliated Huaian No.1 People’s Hospital of Nanjing Medical University “Research and innovation team” project (YCT202301).

Author Contribution

Juan Liu and Zhaofang Tian conceived the project and designed the experiments.Tianping Bao performed the study, analyzed the data, and wrote the paper. Yajuan Zhou and Mengmeng Ma performed the study. Yajuan Zhou analyzed the data.

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No competing interests reported.

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Deficiency of SPP1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Neonatal tracheal aspirate samples

Animal experiment

Lung sample collection and histological analysis

Immunohistochemistry(IHC)

Glutathione (GSH) and Malondialdehyde (MDA) measurement

Enzyme-Linked Immunosorbent Assay (ELISA)

Single-cell RNA sequencing (scRNA-seq) analysis

Cell culture and treatment

Cell transfection

Western blot

Quantitative Real‑Time Polymerase Chain Reaction (qRT-PCR)

Cell viability

Flow cytometry analysis

Statistical analysis

Results

Hyperoxia induces SPP1 up-regulation in mouse lungfibroblasts

SPP1 was up-regulated by hyperoxia and regulated the cell-cell interactions

SPP1 knockdown inhibited the cell fibrosis in vitro

SPP1 knockdown inhibited hyperoxia-induced cell apoptosis, oxidative stress and inflammation response

SPP1 deficiency ameliorated the hyperoxia-induced BPD in neonatal mice

Discussion

Declarations

Author Contribution

References

Additional Declarations

Status:

Version 1