Rapamycin attenuates pyroptosis by suppressing mTOR phosphorylation and promoting autophagy in LPS-induced bronchopulmonary dysplasia

Abstract

Purpose/Aim

Bronchopulmonary dysplasia (BPD) is associated with poor survival in preterm infants. Intrauterine infection can aggravate the degree of obstruction of alveolar development in premature infants; however, the pathogenic mechanism remains unclear. In this study, we sought to determine whether pyroptosis could be inhibited by downregulating mammalian target of rapamycin (mTOR) activation and inducing autophagy in BPD-affected lung tissue.

Materials and Methods

We established a neonatal rat model of BPD induced by intrauterine infection via intraperitoneally injecting pregnant rats with lipopolysaccharide (LPS). Subsequently, mTOR levels and pyroptosis were evaluated using immunohistochemistry, immunofluorescence, TUNEL staining, and western blotting. The Shapiro–Wilk test was employed to assess the normality of the experimental data. Unpaired t-tests were used to compare the means between two groups, and comparisons between multiple groups were performed using analysis of variance.

Results

Pyroptosis of lung epithelial cells increased in BPD lung tissues. After administering an mTOR phosphorylation inhibitor (rapamycin) to neonatal rats with BPD, the level of autophagy increased, while the expression of autophagy cargo adaptors, LC3 and p62, did not differ. Following rapamycin treatment, NLRP3, Pro-caspase-1, caspase-1, pro-IL-1β, IL-1β, IL-18/Pro-IL-18, N-GSDMD/GSDMD, Pro-caspase-11, and caspase-11 were negatively regulated in BPD lung tissues. The opposite results were observed after treatment with the autophagy inhibitor MHY1485, showing an increase in pyroptosis and a significant decrease in the number of alveoli in BPD.

Conclusions

Rapamycin reduces pyroptosis in neonatal rats with LPS-induced BPD by inhibiting mTOR phosphorylation and inducing autophagy; hence, it may represent a potential therapeutic for treating BPD.

Keywords:

• Bronchopulmonary dysplasia
• intrauterine infection
• pyroptosis
• autophagy
• rapamycin

Background

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in premature infants.¹ Although advances in medical technology have improved the survival rates of preterm infants younger than 29 wk gestation (up to 45%), the incidence of BPD remains unchanged.^2–4 BPD is a systemic disease that increases the incidence of infant mortality, short- and long-term respiratory diseases, and neurodevelopmental disorders, while also having lifelong effects on adult health and quality of life.^5,6 Various factors increase the risk of developing BPD, including hyperoxia, mechanical ventilation, prolonged patency of the ductus arteriosus, sepsis, inflammation, and other postpartum factors. Adverse prenatal factors, such as chorioamnionitis, preeclampsia, preexisting hypertensive disorders, gestational diabetes mellitus, and maternal obesity, are also risk factors for BPD.^10,11 Among them, infection and inflammation can become pathogenic factors for BPD before and after delivery. That is, intrauterine infection can directly affect the normal development of alveoli through inflammation and promote preterm birth via chorioamnionitis. Meanwhile, effective methods to prevent or control intrauterine infection are relatively limited,^10–12 resulting in high associated costs.^7–9 Moreover, the precise mechanism by which intrauterine infection influences the pathogenic process of BPD remains unclear and warrants further investigation.

One potential pathway with an important role in the pathological process of intrauterine infection-induced BPD is pyroptosis—a pro-inflammatory form of cell death—which can cause tissue damage.^13,14 Indeed, pyroptosis occurs in hyperoxia-induced BPD and LPS-induced acute lung injury models, while its inhibition can alleviate lung injury.^16–20 Pyroptosis initiation requires at least one pro-inflammatory caspase, including caspase-1 and caspase-11 in mice (caspase-4 and caspase-5 in humans) as well as the participation of various other key factors, including NOD-like receptor pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD), interleukin (IL)-1β, and IL-18. Moreover, the non-classical caspase-11 inflammasome is formed during lipopolysaccharide (LPS) cytoplasmic exposure.^13–15 Within the lung tissues of neonatal mice exposed to hyperoxia, caspase-1 activation, IL-1β expression, and inflammation levels are increased, whereas alveolarization is reduced. However, blocking IL-1β with IL-1 receptor antagonists or the NLRP3 inflammasome with glyburide treatment decreases inflammation and increases alveolarisation.¹⁷ Autophagy can also regulate NLRP3 inflammasome^22,23 and caspase-11 inflammasome activation.^24,25

Autophagy is an intracellular process critical for transporting cytoplasmic components and clearing pathogens.²¹ Promotion of autophagy can inhibit NLRP3 inflammasome activity, reducing acute lung injury in mice caused by sepsis.¹⁶ The protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of autophagy. In fact, within a mouse model of acute lung injury induced by exogenous mitochondrial damage-associated molecular patterns, administration of an autophagy agonist (i.e., rapamycin) inhibited the NLRP3 inflammasome, alleviating associated inflammation and damage.¹⁸ Rapamycin also inhibits IL-1β secretion. Moreover, the anti-inflammatory factor IL-10 negatively regulates inflammasome activation by decreasing mTOR levels in mouse models of colitis and patients with inflammatory bowel disease. IL-10 also inhibits caspase-1-dependent inflammasome activation. However, the relationship between mTOR, autophagy, and pyroptosis in intrauterine infection-induced BPD remains unknown.^26,27

The main objective of the current study is to investigate the role and significance of pyroptosis and mTOR-related autophagy in intrauterine infection-induced BPD. Specifically, we evaluate whether rapamycin administration to a BPD rat model increases mTOR-related autophagy and inhibits pyroptosis, ultimately promoting alveolar development and maturation.

Methods

Animals and BPD model generation

All animal experiments in this study were performed per the ARRIVE guidelines and carried out according to the UK Animals (Scientific Procedures) Act of 1986 and National Research Council’s Guide for the Care and Use of Laboratory Animals. The Institution’s Animal Ethics Committee approved this study (Reference number: CHENGYIDONGLUN2021007).

Female SD rats weighing 300–350 g and male SD rats weighing 350–400 g, aged 3–4 months, were purchased from Chengdu Dossy Laboratory Animal Co., Ltd. (Sichuan, China). According to previous studies, the sex of neonatal rats has little effect on the experimental outcomes; therefore, we did not distinguish between the sexes. All rats were acclimated to standard cages for 1 week before initiating the study procol and were provided ad libitum access to feed and water. Female and male rats were randomly assigned to cages at a 2:1 ratio. The conditions in the animal facility were maintained at a temperature of 22 ± 2 °C, relative humidity of 50–60%, and 12 h/12 h light/dark cycle. A positive vaginal smear was recorded as embryonic time point 0 (i.e., E0) of pregnancy. The necessary precautions were taken throughout the study to minimize pain and stress associated with the experimental treatments.

Finally, 22 pregnant female rats were included, of which 14 were assigned to the BPD model group and 8 to the sham operation group. For the pregnant rats in the intrauterine infection group, LPS (L2880-10MG; Sigma-Aldrich, St. Louis, MO, USA; dissolved in sterile 0.9% sodium chloride solution at a concentration of 0.1 mg/mL) was administered. The dose was 0.4 mg/kg per day for 2 days on E18 and E19, while the control group was injected with sterile 0.9% sodium chloride solution only (volume 4 mL/kg per day for 2 days). The timing and frequency of LPS dosing were based on protocols reported previously.^28,29 LPS doses were determined through preliminary studies and validation to establish concentration gradients. All rats were randomly numbered after surgery using a random number table generated in Excel (Microsoft, Redmond, WA, USA). Random numbers were sorted from small to large and assigned to different groups. Numerical sample identifiers were used during the experimental procedure and data analysis, and the investigators were blinded to the treatment.

Bisex SD rat pups born at E21–E22 were defined as reaching term delivery and included in the study. To ensure consistency between groups, we reduced the number in each group to 7–9 animals at birth. We raised the remaining rats to adulthood or used them for other studies. Finally, 144 full-term neonatal rats were selected for the study, including 88 in the BPD model group and 56 in the control group. Birth was defined as the first time point after birth (i.e., P0, no more than 6 h). Neonatal rats in the LPS-induced BPD group were randomly divided into the BPD, BPD + rapamycin, or BPD + MHY1485 groups. Rapamycin (Cat# S1039; Selleck, USA) and MHY1485 (Cat# S7811; Selleck) were prepared according to the manufacturer’s instructions in a total volume of 50 µL; the neonatal rats from the BPD + rapamycin and BPD + MHY1485 groups received right intraperitoneal injections in the lower abdomen twice (6 h and 24 h after birth). The non-medicated BPD group and the control group were injected with the same volume of sterile water and sterile 0.9% sodium chloride solution. The P1–P5 (i.e., postnatal days 1–5) rodent lungs correspond to 23–32 wk of gestation in humans and therefore are well suited for studying BPD in preterm infants.³⁰ Accordingly, our main study time point was P0–P5.

We followed the ‘Three Rs of Animal Research: (Replace, Reduce, and Refine).’³¹ To minimize pain, intraperitoneal injection of 4% sodium pentobarbital (200 mg/kg body weight) was used for euthanasia.³² Each injection was performed at the level of the right hip and femoral joint of the abdomen and ∼5 mm to the right of the midline, with the needle at a 45° angle to the body wall. Euthanasia was considered successful in the absence of voluntary movement, loss of righting reflex, and respiratory and heartbeat arrest. Each experiment was performed at least three times to improve data quality. Deceased animals were excluded from this study. This study has not been pre-registered.

Histopathology

The left lung tissues of the neonatal rats were fixed and embedded in paraffin for pathological examination. Briefly, the animals were deeply anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde. The tissues were fixed at 22 ± 2 °C for 72 h, dehydrated in graded ethanol solutions, embedded in paraffin, and sliced uniformly horizontally after freezing. Hematoxylin and eosin (H&E) staining (Cat# G1121; Solarbio, Beijing, China) was performed to observe the pathological structure of the placenta and the lung tissues and determine the radial alveolar count (RAC) and mean linear intercept (MLI). Images of lung tissues in each section were captured using a Leica microscope (DM4000B; Leica, Wetzlar, Germany). Six discrete sections were collected from each lung tissue, each section was randomly selected with a 20× objective magnification, and a blinded investigator randomly selected microscopic fields using ImageJ 1.8.0 (RRID: SCR_003070; National Institute of Health [NIH], Bethesda, MD, USA).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

The paraffin-embedded sections were deparaffinized, rehydrated, soaked in a solution of 3% H₂O₂ and methanol for 10 min, and treated with proteinase K (Cat# ST533; Beyotime, Shanghai, China; 20 mg/mL at 22 ± 2 °C). The terminal deoxynucleotidyl transferase enzymatic reaction solution (Cat# KGA700; KeyGen Biotech, Nanjing, China) was added dropwise to the sections and incubated in a humidified chamber at 37 °C for 1 h. Horseradish peroxidase working solution (Cat# KGA700; KeyGen Biotech) was added to the sections and incubated at 37 °C for 30 min. The 3,3′-diaminobenzidine working solution (Cat# KGA700; ZSGB-Bio) was then added after washing to trigger the color reaction. Finally, the sections were observed by a fluorescence microscope (DM4000B; Leica), and TUNEL-positive cells were counted by a researcher blinded to the experimental groups.

Western blotting

Protein concentrations in the right lung tissue of each group were assessed via western blot analysis using the bicinchoninic acid protein assay kit (Cat# PC0020; Solarbio). The samples were separated using 6–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50–70 μg/10 μL), transferred to polyvinylidene difluoride membranes, and incubated overnight at 4 °C with the following primary antibodies: anti-p-Akt (Ser473; 1:1000; RRID: AB_2315049; Cell Signaling Technology), anti-Akt (1:2000; RRID: AB_915783; Cell Signaling Technology), anti-p-mTOR (1:2000; Cat# R25033; ZenBio), anti-mTOR (1:2000; Cat# 380411; ZenBio), anti-p62 (1:2000; RRID: AB_10694431; Proteintech), anti-LC3 (1:1000; RRID: AB_2137737; Proteintech), anti-NLRP3 (1:1000; RRID: AB_2889890; Abcam, Cambridge, UK), anti-caspase-1 (1:1000; RRID: AB_2819181; Santa Cruz Biotechnology), anti-IL-1β (1:1000; Cat# 350193; ZenBio), anti-IL-18 (1:2000; Cat# DF6252; Affinity), anti-GSDMD N-Terminal Antibody (1:1000; Cat# DF13758; Affinity), anti-caspase-11 (1:1000; Cat# R23724; ZenBio), and anti-GAPDH (1:2000; RRID: AB_2107436; Proteintech). The samples were incubated with goat anti-mouse or anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP; 1:5000; RRID: AB_449890; Abcam) for 2 h. Western blot signals were detected using a bioimaging system (ChemiDoc XRS+; Bio-Rad), and the band intensities were analyzed using the ImageJ software.

Immunofluorescence

Immunofluorescence was performed on the paraffin-embedded left lung tissues collected from neonatal rats in each group. Tissue antigen retrieval was performed by blocking with 10% normal goat serum for 20 min. The tissues were then sliced and incubated with anti-p62 (1:2000; RRID: AB_10694431; Proteintech), anti-NLRP3 (1:100; RRID: AB_2889890; Abcam), anti-caspase-11 (1:100; Cat# R23724; ZenBio), and primary antibody solution for immunofluorescence labeling. The sections were conjugated with an appropriate fluorescent fluorescein isothiocyanate or cyanine 3 secondary antibody (1:300; Cat# 550066; ZenBio) and counterstained with 4,6-diamidino-2-phenylindole to label the DNA. The intensities of the fluorescence signals were measured using ImageJ (NIH) and expressed as the mean fluorescence intensity of the lung tissue.

Statistical analysis

Data were presented as the mean ± standard error of the mean (SEM). Owing to the small sample size, no outlier tests were conducted. The Shapiro–Wilk test was used to assess the normality of the experimental data. Unpaired t-tests were used to compare the means between two groups. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) and the least significant difference post-hoc test. All graphs were prepared using the Prism 8.02 software (RRID: SCR_002798; GraphPad, San Diego, CA, USA). SPSS 25.0 (RRID: SCR_002865; IBM, Armonk, NY, USA) was used for statistical analyses. A p < 0.05 indicated a significant difference.

Results

Effect of LPS on placenta and alveolar development in neonatal rats

The placentas of the control group were normal after delivery, with no signs of inflammation or obvious neutrophil infiltration. H&E staining of the placenta of rats in the LPS group showed a significantly higher degree of neutrophil infiltration in each layer of tissue than in the control group (p < 0.0001; Figure 1a, b).

Figure 1. Effect of LPS on placenta and alveolar development in neonatal rats. (a, b) H&E staining of placental tissue and comparison of neutrophil counts between two groups. The data were expressed as mean ± SEM for n = 6 rats/group (****p < 0.0001). (c–e) Comparison of pathological manifestations, RAC and MLI values in lung tissues at each postnatal time point (P0, P1.5, P3, P5, and P7) between the control and LPS groups. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05 at P3, **p < 0.01 at P5, and ***p < 0.05 at P7). P0, P1.5, P3, P5, and P7 are postnatal time points.

Additionally, H&E staining was used to observe the alveolar development of the LPS and control groups at various birth time points (P0, P1.5, P3, P5, and P7). The RAC was evaluated to compare the number of alveoli between the LPS and control groups; no significant differences were observed at P0 or P1.5. This may be attributed to the number of alveoli approaching 1, which occurs at the beginning of development. However, emphysema was more pronounced, and the alveolar volume was larger in the LPS group than in the control group (Figure 1c, d). At P3 (p < 0.05), P5 (p < 0.01), and P7 (p < 0.001), the LPS group had a lower RAC value than the control group. Meanwhile, the MLI was significantly higher in the LPS group (P3, p < 0.01; P5, p < 0.0001; P7, p < 0.001), suggesting increased alveolar cross section and volume, which was consistent with the RAC results. Hence, overall, the number of alveoli was significantly reduced, the distribution of alveoli was more disordered, and the degree of emphysema was higher in the LPS group than in the control group. These results suggest that LPS induced BPD development (Figure 1c–e).

Effects of LPS on pyroptosis in neonatal rat lung tissues

The dead cells in BPD and control lung tissues were counted via TUNEL staining at various birth time points (P0, P1.5, P3, P5, and P7). The results showed that the rate of abnormal cell death in lung tissues of the BPD group significantly increased at P0, and the degree of increase was greater. There was an unexpected decrease in the number of cell death at P1.5 compared to other time points. Cell death significantly increased at P3, P5, and P7 (Figure 2a, b). The relative levels of the pyroptosis-related proteins Pro-caspase-1, caspase-1, NLRP3, Pro-IL-1β, IL-1β, IL-18/Pro-IL-18, GSDMD-N/GSDMD, Pro-caspase-11 and caspase-11 (Figure 2c) in the lung tissue homogenates of the two groups was compared via western blotting. The levels of Pro-caspase-1 (P0, p < 0.05; P3, p < 0.01), caspase-1 (P0, p < 0.001; P3, p < 0.0001), NLRP3 (P0, p < 0.01; P3, p < 0.001), Pro-IL-1β (P0, p < 0.05; P3, p < 0.01), IL-1β (P0, p < 0.0001; P3, p < 0.001), IL-18/Pro-IL-18 (P0, p < 0.001; P3, p < 0.0001), GSDMD-N/GSDMD (P0, p < 0.05; P3, p < 0.0001), Pro-caspase-11 (P0, p < 0.01; P3, p < 0.05), and caspase-11 (P3, p < 0.01) (Figure 2c–e) were significantly increased at P0 and P3 in the BPD group. Furthermore, significant differences in caspase-1 (p < 0.001), IL-1β (p < 0.01), GSDMD-N/GSDMD (p < 0.01), caspase-11 (p < 0.05) (Figure 2c, f) were found at P5. Caspase-11 (P0), Pro-caspase-1, Pro-IL-1β, IL-18/Pro-IL-18, and Pro-caspase-11 (P5) had no significant differences. These results demonstrate that the level of pyroptosis induced by the formation of the NLRP3 inflammasome and caspase-11 inflammasome may be increased primarily during early alveolar development (P0–P3).

Figure 2. Effects of LPS on pyroptosis in neonatal rat lung tissues. (a, b) The ratio of dead cells to all cells (per 1000 cells) at each postnatal time point (P0, P1.5, P3, P5, and P7) in the BPD and control groups was compared using TUNEL staining (n = 6 rats/group; ***p < 0.001 and ****p < 0.0001). (c–f) Relative levels of the indicators related to pyroptosis: Pro-caspase-1, caspase-1, NLRP3, Pro-IL-1β, IL-1β, IL-18/Pro-IL-18, GSDMD-N/GSDMD, Pro-caspase-11, and caspase-11 in lung tissues of the BPD and LPS groups, measured via western blotting. Data are expressed as mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Rapamycin promotes alveolar development in BPD

Compared with that of the BPD group, the RAC value of the BPD + rapamycin group was increased at P2, and significantly increased at P5 (p < 0.05) and P14 (p < 0.05) (Figure 3a, b, d, f). RAC values decreased at P2, P5 and P14 following MHY1485 treatment, although not significantly. Compared with that of the BPD group, the MLI level of the BPD + rapamycin group was significantly decreased at P2 (p < 0.05), P5 (p < 0.001), and P14 (p < 0.01) (Figure 3a, c, e, g). The MLI level increased at P14 following MHY1485 treatment (p < 0.05) (Figure 3a, c, e, g), and at P2 and P5, however, not significantly. After rapamycin treatment, the congestion of the left lung tissue was improved, and the volume of the lung tissue was increased. However, the lung tissue congestion of the MHY1485 + BPD group was more serious, and the lung tissue volume was low (Figure 3h). These results confirm that rapamycin can promote alveolar development in BPD and has therapeutic potential.

Figure 3. Rapamycin promotes alveolar development in BPD. (a-g) RAC and MLI values of the four groups (control, BPD, BPD + rapamycin, and BPD + MHY1485) were compared at P2, P5, and P14 using hematoxylin and eosin staining. Data are expressed as mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). (h) Macroscopic specimens of lung tissue from the four groups are also shown. The morphology, including the size, of the left lung of neonatal rats was observed mainly from the dorsal side. *P represents the statistical data values of the control and BPD groups, BPD and BPD + rapamycin groups, and BPD and BPD + MHY1485 groups.

Rapamycin increases autophagy levels in BPD

Compared with those of the control group, activation of the Akt/mTOR signaling pathway in the BPD group was significantly inhibited at P0 and P3, p-Akt/Akt (p < 0.05) and p-mTOR/mTOR (p < 0.01) levels decreased at P0, and were very low at P3 (p-Akt/Akt: p < 0.001; and p-mTOR/mTOR: p < 0.05). However, no significant differences were observed in these indicators at P5 (Figure 4a–d). This may be a feedback and defense mechanism in neonatal rats. The decreased activation of Akt/mTOR increased the levels of autophagy, which may have had an inhibitory effect on pyroptosis.

Figure 4. Rapamycin upregulates autophagy levels in BPD. (a–d) The phosphorylation levels of Akt and mTOR in lung tissues of the BPD and control groups were detected using western blotting at P0 and P3. Quantitative comparisons of p-Akt/Akt and p-mTOR/mTOR levels were performed. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, and ***p < 0.001). (e–h) The effects of rapamycin and MHY1485 treatments on p-mTOR/mTOR, p62, and LC3 II/I levels in BPD at P2 were examined. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05 and **p < 0.01). (i, j) The p62 immunofluorescence images and mean fluorescence intensity analysis showed the differences observed at P2 following rapamycin and MHY1485 treatment. Data are expressed as the mean ± SEM for n = 6 rats/group (****p < 0.0001).

We further explored the relationship between the Akt/mTOR signaling pathway and autophagy in BPD. Western blotting showed that the p-mTOR/mTOR (p < 0.01) and p62 (p < 0.05) levels were decreased, while that of LC3 II/I (p < 0.01) was increased in the BPD group at P2 (48 h; Figure 4e–h). The mean fluorescence intensity of p62 (p < 0.0001) in the lungs of the BPD group decreased at P2 (Figure 4i, j), indicating that autophagy was increased in the lung tissue, consistent with our speculation. After treating newborn rats with BPD twice with rapamycin at 6 and 24 h (P1) post-birth, mTOR phosphorylation (p < 0.01) in the lung tissues was further reduced at P2, while the level of p-mTOR/mTOR (p < 0.05) was restored after administering MHY1485 (Figure 4e–h). Following rapamycin treatment, along with the decrease in p-mTOR/mTOR levels, the western blotting results showed further decreased levels of p62 (p < 0.05) and LC3 II/I (p < 0.05) in the lung tissues of the BPD group (Figure 4e–h). The opposite results were observed for p62 (p < 0.05) and LC3 II/I (p < 0.05) following MHY1485 treatment (Figure 4e–h). Tissue fluorescence images showed that the mean fluorescence intensity of p62 in the BPD + rapamycin group was further decreased, while the level of p62 in the lung tissues significantly increased (p < 0.0001; Figure 4i, j) following MHY1485 treatment, which was consistent with the western blot results.

These results suggest that rapamycin can maintain and reduce mTOR activation and upregulate p62 and LC3 II/I-related autophagy levels, while MHY1485 has the opposite effect, breaking the hypophosphorylation state of mTOR.

Rapamycin reduces the activation of the NLRP3 inflammasome and attenuates pyroptosis levels in BPD

TUNEL staining of the lung tissues revealed an increased rate of cell death in the BPD group at P2 (p < 0.01). Meanwhile, rapamycin reduced cell death (p < 0.05) and abnormal cell death was more evident following MHY1485 treatment (p < 0.0001; Figure 5a, b). The levels of pyroptosis-related proteins NLRP3 (p < 0.0001), Pro-caspase-1 (p < 0.01), caspase-1 (p < 0.05), Pro-IL-1β (p < 0.0001), IL-1β (p < 0.05), IL-18/pro-IL-18 (p < 0.05) and GSDMD-N/GSDMD (p < 0.01; Figure 5c–j) were increased in the lung tissues of the BPD group at P2, indicating that the NLRP3 inflammasome was activated and pyroptosis was initiated. The levels of NLRP3 (p < 0.001), Pro-caspase-1 (p < 0.05), caspase-1 (p < 0.05), Pro-IL-1β (p < 0.001), IL-1β (p < 0.05), IL-18/Pro-IL-18 (p < 0.05), and GSDMD-N/GSDMD (p < 0.05; Figure 5c–j) decreased in the BPD + rapamycin group compared with the BPD group. Meanwhile, the BPD + MHY1485 group showed increased levels and activation of NLRP3 (p < 0.05), Pro-caspase-1 (p < 0.05), caspase-1 (p < 0.001), IL-1β (p < 0.05), and IL-18/Pro-IL-18 (p < 0.01), whereas no differences were observed in Pro-IL-1β and GSDMD-N/GSDMD expression (Figure 5c–j). Moreover, immunofluorescence staining showed that NLRP3 levels in the BPD group increased significantly at P2 (p < 0.0001). Rapamycin significantly reduced NLRP3 inflammasome activation (p < 0.05), while MHY1485 had the opposite effect (p < 0.001; Figure 5j, k). These results suggest that rapamycin attenuated NLRP3 inflammasome-induced pyroptosis and reduced the inflammatory response in BPD lung tissues at P2, while MHY1485 aggravated pyroptosis.

Figure 5. Rapamycin reduces NLRP3 inflammasome activation and attenuates pyroptosis levels in BPD. (a, b) TUNEL-positive cells rates (per 1000 cells) were counted to evaluate the degree of pyroptosis in lung tissues of four groups (control, BPD, BPD + rapamycin, and BPD + MHY1485) at P2. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, and ****p < 0.0001). (c–j) The relative levels of Pro-caspase-1, caspase-1, NLRP3, Pro-IL-1β, IL-1β, IL-18/Pro-IL-18, GSDMD-N/GSDMD, Pro-caspase-11, and caspase-11 in these four groups was detected using western blotting; the grey value was determined. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). (k l) Tissue immunofluorescence was used to analyze differences in NLRP3 levels among groups. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05, ***p < 0.001, and ****p < 0.0001).

Rapamycin reduces caspase-11 inflammasome production in BPD

Western blotting showed that Pro-caspase-11 (p < 0.0001) and caspase-11 (p < 0.0001) levels (Figure 6a–c) in neonatal lung tissues from rats with BPD significantly increased at P2, consistent with the immunological results (p < 0.0001; Figure 6d, e). The levels of Pro-caspase-11 (p < 0.01) and caspase-11 (p < 0.01) were decreased in the BPD + rapamycin group (Figure 6a–c), consistent with the immunofluorescence results (p < 0.001; Figure 6d, e). Activation of the caspase-11 inflammasome was aggravated in the BPD + MHY1485 group (p < 0.05) (Figure 6a–c), while the average immune strength value of caspase-11 increased (p < 0.0001; Figure 6d, e). These results indicated that rapamycin also inhibited the non-canonical pathway of pyroptosis induced by the caspase-11 inflammasome in the lung tissues of neonatal rats with BPD at P2, while MHY1485 had the opposite effect.

Figure 6. Rapamycin reduces caspase-11 inflammasome production in BPD. (a–c) The relative levels of Pro-caspase-11 and caspase-11 in the four groups (control, BPD, BPD + rapamycin, and BPD + MHY1485) was detected using western blotting; the differences in grey value among the groups were compared. Data are expressed as the mean ± SEM for n = 6 rats/group (*p < 0.05, **p < 0.01, and ****p < 0.0001). (d, e) Tissue immunofluorescence was used to analyze differences in caspase-11 levels among groups. Data are expressed as the mean ± SEM for n = 6 rats/group (***p < 0.001, and ****p < 0.0001).

Discussion

BPD is a serious chronic lung disease in premature infants with a prevalence rate of 11–50% across developed and developing countries.¹ Nearly 50 000 extremely preterm infants are born annually in the United States, of which approximately 35% (18 000) present with BPD.³³ BPD is also a risk factor for serious complications, such as pulmonary hypertension, cardiac dysfunction, chronic obstructive pulmonary disease, and neurodevelopmental disorders.^1,2,34,35 Children with BPD clinically exhibit fewer, larger, and more simplified alveoli, mild thickening of the smooth muscle, and pathological findings of fewer but deformed arteries.^36,37 BPD pathogenesis includes damage to the immature lung caused by various unfavorable factors, including oxygen toxicity, barotrauma or volume trauma, intrauterine inflammation, and sepsis, based on genetic susceptibility, low gestational age, and abnormal repair of lung tissue after injury.^36,38,39 Intrauterine infection can lead to intrauterine inflammation, which is not only an important independent risk factor for preterm birth, but also plays an important role in the occurrence and development of BPD.^38,39 However, the pathogenic mechanism of intrauterine infection remains unclear; therefore, it is necessary to clarify its mechanism and seek possible treatment strategies.

In this study, a neonatal model of BPD was successfully established via intraperitoneal injection of LPS to pregnant rats. Our results suggest that in the critical period of alveolar development (P1–P5),³⁰ LPS can significantly reduce the RAC value and increase the MLI level (Figure 1c–e) of the BPD group, causing a decreased number of alveoli, a simpler alveolar structure, smaller blood vessels in the alveolar wall, and interstitial pulmonary hyperplasia in the BPD group. Previous studies have found increased pyroptosis in animal models of hyperoxia lung injury and LPS-induced acute lung injury.^16–20 Pyroptosis induced by the NLRP3 inflammasome^40,41 and the caspase-11 inflammasome^42,43 have also been reported in several other inflammatory models as a detrimental factor for normal tissue differentiation and development. Our study found that cell death significantly increased in the lung tissues of neonatal rats in the BPD group at each time point (P0, P1.5, P3, P5, and P7; Figure 2a, b). The levels of Pro-caspase-1, caspase-1, NLRP3, Pro-IL-1β, IL-1β, IL-18/Pro-IL-18, GSDMD-N/GSDMD, Pro-caspase-11, and caspase-11 increased at P0 and P3 (Figure 2c–e), while those of caspase-11 (P0), Pro-caspase-1, Pro-IL-1β, IL-18/Pro-IL-18, and Pro-caspase-11 (P5) did not differ significantly (Figure 2c, f). Only significant differences in caspase-1, IL-1β, GSDMD-N/GSDMD, caspase-11 were detected (Figure 2c, f). Hence, pyroptosis occurred at the early stage of alveolar development (P0–P3) in the lung tissues of the LPS-induced BPD neonatal rat model, which may account for the slow increase in = alveoli number. At P3–P5, the pyroptosis level gradually decreased with a weakening of the influence imposed by intrauterine inflammation and the recovery of neonatal rats; accordingly, we did not focus on this period.

The Akt/mTOR signaling pathway was significantly inhibited during the early stage of alveolar development (P0, P2, and P3; Figure 4a–j) in the LPS-induced neonatal rat model of BPD. LPS can significantly inhibit Akt/mTOR signaling and reduce mTOR activation.^44,45 With the reduction in p-mTOR levels, the level of autophagy in tissues increases. During autophagy, the light chain of the microtubule-associated protein 3 (LC3) is converted from Pro-LC3 to LC3-I, which is processed into LC3-II and converted to LC3-I.^46–48 Localized on the autophagosome membrane, LC3-II is degraded and recycled via autophagy. The interaction of p62 with LC3 plays a key role in the formation and degradation of autophagosomes; the higher the level of autophagy, the more p62 is degraded. Therefore, determining LC3-II/LC3-I and p62 levels is a reliable method for monitoring the entire autophagy process.^46–48 In our study, the level of autophagy in the lung tissues of neonatal rats with BPD increased at P2, while those of p62 decreased, and the ratio of LC3 II/I increased. Autophagy inhibits pyroptosis;^22–27 therefore, we speculate that it may be a self-defense or feedback mechanism of neonatal rats to cope with pyroptosis. Accordingly, we explored this possible mechanism using the mTOR inhibitor rapamycin and the mTOR agonist MHY1485 in early alveolar development (6 and 24 h post birth) in BPD pups.

Our results show that rapamycin, as an enhancer of autophagy, can promote alveolar development to some extent (at P14; Figure 3a–g), while MHY1485 has the opposite effect on alveolar development (at P14) (Figure 3a–g). Following administration of rapamycin, p62 levels in BPD lung tissues further decreased at P2, while the LC3 II/I ratio increased, suggesting that the level of autophagy increased, and MHY1485 had the opposite effect (Figure 4e–j). The protective effect of rapamycin on BPD can be attributed to its inhibition of the NLRP3 inflammasome-induced classical pyroptosis pathway and the caspase-11 inflammasome-induced non-canonical pyroptosis pathway by continuously enhancing the level of autophagy in the early stage of alveolar development. This study showed that, while the level of autophagy increased after using rapamycin, those of Pro-caspase-1, caspase-1, NLRP3, Pro-IL-1β, IL-1β, IL-18/Pro-IL-18, GSDMD-N/GSDMD (Figure 5c–l), Pro-caspase-11, and caspase-11 (Figure 6a–e) were significantly inhibited, and the number of cells undergoing pyroptosis was reduced (Figure 5a, b). Interestingly, MHY1485 can suppress the level of autophagy upregulated in the lung tissues of neonatal rats with BPD due to decreased mTOR phosphorylation, which also weakens the feedback protection mechanism of offspring against pyroptosis and increases pyroptosis. Our findings suggest that the administration of MHY1485 early in alveolar development may deleteriously affect BPD. Rapamycin can prevent the development of lung tissues of neonatal rats from being affected by pyroptosis in the early stage of alveolar development to some extent.

Conclusions

Our results indicate that rapamycin can reduce the occurrence of NLRP3 inflammasome- and caspase-11 inflammasome-mediated pyroptosis by increasing the level of mTOR-induced autophagy in the early stage of alveolar development in the neonatal rat model of BPD caused by LPS-induced intrauterine infection and ultimately promote the normal development and maturation of alveolar tissue (Figure 7). These findings suggest rapamycin as a potential drug to prevent BPD after intrauterine infection.

Figure 7. Schematic of the mechanism by which rapamycin attenuates pyroptosis induced by LPS in bronchopulmonary dysplasia rats. mTOR phosphorylation is inhibited and autophagy promoted.

Authors’ contributions

FZ conceptualized and designed the study, validated the data, analyzed and curated the data, performed the experiments, performed software-related analysis, and wrote and revised the manuscript. LL conceptualized the study, validated and curated the data, acquired funding, obtained resources, revised the manuscript, supervised the study, and performed visualization and administration of the project. MW, ZL, YF, ZG, YZ, and LH performed the experiments and wrote the original draft of the manuscript. JD performed the experiment and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Editage for their assistance with English language editing.

Disclosure statement

The authors report there are no competing interests to declare.

Additional information

Funding

This research was supported by the Sichuan Province Medical Research Project (grant number S21007), the Sichuan Province Applied Basic Research Project (grant number 2022NSFSC0787), the Chengdu Medical Research Project (grant number 2021216), the Research Fund Project of Chengdu Medical College (grant number CYZYB22-11), the Chengdu Medical College Graduate Innovation Fund Key Project (grant number YCX2021-11), the Foundation of Development and Regeneration Laboratory (SYS19-05), and the Fundamental Research Funds for the Central University (SCU2022D006).