Activation of LXR signaling ameliorates apoptosis of alveolar epithelial cells in Bronchopulmonary dysplasia

Abstract

Background and purposes

Liver X receptors (LXRs) are specialized nuclear receptors essential for maintaining cholesterol homeostasis, modulating LXR activity could have therapeutic potential in lung diseases. Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by impaired alveolar development, in which apoptosis of alveolar epithelial cells is a key contributing factor. The current research focuses on exploring the potential mechanism by which the LXR pathway regulating alveolar epithelial type II cell apoptosis in response to hyperoxia exposure.

Methods

BPD infants and non-BPD preterm infants were enrolled to measure serum total cholesterol (TC) levels. To further investigate the role of cholesterol metabolism in BPD, a neonatal rat model of BPD was established, and in vitro studies were conducted using mouse lung epithelial cells (MLE12). These experiments aimed to explore the impact of hyperoxia on cholesterol metabolism and assess the effects of LXR agonist intervention.

Results

Elevated serum TC levels in BPD infants were observed, accompanied by lung cholesterol overload in BPD rats. Hyperoxia exposure also led to intracellular cholesterol accumulation in MLE12 cells, which may be attributed to the downregulated LXR signaling pathway. Activation of the LXR pathway prevented apoptosis and mitochondrial dysfunction in MLE12 cell. In BPD rats, intervention with the LXR agonist restored alveolar architecture and reduced alveolar epithelial type II cell apoptosis, which was associated with decreased oxidative stress and lung cholesterol accumulation.

Conclusions

Disrupted cholesterol metabolism and impaired homeostasis in premature infants may contribute to the development of BPD. Targeting LXR signaling may provide potential therapeutic targets in BPD.

Clinical trial number

Not applicable.

Introduction

Bronchopulmonary dysplasia (BPD) is a prevalent chronic respiratory condition that primarily impacts very low birth weight and extremely premature infants [1]. Despite advancements in neonatal care, the incidence of BPD remains high, with limited effective preventive or therapeutic interventions [1]. Infants with BPD often experience adverse long-term outcomes, including increased infections, impaired lung function, pulmonary hypertension, abnormal neurodevelopment, and high mortality rates [2]. The exact etiology and mechanisms of BPD are not fully elucidated, but it is believed to be linked to a halt in alveolar formation and angiogenesis [3]. Current pharmacological treatments only provide symptomatic relief rather than promoting lung recovery. It is well evidenced that the development of BPD is associated with oxygen supplementation and mechanical ventilation, which can lead to oxidative stress and inflammation, as well as pulmonary cell injury and death [4]. Alveolar epithelial type I (ATI) and type II (ATII) cells lining the alveoli are injured when exposed to hyperoxia [5]. Alveolar regeneration is primarily directed by ATII, which has the capacity for self-renewal and gives rise to ATI after lung injury [6]. Apoptosis is an evolutionarily conserved and highly regulated pathway of cell death, prior research has shown that excessive apoptosis induced by hyperoxia in ATII cells plays a significant role in the development of BPD [7, 8]. Thus, changes in the pulmonary microenvironment after hyperoxia may complicate the course of BPD by inducing ATII cell apoptosis and hindering alveolar growth [9].

Oxidative stress is intricately linked to disruptions in cholesterol metabolism [10]. Studies have found that preterm infants typically exhibit higher cholesterol levels in their blood compared to their full-term counterparts [11]. Elevated cholesterol levels, or hypercholesterolemia, may result in cholesterol accumulation in various cell types [12]. Recent research suggests that cholesterol affects inflammatory and mitochondrial responses in lung epithelial cells in lung disease conditions, such as chronic obstructive pulmonary disease [12]. Reverse cholesterol transport (RCT) is a process mediating the removal of excess cholesterol from peripheral tissues [13]. Animal studies have reported that mice lacking ATP-binding cassette (ABC) transporter-ABCA1, an important player in RCT, exhibited cholesterol overload, abnormal pulmonary morphology and physiology, and a pro-inflammatory phenotype [14]. The Liver X receptor (LXR), recognized as a key regulator for RCT [15], has been identified in several studies as a promising target for improving pulmonary outcomes in lung diseases when activated by LXR agonists [16, 17].

In this study, we presented evidence that excess oxygen exposure was associated with cholesterol metabolism disorder in the early life of BPD for the first time and raised a novel hypothesis by which hyperoxia adversely affects pulmonary cholesterol homeostasis leading to oxidative stress and ATII apoptosis, which can be alleviated by LXR pathway activation.

Methods

Study population

A total of 65 infants born at < 32 weeks gestation age (GA) were enrolled in this study, with informed parental consent obtained for each participant. Infants were followed up to 36 weeks post-menstrual age. The definition of BPD was according to the National Institute of Child Health and Human Development in 2018 [18]. A premature infant (born < 32 weeks’ GA) with BPD was characterized by persistent parenchymal lung disease with radiographic evidence. By 36 weeks postmenstrual age, the infant necessitated extra respiratory assistance, with a fraction of inspiration O₂ (FiO₂) ≥ 21%, over 3 successive days, to sustain arterial oxygen saturation within 90–95% [18].

BPD rat model establishment and intervention

Sprague-Dawley (SD) rats that were 6 to 8 weeks old were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All procedures were conducted following the NIH Guidelines concerning the treatment and utilization of lab animals, approved by Jiangnan University (approval No. JN. No20240430S0241230 [216]). Newborn rat offspring were subjected to either 85% O₂ (BPD group) or 21% O₂ room air (RA group) starting from birth (PN0) up until postnatal day 14 (PN14). At PN8, the newborn rats of the BPD group were further divided into 2 groups by means of intervention: intraperitoneal injection with PBS at PN8 - PN14 (BPD + PBS group) and intraperitoneal injection with LXR agonist T0901317 (Cayman Chemical, Cat#71810) group (BPD + T09 group). Based on previous works, T0901317 was dissolved in DMSO and intraperitoneal administrated with a dose of 25 mg/kg as reported in other lung disease conditions [19,20,21,22]. Four-micrometer sections of formalin-fixed and paraffin-embedded lung tissue were stained with hematoxylin and eosin (HE) for histopathological examination. The sections were examined under light microscopy by two independent investigators who were blinded to the treatment groups. Total cholesterol (TC) concentrations were quantified using a commercial cholesterol kit (Nanjing Jiancheng Bioengineering Institute, Cat#A111-1-1, Nanjing, China). Lung tissues were used to determine TC following the manufacturer’s instructions. Final data were presented as TC contents in 1 mg protein (mmol/mg pro).

In vitro studies

Murine lung epithelial cells-MLE12 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Gibco, Thermo Fisher Scientifc, Inc.) supplemented with 10% fetal bovine serum (Yeasen, Shanghai, China) and exposed to 85% O₂ (HYX group) or 21% O₂ room air (NOX group) for 24–48 h. MLE12 cells were treated with cholesterol (MCE, Cat#HY-N0322) under the NOX environment or HYX stimulation. MLE12 cells were treated with or without LXR agonist GW3965 (MCE, Cat#HY-10627 A) under HYX stimulation. Proliferation was assessed using CCK8 assay (Apex Bio, Cat#1018) according to the manufacturer’s instructions. The cells were harvested and incubated in the binding buffer containing 5 µl Annexin V-FITC and 10 µl PI in the dark for 15 min to analyze the percentage of apoptotic cells by the inverted fluorescence microscope, according to the manufacturer’s instructions (MCE, Cat# HY-K1073). The mitochondrial membrane potential was measured using the JC-1 Apoptosis Detection Kit (MCE, Cat#HY-K0601), according to the manufacturer’s instructions. The treated cells were stained with JC-1 for 20 min at 37 °C. Next, the stained cells were collected and analyzed using a Leica fluorescence microscope (Beckman Coulter Gallios, USA). The intracellular reactive oxygen species (ROS) formation was detected using 2,7-dichlorofluorescein diacetate as a fluorescent probe, following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Cat#E004-1-1, Nanjing, China).

Western blot analysis and quantitative real-time PCR

Cell lysates were prepared using RIPA buffer, and protein levels were determined with a BCA protein analysis kit (Apex Bio, Cat#K4101). About 30 µg of proteins were fractionated on 10% or 12% SDS-PAGE gels and transferred to PVDF membranes. Following blocking with 5% non-fat milk, the membranes were exposed to specific primary antibodies overnight at 4 °C, including anti-BAX (1:500, MCE, Cat#HY-P80929), anti-BCL2 (1:500, MCE, Cat#HY-P80566), anti-VEGFA (1:500, MCE, Cat#HY-P80929), anti-CD31 (1:5000, Proteintech, Cat# 11265-1-AP), anti-β-catenin (1:500, MCE, Cat#HY-P81261), anti-SPC (1:1000, Proteintech, Cat#10774-1-AP), anti-LXRα (1:5000, Proteintech, Cat#60134-1-Ig), and anti-GAPDH (1:50000, Proteintech). Subsequently, the membranes were treated with appropriate secondary antibodies for 1 h at room temperature, and the protein bands were detected and analyzed using a laser-scanning imaging system (Bio-Rad Molecular Imager, USA). Image J software was used to calculate the average gray value of the protein, and the average gray value of the target protein was divided by the average gray value of the corresponding GAPDH to normalize the relative intensity. TRIzol was used to extract total RNA. Then, mRNA was reverse-transcribed to cDNA by HiScript III U + One Step quantitative real-time (qRT) PCR Probe 5 × Master Mix (Vazyme Biotech, Cat# Q611). The qRT-PCR was performed by HiScript II One Step qRT-PCR Probe Kit (Vazyme Biotech, China, Cat#Q222-01) with primers listed in Table S1 and Table S2.

Immunofluorescence

MLE12 cells affixed to coverslips within a 12-well plate were rinsed twice with PBS before being fixed with 4% paraformaldehyde (400 µL per well) for a half-hour at ambient temperature. These cells were then treated with 0.5% Triton X-100 in PBS for 10 min and afterward blocked with 2% BSA in PBS for 30 min at room temperature. The blocking step was followed by overnight incubation with specific primary antibodies at 4 °C including LXRα antibodies (1:100, Proteintech, Cat#60134-1-Ig), SFTPC antibody (1:100, Proteintech, Cat#10774-1-AP). The following day, the cells underwent three PBS washes at 4 °C, each lasting 5 min, and were then treated with a fluorescent secondary antibody for a duration of two hours. Afterward, the cells were washed with PBS three times, and stained with DAPI for 5 min, followed by an additional three PBS washes. Images were acquired using a fluorescence microscope (Nikon Co, Tokyo, Japan) [23].

Statistical analysis

The study cohort’s characteristics were presented through descriptive statistics, including medians, means, standard deviation, frequency, and percentage. SPSS 21.0 software was utilized for statistical analysis of the data. Continuous variables were assessed using the T-test or One-way ANOVA test if they had a normal distribution, while the Mann-Whitney U test was used for continuous variables without normal distribution. Categorical variables were analyzed using the Chi-square test or Fisher’s Exact test, if appropriate. Statistical significance was defined as a P-value < 0.05.

Results

Hyperoxia exposure increased circulating total cholesterol level and disrupted lung cholesterol homeostasis

To investigate the alternations of cholesterol metabolism after hyperoxia exposure, a total of 65 infants born at < 32 weeks gestation who needed respiratory support were enrolled in this study, and clinical features were summarized in Table 1. The serum TC levels of the preterm infants who developed BPD (n = 40) at 1 week after birth were significantly higher than that of the preterm infants who did not develop BPD (n = 25), while there was no significant difference in the serum TC levels of the preterm infants at 2 weeks after birth (Fig. 1A). Meanwhile, the median inhaled FiO₂ was significantly higher and the proportion of mean FiO₂ > 21% was higher in the BPD group, suggesting that hyperoxia exposure may cause cholesterol metabolism disorder in preterm infants, which may be related to the development of BPD.

Table 1 The clinical features of the BPD and non-BPD infants

Fig. 1

Hyperoxia exposure increased serum total cholesterol level and disrupted lung cholesterol homeostasis. (A) serum total cholesterol (TC) levels and Fraction of inspiration O₂ concentration (FiO₂) of the preterm infants who developed bronchopulmonary dysplasia (BPD) (n = 40) or not (n = 25). (B) Representative photographs of HE-stained lung sections from rats exposed to room air (RA group) or hyperoxia (BPD group). Scale bars, 100 μm (100X) and 50 μm (200X), n = 5/group. (C) Representative pictures of the rats and lung tissues, and the quantification of the HE-stained lung sections of the rats, shown as mean linear intercept (MLI) and radial alveolar counts (RAC), n = 5/group. (D) Weight, serum TC, lung TC content, and bronchoalveolar lavage fluid (BALF) APOA of the BPD rats and RA group. n = 8–10/group. (E) Relative mRNA expression of Abca1, Abcg1, Ldlr, Hmgcr, normalized to GAPDH. n = 6/group. The box represents mean ± SD/mean ± SEM and the whiskers represent maximum and minimum values. The experiments were repeated at least 3 times. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

We subsequently established a BPD rat model through hyperoxia exposure to delve deeper into cholesterol metabolism. Using HE staining, we evaluated pathological alterations in lung tissue, quantified by the mean linear intercept (MLI) and radial alveolar counts (RAC). As previously reported [24], the BPD rat model induced by hyperoxia exposure exhibited features of alveolar enlargement, evidenced by higher MLI values and lower RAC (Fig. 1B and C). Furthermore, in the BPD rat group, the lung volume was diminished (Fig. 1C). When compared to the control group under the normoxia environment, the BPD group exhibited a significant decrease in body weight, along with elevated levels of plasma TC and pulmonary TC content (Fig. 1D). Additionally, the levels of serum TC were found to correlate with oxygen concentration administered (Supplementary Fig. 1). ELISA analysis revealed that the levels of bronchoalveolar lavage fluid (BALF) APOA, which is associated with RCT, were notably reduced in the BPD group (Fig. 1D). Next, to further investigate the disrupted lung cholesterol homeostasis caused by hyperoxia, lung RNA was isolated from both groups to target genes related to cholesterol influx, efflux, and synthesis. The results indicated a significant decrease in Abca1 and Abcg1 mRNA expression, involved in RCT, in the BPD group compared to the RA group (Fig. 1E). A trend toward decline with no significant difference in Hmgcr mRNA expression indicating cholesterol synthesis was observed while there was no significant difference in mRNA level of Ldlr responsible for cholesterol intake in the BPD rat lung tissue (Fig. 1E). These above data suggested that hyperoxia exposure may also lead to lung cholesterol overload, which may be linked to circulating cholesterol metabolism disorder, contributing to BPD development.

Hyperoxia exposure led to intracellular cholesterol accumulation and oxidative stress

To further evaluate the effect of hyperoxia exposure and exogenous cholesterol overload on lung tissue, we stimulated murine lung epithelial cells (MLE12) with cholesterol at different concentrations under a hyperoxia environment in vitro. Our findings revealed an increase in intracellular cholesterol content due to external cholesterol stimulation. Additionally, hyperoxia exposure also up-regulated intracellular cholesterol accumulation (Fig. 2A), which indicated that both a cholesterol-overload environment and hyperoxia exposure have a synergistic impact on cellular cholesterol accumulation. Cellular cholesterol homeostasis is associated with mitochondrial oxidative injury [25], we next analyzed ROS production in MLE12 cells. Although with no statistical difference, we also noted a combination effect of ROS production under exogenous cholesterol and hyperoxia stimulation, but there were no differences in ROS production between different cholesterol concentrations under the normoxia environment (Fig. 2B). It revealed that hyperoxia exposure upregulated the accumulation of intracellular cholesterol, which may be associated with cellular oxidative stress, while exogenous cholesterol exacerbated this condition.

Fig. 2

Hyperoxia exposure leads to intracellular cholesterol accumulation and increased ROS production. (A) Representative pictures of MLE12 cells exposed to normoxia (NOX) or hyperoxia (HYX) for 24 h with or without 100 ¼M cholesterol stained by Filipin III and the quantification of the mean fluorescence intensity of Filipin III staining. Scale bars, 50 μm, n = 5/group. (B) Reactive oxygen species (ROS) production of the MLE12 cells exposed to NOX or HYX for 24 h with or without 100 µM cholesterol. Scale bars, 50 μm, n = 5/group. (C) Relative mRNA expression of Abca1, Abcg1, Ldlr, Hmgcr, normalized to GAPDH. n = 5/group. (D) Representative pictures of MLE 12 cells stained with LXRα antibody exposed to NOX or HYX for 24 h, and the quantification of the mean fluorescence intensity of LXRα. Scale bars, 50 μm, n = 5/group. The experiments were repeated at least 3 times. *P < 0.05; ** P < 0.01; *** P < 0.001

Cellular cholesterol homeostasis is primarily regulated by two key transcription factors, SREBP2 and LXR, responsible for upregulating cholesterol content and downregulating cholesterol levels, respectively [26]. We next analyzed mRNA levels of SREBP2-regulated genes and LXR-regulated genes under the hyperoxia condition, at the transcriptional level, hyperoxia exposure led to a prominent downregulation of LXR-transcribed genes including Abca1 and Abcg1 but only minor changes in SREBP2-transcribed genes (Ldlr and Hmgcr) with no significant difference (Fig. 2C). Immunofluorescence of MLE12 also showed that hyperoxia exposure decreased protein expression of LXRα (Fig. 2D), a subtype of LXR, mainly regulating the expression of ABCA1 and ABCG1 transporters, which are highly expressed in pneumocytes [27, 28].

LXR agonist attenuated hyperoxia-induced ROS production and mitochondrial dysfunction

To evaluate the roles of the LXR pathway under hyperoxia exposure, which induces cholesterol efflux, LXR agonists GW3965 were applied. MLE12 cells were treated under hyperoxia stimulation with different doses of GW3965 for 24 h to evaluate the cell viability by a CCK8 assay. As shown in Fig. 3A, the cell viability of MLE12 cells was decreased significantly and LXR agonist GW3965 5µM exerted around a 10% increase in cell proliferation under hyperoxia exposure.

Fig. 3

The LXR agonist attenuated hyperoxia-induced mitochondrial dysfunction and reduced reactive oxygen species (ROS). (A) MLE12 cells were cultured under normoxia (NOX) or hyperoxia (HYX) conditions with different doses of GW3965 for 24 h, and cell viability was determined by CCK-8 assays. n = 4/group. (B) Relative mRNA expression of Abca1 normalized to Gapdh. n = 4/group. (C-D) Representative pictures of MLE12 cells exposed to NOX or HYX for 24 h treated with or without 5 µM GW3965 stained by Filipin III and the quantification of the mean fluorescence intensity of Filipin III staining. Scale bars,50 μm, n = 4/group. (E) Reactive oxygen species (ROS) production of the MLE12 cells exposed to NOX or HYX environment. Scale bars, 50 μm, n = 4/group. (F-G) Representative pictures of MLE 12 cells stained with JC-1 exposed to NOX or HYX for 24 h, and the quantification of the mean fluorescence intensity of JC-1. Dye accumulation in mitochondria was detected by fluorescence microscopy (aggregate red form with absorption/emission of 585/590 nm and green monomeric form with absorption/emission of 510/527 nm). The red/green fluorescence ratio of JC-1 dimers to monomers in MLE12 cells stained with JC-1. The histogram showed the red/green fluorescence ratio of JC-1 dimers to monomers in MLE12 cells stained with JC-1. Scale bars, 50 μm, n = 5/group. The experiments were repeated at least 3 times. *P < 0.05; ** P < 0.01; *** P < 0.001

Hyperoxia stimulation resulted in a decrease of approximately 70% in the mRNA level of Abca1, whereas the LXR agonist GW3965 5µM notably increased the mRNA expression of Abca1 in a dose-dependent manner (Fig. 3B). Additionally, GW3965 5µM exhibited the ability to diminish intracellular cholesterol accumulation in response to hyperoxia stimulation (Fig. 3C and D), leading to a reduction in ROS production under the hyperoxic environment (Fig. 3E). Elevated ROS levels are associated with reduced membrane potential and cell apoptosis [29]. Lower numbers of MLE 12 cells with the red dimeric form of JC-1 (indicative of normal status) in contrast to the ones with green JC-1 monomers (representing a depolarized mitochondrial membrane) were observed after hyperoxia exposure, indicating loss of mitochondrial membrane potential (Fig. 3F and G). Interestingly, this trend was reversed when treated with 5µM GW3965 (Fig. 3F and G). These findings suggested that activation of the LXR pathway can reduce hyperoxia-induced ROS production and alleviate mitochondria dysfunction by reducing cholesterol overaccumulation in MLE12 cells.

LXR agonist protected against hyperoxia-induced cell apoptosis

Hyperoxia increased the relative protein expression of the apoptosis-related protein BAX whereas there was a decrease in the expression of anti-apoptosis protein BCL2 in MLE12 cells exposed to hyperoxia (Fig. 4A). To investigate the effect of LXR agonist on hyperoxia-induced apoptosis in MLE12 cells, we further analyzed the alternation of the BCL2 and BAX protein expression. Results from western blot and qRT-PCR analysis revealed that LXR agonist GW3965 administration reversed the elevated BAX/GAPDH ratio associated with proapoptotic effects and amplified the reduced BCL2/GAPDH ratio linked to antiapoptotic effects triggered by hyperoxia, which may be associated with the activation of β-catenin pathway (Fig. 4A and B). In order to conduct a more thorough examination of the impact of GW3965 treatment on MLE12 cells in the presence of hyperoxia, apoptosis staining results revealed that GW3965 reduced the percentage of FITC V-positive MLE12 cells, along with the proportion of FITC V-positive /PI-positive MLE12 cells under hyperoxic stimulation, demonstrating an anti-apoptotic influence (Fig. 4C and D). These results suggested that LXR agonist inhibits hyperoxia-induced apoptosis in MLE12 cells. At the mRNA and protein level, hyperoxia lowered the expression of the epithelial cell marker SFTPC in MLE12 cells as an indicator of ATII (Fig. 4D and E). Treatment of MLE12 with GW3965 had a positive effect on the expression of SFTPC under hyperoxic stimulation (Fig. 4D and E). We also found that the increased mRNA expression of inflammatory cytokines including IL-6, and TNF-α induced by hyperoxia exposure was downregulated by GW3965 treatment (Fig. 4F).

Fig. 4

LXR agonist protected against hyperoxia-induced cell apoptosis. (A) The relative protein levels of BCL2, BAX, and β-catenin were determined by western blot analysis and quantified. n = 4/group. (B) The relative mRNA expression of Bcl2 and Bax was determined by qRT-PCR, normalized to Gapdh. n = 4/group. (C) Representative pictures of MLE12 cells stained with Annexin FITC and PI. Scale bars, 50 μm, n = 4/group. (D) The immunofluorescence staining and fluorescent quantitation of SFTPC in MLE12 cells exposed to normoxia (NOX) or hyperoxia (HYX), and HYX with GW3965 intervention. Scale bars, 50 μm, n = 4/group. (E) The relative mRNA expression of Sftpc, normalized to Gapdh. (F) The relative mRNA expression of Il-6 and Tnf-α, normalized to Gapdh. n = 4/group. The experiments were repeated at least 3 times. *P < 0.05; ** P < 0.01; *** P < 0.001

LXR agonist exhibited protective effects on lung injury in BPD rat model

To explore whether LXR pathway plays a role in the development of BPD in vivo, we activated LXR signal pathway with LXR agonist T09 in the BPD rat model. SD newborn rats were assigned to the normoxia environment (RA group), hyperoxia with PBS (BPD + PBS group), or hyperoxia with LXR agonists (BPD + T09 group). Morphological analysis of lung tissue sections stained with HE revealed that the BPD + PBS group had higher MLI and lower RAC compared to the RA group, which was reversed by administration of T09 (Fig. 5A and B). It indicated that the LXR agonist improved alveolarization in newborn rats exposed to hyperoxia. Western blot analysis also showed that T09 supplementation increased protein levels of CD31, VEGFA (vascular endothelial cell markers), and SFTPC (markers of ATII) reduced by hyperoxia exposure (Fig. 5C).

We also observed abnormal α-smooth muscle actin (α-SMA) distribution throughout the interstitium in lung tissues of the BPD group. The increased expression of the fibrosis marker α-SMA induced by hyperoxia was reversed after T09 administration (Fig. 5C and D). Additionally, the enhanced mRNA expression of inflammatory cytokines including IL-6, TNF-α, and CXCL1 induced by hyperoxia exposure was downregulated by T09 intervention, regardless of statistical difference (Fig. 5E). Nevertheless, the LXR agonist did not appear to influence the differentiation of ATII cells into ATI cells, as there was no significant difference in aquaporin 5 gene expression after T09 intervention (Fig. 5E). There was no significant difference in body weight between the BPD + T09 treatment group and the BPD group (Supplementary Fig. 2A). These findings suggested that T09 treatment may alleviate BPD by repairing damage to ATII cells caused by high oxygen concentration exposure, potentially leading to improvement in vascular development and inflammation.

Fig. 5

LXR agonist exhibited protective effects against lung injury in BPD rat model. (A) Representative photographs of HE-stained lung sections from rats exposed to room air (RA), hyperoxia with PBS administration (BPD + PBS) and hyperoxia with T0901317 administration (BPD + T0901317 group). Scale bars, 100 μm (100X) and 50 μm (200X), n = 5/group. (B) Quantification of the HE-stained lung sections of the rats, shown as mean linear intercept (MLI) and radial alveolar counts (RAC), n = 5/group. (C) The relative protein levels of CD31, VEGFA, SFTPC, α-SMA and GAPDH were determined by western blot analysis, with quantification shown as histograms. n = 4/group. (D) Lung tissue sections were stained for α-SMA depicted in red and DAPI (shown in blue). In the RA group, the α-SMA was mainly located at the tips of the secondary crests while in the BPD group, α-SMA was extensively present throughout the interstitial spaces. Scale bar, 20 μm. n = 4/group. (E) The relative mRNA expression of Il-6, Tnf-α, Cxcl1, aquaporin 5 (Aqp5) and α-SMA, normalized to Gapdh. n = 5/group. The experiments were repeated at least 3 times. *P < 0.05; ** P < 0.01; *** P < 0.001

LXR pathway activation reduced hyperoxia-induced accumulation of lung cholesterol and alleviated oxidative stress-induced apoptosis

To further explore the role and potential mechanism of LXR activation, by using immunofluorescence staining for SFTPC, we demonstrated that hyperoxia decreased the number of SFTPC⁺ cells, while T09 administration significantly increased the proportion of SFTPC⁺ cells (Fig. 6A and B). Besides, we used TUNEL staining to determine the cell survival in ATII in each group after the LXR agonist administration. The results showed that the relative number of SFTPC⁺/TUNEL⁺ cells was significantly reduced in the BPD + T09 intervention group compared with the BPD + PBS group (Fig. 6A and B), indicating that LXR agonist administration prevented ATII from cell apoptosis in hyperoxia-induced lung injury.

It was reported that the effects of LXR agonists on apoptosis alleviation were dependent on LXRα, but Not LXRβ [30]. LXRα mainly targets the genes involved in RCT, including ABCA1 and ABCG1 transporters [28], highly expressed in pneumocytes [27, 31]. We noted a significant decrease in LXRα protein levels after hyperoxia exposure by immunofluorescence while T09 intervention increased the LXRα protein level in lung sections (Fig. 6C and D). These results were further confirmed by western blot analysis which revealed that T09 administration reversed the hyperoxia-induced increase in the BAX/GAPDH ratio and decrease in the BCL2/GAPDH ratio, which may be linked to the activation of the β-catenin pathway (Fig. 6E). We also observed that T09 administration reduced lung TC content (Fig. 6F), and significantly decreased serum TC levels (Supplementary Fig. 2B). In addition, the malondialdehyde (MDA) levels as biomarkers of oxidative stress were also noted to be diminished by T09 administration (Fig. 6G). The above data revealed that LXR agonist treatment could alleviate hyperoxia-induced lung injury, which may be partially explained by reduced lung cholesterol overaccumulation and alleviated oxidative stress.

Fig. 6

LXR agonist reduced hyperoxia-induced accumulation of lung cholesterol and alleviated alveolar type II (ATII) apoptosis. (A) Representative photographs of SFTPC (green) /TUNEL (red) /DAPI (blue) -stained lung sections from rats exposed to room air (RA), hyperoxia with PBS administration (BPD + PBS) and hyperoxia with T0901317 administration (BPD + T0901317). Scale bars, 50 μm, n = 5/group. (B) Quantification of the SFTPC ⁺ cells and SFTPC⁺TUNEL⁺ cells of the lung sections from rats. (C-D) The representative pictures of lung sections stained with LXRα antibody and the quantification of the fluorescence. Scale bars, 20 μm, n = 5/group. (E) The relative protein levels of BCL2, BAX, β-catenin, and GAPDH were determined by western blot analysis. n = 5/group. (F) The lung total cholesterol (TC) content of the three groups of rats. n = 5/group. (G) The malondialdehyde (MDA) of the three groups of rats. n=5/group. The experiments were repeated at least 3 times. *P < 0.05; ** P < 0.01; *** P < 0.001

Discussion

Arrested alveolar development is the main pathological characteristic of neonatal BPD, numerous studies have aimed to improve alveolarization and have focused on alveolar epithelial cell damage and impairment [32]. To the best of our knowledge, we demonstrated that hyperoxia exposure led to increased circulating TC contents for the first time. A previous study reported a marked increase in small high-density lipoprotein (HDL) and total low-density lipoprotein (LDL) and a decrease in large HDL in most premature infants in the first weeks of life [33]. The authors found that infants with a lower percentage of plasma large HDL and a higher percentage of plasma small HDL at 2 weeks of age had a longer duration of respiratory support [34]. BPD often occurs in preterm infants who have received substantial respiratory support with either mechanical ventilation or supplementation with oxygen [35]. Based on our data of BPD infants, the elevated circulating TC level may be associated with FiO₂, parallelized with the results noted in the BPD animal model in which higher levels of oxygen concentration led to higher serum TC levels. These findings indicate that disrupted cholesterol metabolism is closely related to excessive oxygen supplementation and the pathogenesis of BPD. Besides, we also found overaccumulation of cholesterol induced by hyperoxia stimulation in the lung tissue of the BPD rat model and MLE cells in vitro. Previous studies have indicated that cholesterol accumulation may lead to excessive inflammation and progression of lung lesions in chronic obstructive pulmonary disease [31]. Therefore, we supposed that an abnormal lipid profile in premature infants may be associated with the development of BPD, which means the circulating cholesterol-rich environment combined with hyperoxia stimulation may lead to the accumulation of cholesterol within the lung epithelial cells, but the dual role of cholesterol in cellular signaling and membrane dynamics necessitates a deeper exploration of how its dysregulation under hyperoxic conditions triggers cell death pathways and exacerbates lung injury.

Ye et al. have reported that H₂O₂-induced oxidative stress could deregulate the expressions of LXRα and LXRβ and trigger the ER stress resulting thereafter in the accumulation of cholesterol in endothelial cells [36]. The LXRs, are responsible for the efflux of excess cholesterol out of cells onto HDL particles by inducing the expression of the key cholesterol transporters ABCA1 and ABCG1 [37]. Our results in vitro and in vivo showed that lung cholesterol overload may be attributed to the downregulation of the LXR pathway, evidenced by decreased mRNA expression of LXR-regulated genes Abca1 and Abcg1 as well as the diminished protein level of LXRα in MLE12 cells and BPD rat lung sections. Moreover, our research revealed an interesting interaction between hyperoxia exposure and cholesterol overload in MLE12 cells, resulting in increased ROS production. It is known to all that mitochondria dysfunction leads to increased generation of ROS and the production of ROS represents a major cause of oxidative stress [38]. Our results indicated that LXR activation can ameliorate hyperoxia-induced aberrant mitochondrial dynamics and reduce ROS formation, partially explained by enhanced cholesterol efflux and alleviated intracellular cholesterol accumulation. It is also well-accepted that excessive intracellular lipid accumulation triggers endoplasmic reticulum stress and apoptosis in macrophages [29]. Apoptosis is programmed cell death that is regulated by specific biochemical and molecular factors and occurs under both normal and abnormal physiological conditions [39]. It was well-documented that apoptosis of alveolar epithelial cells plays an important role in the occurrence and development of BPD [40]. Our results suggested that LXR agonists improved ATII cell survival both for lung injuries in the BPD rat model, and in vitro, for MLE-12 cells after hyperoxic exposure. The observed protective effect was strongly associated with the attenuation of cholesterol accumulation induced by hyperoxia in MLE12 and rat lungs. Bax and Bcl2 are well-known apoptotic proteins. Bax induces cell apoptosis while Bcl2 inhibits the release of cytochrome c from mitochondria and inhibits apoptosis [15]. We found that LXR activation reversed the decreased Bcl2/Bax ratio triggered by hyperoxia exposure, thus increasing survival rates of alveolar epithelial cells in vivo and in vitro. The β-catenin pathway was reported to mitigate cell death and restore mitochondrial homeostasis in LPS-exposed HK-2 cells [41]. It was also reported that the β-catenin pathway may participate in the development of BPD [42]. We also found the LXR agonist intervention might exert protection from BPD by activation of the β-catenin pathway. However, we still lack of direct evidence regarding the mechanism by which external cholesterol induces the secondary accumulation of intra-epithelial cholesterol and cell apoptosis.

In conclusion, the current study demonstrated the existence of cholesterol disorder in early life of preterm infants with hyperoxia supplementation associated with BPD development for the first time, which raised a possibility that serum lipid profile may be a predictable marker for BPD progression and disease severity. We also presented a novel mechanism by which hyperoxia adversely affects pulmonary cholesterol homeostasis as well as oxidative stress and AT II apoptosis, which can be alleviated by LXR pathway activation. There are also several limitations in our investigation. We mainly focused on AT II cells, but there are other lung cells participative in the BPD development. LXRs are also widely reported to be involved in the regulation of inflammation and the immune response. Especially, LXR inactivation or deficiency was significantly associated with the expression of proinflammatory cytokines IL-1β, and IL-6 in irradiated macrophages [43]. In the context of BPD, inflammation is also key for disease pathogenesis, as it often develops as a result of lung injury and is an aggravating factor for BPD development. We observed alleviation of inflammation but we did not further detect the distribution of pulmonary alveolar macrophages. Pulmonary hypertension accounts for the majority of mortality in infants with BPD [44], the in vivo results also indicated improvement in vascular dysplasia and fibrosis after T09 intervention, but we did not further explore the symptoms of pulmonary hypertension in the BPD rats. Although LXR agonists seem promising in the treatment of BPD and BPD-associated pulmonary hypertension, problems remain. In animal models, LXR agonists were also reported to be linked to hypertriglyceridemia, and hepatic steatosis, so the translatability to the clinical setting of LXR agonist positive and adverse effects on lipid metabolism and immune function is uncertain [37]. Additionally, it is difficult to mimic the dynamic changes in cholesterol metabolism in vitro, so we focused on the role of LXR activation on cell apoptosis under hyperoxia condition without exogenous cholesterol stimulation in MLE12 cells. In conclusion, the protective effect of LXR agonists highlights the potential of targeting this pathway for therapeutic intervention in BPD management, further research is still needed to fully elucidate the specific role of LXR signaling in the context of BPD.