Szeto-Schiller 31 eases acute lung injury in neonatal mice with acute respiratory distress syndrome by mediating TXNIP expression and NLRP3 inflammasome activation

Introduction

Neonatal acute respiratory distress syndrome (ARDS), a severe respiratory crisis driven by an acute inflammatory response in the lungs, is characterized by intractable hypoxemia, respiratory distress, and decreased lung compliance (1). Currently, management of exogenous surfactant proteins, mechanical ventilation, assisted neuromodulated ventilation, ensuring appropriate ventilation, and nebulized administration are able to alleviate the symptoms of neonatal ARDS, but there are still many challenges and limitations (2,3). Therefore, studying and exploring the therapeutic targets for neonatal ARDS in depth hold great significance in improving neonatal ARDS.

Neonatal ARDS arises from a complex mechanism that involves the interaction of numerous factors. Oxidative stress (OxS) is a consequence of the imbalance between oxidative and anti-oxidant, and OxS is a key driver of ARDS in newborns (4). OxS may be elicited by multiple factors in ARDS, including infection, mechanical ventilation, and ischemia-reperfusion injury, leading to an elevation in reactive oxygen species (ROS) production, thus impairing cells and giving rise to cell dysfunction and tissue injury (5). The increase in ROS can activate inflammatory signaling pathways, thereby exacerbating pulmonary inflammation and injury (6). Therefore, therapeutic strategies targeting OxS and inflammatory responses hold promising values for managing neonatal ARDS.

Thioredoxin-interacting protein (TXNIP) functions as a multifunctional protein involved in multiple cell processes (7). It has been demonstrated that TXNIP serves as a vital bridge between OxS and inflammation (8). TXNIP dissociates from thioredoxin and interacts with nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the presence of ROS, resulting in the activation of NLRP3 inflammasomes (9). Earlier studies have demonstrated that inhibiting the TXNIP/NLRP3 pathway can protect against lipopolysaccharide (LPS)-induced lung injury (10,11). Thus, targeting the TXNIP/NLRP3 pathway may improve OxS and inflammatory response in neonatal ARDS.

Szeto-Schiller 31 (SS-31), a drug with anti-oxidant and protective mitochondrial functions, has been reported to ameliorate diseases or injuries associated with OxS and mitochondrial dysfunction (12). Nie et al. reported that SS-31 represses the nuclear factor erythroid 2-related factor 2-dependent NLRP3 inflammasomes in macrophages, thereby alleviating idiopathic pulmonary fibrosis (13). In addition, SS-31 suppresses inflammation and OxS in airways caused by cigarette smoke, suggesting that SS-31 can safeguard respiratory health (14). The S100 calcium-binding protein A8/NLRP3 pathway is targeted by SS-31 to inhibit inflammatory responses and decrease LPS-induced acute lung injury (ALI) (15). Notably, SS-31 inhibits TXNIP expression and attenuates renal injury in diabetic kidneys (16). However, whether SS-31 ameliorates ALI in neonatal ARDS by mediating the TXNIP/NLRP3 pathway-dependent OxS and inflammatory responses is unclear.

Therefore, the present study explored whether SS-31 protects against ALI by mediating the TXNIP/NLRP3 pathway using LPS-induced ARDS cell models and animal models, which helps establish a theoretical foundation for SS-31 as a potential therapy for neonatal ARDS. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-165/rc).

Methods

Data collection and analysis

Differentially expressed genes (DEGs) in ARDS patients were analyzed based on the Gene Expression Omnibus (GEO) database (GSE5883). Data from all arrays were processed to eliminate batch bias using the “sva” R package. A screening criterion for DEGs was log₂fold change >1 and P<0.05. Signal transduction pathways involved in DEGs were acquired via Kyoto Encyclopedia of Gene Genomes (KEGG) pathway enrichment analysis. Gene Ontology (GO) pathway enrichment analysis of DEGs was performed using the for Annotation, Visualization and Integrated Discovery​ online bioinformatics tool and the Cluster Profiler package. DEGs in the enrichment of GO-cellular component (CC), GO-biological process (BP), GO-molecular function (MF), and KEGG were subjected to Venn analysis. Lung-related drugs that work via mediating TXNIP expression were screened based on the Search Tool for Interactions of Chemicals (STITCH) database (http://stitch.embl.de/).

Clinical sample collection

Newborns with ARDS (n=42) admitted to the Neonatal Unit of Nantong First People’s Hospital between January 2021 and December 2023 were recruited in this study. Inclusion criteria were as follows: newborns were diagnosed with ARDS as per the Montreux definition [2017], newborns possessed complete clinical information, and newborns did not receive any treatment. Newborns hospitalized for other diseases during the same period were enrolled as controls (n=20). Inclusion criteria for control newborns: gestational age 37–42 weeks; no history of resuscitation for asphyxia at birth; no meconium aspiration or congenital anomalies; no cardiopulmonary disease or respiratory distress after birth. The study was reviewed and approved by the ethics committee of Nantong First People’s Hospital (No. 2021KT112). Parents of all participants had provided informed written consent. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Collection of venous blood (2 mL) from both groups of newborns was done within 24 hours of diagnosis using blood-collecting tubes containing inert separator gels and procoagulants. Blood samples were centrifuged within 2 hours (3,000 rpm, 10 minutes) to obtain serum samples and stored at −80 ℃.

Cell culture and grouping

Human lung microvascular epithelial cells (HLMVECs; #CP-H001; Procell, Wuhan, China) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (#11965092; Thermo, Waltham, MA, USA) and 1% penicillin and streptomycin (#15140148; Thermo) at 37 ℃ in an incubator with 95% humidity and 5% CO₂. To construct ARDS models in vitro, different doses of LPS (10 mg/mL, 1 mg/mL, 500 µg/mL, 400 µg/mL, 300 µg/mL, 200 µg/mL, 100 µg/mL, 50 µg/mL, 10 µg/mL, and 1 µg/mL; #KGA8127-51; KeyGEN BioTech, Nanjing, China) were used to stimulate HLMVECs for 24 hours. To assess the cytotoxicity of SS-31 (#HY-P0125; MedChemExpress; Shanghai, China), HLMVECs were treated with different doses of SS-31 (10, 5, 2.5, 1.25, 0.625, and 0 µM) for 24 hours. HLMVECs were set up in 4 sub-groups, including control, LPS, SS-31, and LPS + SS-31. For the above sub-groups, HLMVECs were cultured for 24 hours, followed by stimulating with phosphate-buffered saline (PBS; 10 mg/mL), LPS (300 µg/mL), SS-31 (2.5 µM), or LPS (300 µg/mL) + SS-31 (2.5 µM) for another 24 hours.

Cell viability analysis

HLMVECs (100 µL/1×10⁵ cells/mL) were inoculated in 96-well plates for 24 hours, followed by stimulating with LPS, SS-31, or LPS + SS-31. Twenty-four hours later, the cell counting kit-8​ (CCK-8) reagent (#CP736; 5 µL; Dojindo, Kumamoto, Japan) was added and incubated for 2 hours. Measurement of the optical density​(OD) was performed at 450 nm using a multi-mode spectrophotometer (Spectramax M3; Molecular Devices, San Jose, CA, USA). Cell viability rate (%) = [OD₄₅₀ (experimental) − OD₄₅₀ (blank)/OD₄₅₀ (control) − OD₄₅₀ (blank)] × 100%. Cell inhibitory rate (%) = [1 − OD₄₅₀ (experimental)/OD₄₅₀ (control)] × 100%.

Cell proliferation analysis

For cell proliferation analysis, the BeyoClick™ 5-ethynyl-2'-deoxyuridine (EDU)-594 cell proliferation assay kit (#C0078S; Beyotime, Shanghai, China) was used as per the operating guidelines incorporated in the kit. In brief, HLMVECs (500 µL/1×10⁵ cells/mL) were treated with different regimens. After 24 hours, HLMVECs were incubated with EDU (50 µM) for 2 hours. After washing, HLMVECs were fixed by 4% paraformaldehyde (500 µL) for 30 minutes and then incubated in a solution of glycine (200 µL/2 mg/mL) for 5 minutes. After washing, HLMVECs were incubated in 1× Apollo^® Staining Reaction Solution (500 µL) for 30 minutes away from light, followed by processing with TritonX-100 (500 µL/0.5%). Staining of nuclei was made successfully with 4',6-diamidino-2-phenylindole (DAPI; #C1002; Beyotime). Photographs were taken and observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Lactate dehydrogenase (LDH) release assay

Assessment of cell injury was carried out by measuring the LDH activity in cell culture supernatants. HLMVECs (100 µL/1×10⁵ cells/mL) were stimulated with different projects, and detection of the LDH activity in the supernatant was achieved using the LDH cytotoxicity assay kit (#C0016; Beyotime) following the guidelines for use provided by the manufacturer. Measurement of OD values at 490 nm was conducted by a multi-mode spectrophotometer.

Cell apoptosis analysis

HLMVECs were incubated for 24 hours under different treatments, followed by digestion with trypsin without ethylene diamine tetraacetic acid. After washing twice with PBS, HLMVECs were collected. HLMVECs (about 5×10⁵) were made into single-cell suspension with 500 µL of binding buffer, followed by the addition of Annexin V-allophycocyanin​(APC) (5 µL) and 7-aminoactinomycin D (7-AAD; 5 µL). After mixing gently, HLMVECs were reacted away from light for 10 minutes. HLMVECs were subjected to flow cytometry (CytoFLEX Flow Cytometer, Beckman Coulter, Brea, CA, USA) within 1 hour.

Animal experiments

Mice with a C57BL/6 background were purchased from Vital River Co., Ltd. [Beijing, China; animal certificate number SCXK (Jing)2021-0011] for breeding, consisting of 15 female mice and five male mice. These mice were housed under controlled conditions with a light/dark cycle of 12 hours, humidity of 45–60%, and temperature of 24–25 ℃. Water and food were offered ad libitum. Animal care and experimental procedures were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committees of Nantong University (No. P20230218-024; date: March 8, 2023). The experimental unit was the individual mouse, C57BL/6 mice (6-day-old) were used for animal experiments and randomly assigned control, LPS, LPS + SS-31 5 mg/kg (SS-31-5 mg/kg), and LPS + SS-31 10 mg/kg (SS-31-10 mg/kg) groups (n=5 mice/group). To establish ARDS animal models C57BL/6 mice were injected intraperitoneally with LPS (2 mg/kg). After 30 minutes of LPS injection, mice were treated with different doses of SS-31 (5 or 10 mg/kg) by intraperitoneal administration, respectively. Control mice were administered equal amounts of sterile saline. A sample size of 20 mice (n=5 mice/group) was determined using the G*Power software package for a priori one-way analysis of variance (ANOVA) of lung wet/dry weight ratio, with a large effect size (Cohen’s f=0.72) derived from pilot experiments, achieving 87.1% power at α=0.05. Twelve hours later, mouse eyeballs were removed for collection of blood samples, and serums were separated through centrifugation (3,500 rpm, 4 ℃, 15 minutes). All mice were euthanized and their lung tissues were removed for subsequent experiments. A protocol was prepared before the study without registration.

Histopathological analysis

Mouse lung tissues were fixed overnight with 4% paraformaldehyde (#P0099; Beyotime) and embedded in paraffin wax. Mouse lung tissues were then sliced into sections with 5 µm thickness and then stained with the hematoxylin and eosin (HE) staining kit (#C0105; Beyotime) according to the instructions of manufacturer. After staining, lung sections were photographed by a microscope (Olympus).

Wet-to-dry weight ratio

Pulmonary oedema was assessed by analyzing the ratio of wet-to-dry weight. Briefly, a portion of tissue was excised from the right upper lung and flushed with saline. After drying on filter paper, the lung tissue was weighed on a piece of aluminum foil (wet weight), followed by drying in an oven (60 ℃, 48 hours), and then reweighed (dry weight). The wet-to-dry weight ratio was calculated as wet weight/dry weight (17).

Western blot

Total protein was extracted from HLMVECs and lung tissues with a whole protein extraction kit (#KGB5303; KeyGEN BioTech) and then quantified using a bicinchoninic acid (BCA) protein assay kit (#KGB2101; KeyGEN BioTech). Equal amounts of proteins (25 µg) were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were incubated with appropriate primary antibodies at 4 ℃ overnight after sealing with 5% bovine serum albumin. Subsequently, the membranes enhanced electrochemiluminescence​ assay kit (#KGC4603; KeyGEN BioTech). Information on primary antibodies is exhibited in Table S1.

Enzyme-linked immunosorbent assay (ELISA)

The levels of TXNIP, NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell culture supernatants and/or serum samples were measured using the corresponding ELISA kits based on guidelines from the manufacturer. Information on the ELISA kits is presented in Table S2.

ROS assay

Intracellular ROS detection was carried out with a ROS assay kit (#KGA7308; KeyGEN BioTech). Briefly, different cultured HLMVECs were harvested and washed with serum-free medium for three times, followed by incubation with 2',7'-dichlorodihydrofluorescein diacetate (10 µM) at 37 ℃ for 20 minutes. After washing with serum-free medium, HLMVECs were analyzed by using flow cytometry (CytoFLEX Flow Cytometer).

Intraorganizational ROS levels were detected with a slice ROS assay kit (green fluorescence) (#BB-460513; Bestbio, Shanghai, China). In brief, 1× washing solution (200 µL) was added to frozen tissue sections (10 µm) at room temperature and allowed to stand for 5 minutes. After aspirating the washing solution, the sections were incubated in staining working solution (100 µL) for 30 minutes. The sections were rinsed twice with PBS and loaded with coverslips, followed by detection of fluorescence intensity under a fluorescence microscope (Olympus).

Detection of OxS-related parameters

Intracellular malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities were measured with cell MDA assay kit (#A003-4-1; Jiancheng Bioengineering Institute, Nanjing, China) and the SOD assay kit (#A001-3; Jiancheng Bioengineering Institute) based on manufacturer’s guidelines.

For tissue samples, mouse lung tissues were homogenized on ice and centrifuged (10,000 ×g, 4 ℃, 15 minutes) to obtain supernatants. Re-centrifugation (35,000 ×g, 4 ℃, 8 minutes) was performed to obtain the supernatant. The MDA levels and SOD activities were detected with mouse the MDA/SOD ELISA kit (#JL13329/JL12237; Jianglai Biotechnology Co., Ltd., Shanghai, China).

TdTmediated dUTP nick end labeling (TUNEL) staining

An apoptosis detection kit (#C1088; Beyotime) was used to assess cell death in lung tissues. In brief, lung tissue sections were processed with xylene and ethanol, followed by incubation with proteinase K (20 µg/mL; #ST533; Beyotime) without DNase at 37 ℃. Thirty minutes later, the TUNEL detection solution (50 µL) was added and incubated for 60 minutes. For nuclear re-staining, DAPI was utilized, and the sections were observed under a microscope (Olympus).

Statistical analysis

Statistical analyses were performed using Prism 9 software (GraphPad, San Diego, CA, USA). Data from at least three independent experiments were presented as mean ± standard error of the mean (SEM). Normality was assessed using the Shapiro-Wilk method. When comparing the two groups, an unpaired t-test was adopted. Comparisons were made among multiple groups using one-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was set at a P value <0.05.

Results

Screening for ARDS-related genes and target drugs

To gain insight into the molecular mechanisms of ARDS pathogenesis, we analyzed the GSE5883 dataset which contains DEGs derived from HLMVECs with and without exposure to LPS for 24 hours. A total of 1,723 DEGs were screened, including 1,053 down-regulated DEGs and 670 up-regulated DEGs (|log₂fold change| >1, P<0.05) (Figure 1A). These DEGs were mainly associated with the TNF signaling pathway, lipid and atherosclerosis, IL-17 signaling pathway, cytokine-cytokine receptor interaction, and NOD-like receptor signaling pathway (Figure 1B). DEGs co-enriched in GO-BP, GO-CC, GO-MF, and KEGG were obtained by Venn analysis. We discovered that C3, TXNIP, and IL-6 were co-enriched in the above four subsets (Figure 1C). Among them, TXNIP caught our eye because of its association with the activation of NLRP3 inflammasomes. Next, we screened the drugs associated with TXNIP with the aid of the STITCH database. The results showed that TXNIP may be a target for Bendavia (also called SS-31) and Metformin. However, Metformin has other target proteins in addition to TXNIP, so SS-31 was chosen for subsequent analysis (Figure 1D). These results manifested that SS-31 may be a potential drug for ARDS treatment by targeting TXNIP.

Figure 1 Looking for ARDS-associated DEGs and targeted drugs. (A) A volcano plot showing DEGs in the GSE5883 dataset (|log₂fold change| >1, P<0.05). (B) KEGG pathway enrichment analysis of DEGs in the GSE5883 dataset. (C) Venn analysis of DEGs co-enriched in GO-BP, GO-CC, GO-MF, and KEGG. (D) Analysis of TXNIP-related drugs based on the STITCH database. ARDS, acute respiratory distress syndrome; BP, biological process; CC, cellular component; CTRL, control; DEGs, differentially expressed genes; GO, Gene Ontology; IL, interleukin; KEGG, Kyoto Encyclopedia of Gene Genomes; MF, molecular function; STITCH, Search Tool for Interactions of Chemicals; TXNIP, thioredoxin-interacting protein.

Serum TXNIP levels are positively correlated with NLRP3 in newborns with ARDS

Based on these results, we further validated expression patterns of TXNIP and NLRP3 inflammasome-related parameters in neonatal sera with ARDS. Results of ELISA showed that serum TXNIP, NLRP3, ASC, and caspase-1 levels were significantly higher in newborns with ARDS compared to healthy controls (Figure 2A-2D). Western blot results for these parameters were consistent with ELISA results (Figure 2E,2F). These results manifested that circulating TXNIP levels were elevated in newborns with ARDS, accompanied by activation of NLRP3 inflammasomes. Serum TXNIP levels were positively correlated with NLRP3 in newborns with ARDS (Figure 2G). Collectively, these results suggested that abnormal TXNIP up-regulation and NLRP3 inflammasome activation may be associated with neonatal ARDS.

Figure 2 Serum TXNIP levels are associated with NLRP3 inflammasomes in newborns with ARDS. (A-D) Serum TXNIP, ASC, NLRP3, and caspase-1 levels between control subjects and ARDS were detected by ELISA. (E,F) Protein levels of TXNIP, ASC, NLRP3, and caspase-1 in neonatal sera were assessed by western blot. (G) The correlation between serum TXNIP and NLRP3 levels in newborns with ARDS were determined by Pearson’s correlation analysis. Data are represented as mean ± SEM. Statistical analysis was performed using unpaired t-test (A-D) or two-way ANOVA (E). ***, P<0.001. ANOVA, analysis of variance; ARDS, acute respiratory distress syndrome; ASC, apoptosis-associated speck-like protein; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; SEM, standard error of the mean; TXNIP, thioredoxin-interacting protein.

SS-31 lightens LPS-induced HLMVEC injury

To investigate the efficacy of SS-31 on ARDS treatment, we exposed HLMVECs to LPS to establish ARDS cell models in vitro. Different doses of LPS were used to treat HLMVECs. As exhibited in Figure S1A, the viability of HLMVECs showed a dose-dependent decrease after LPS treatment, and the inhibitory rate of LPS on HLMVECs was more than 60% when its concentration was 400 µg/mL, so 300 µg/mL of LPS was chosen to induce HLMVECs for ARDS cell models. Next, we explored the cytotoxicity of SS-31 on HLMVECs. The chemical structural formula of MTP-131 is presented in Figure S1B. Moreover, we observed that SS-31 treatment had no significant effect on the activity of HLMVECs (Figure S1C). However, SS-31 treatment impaired the inhibitory effect of LPS on HLMVECs, and 2.5 µM of SS-31 was selected for further study (Figure S1D).

To verify whether SS-31 plays a protective role on LPS-stimulated HLMVECs by regulating TXNIP and NLRP3 inflammasome activation, we detected TXNIP and NLRP3 protein levels in HLMVECs co-treated with LPS and SS-31. Stimulation with LPS elevated TXNIP and NLRP3 protein levels in HLMVECs, yet these changes were overturned partly following SS-31 treatment (Figure 3A). After that, we estimated the efficacy of SS-31 on LPS-stimulated HLMVEC injury. EDU assays exhibited a significant inhibitory effect of LPS on the proliferation of HLMVECs, but LPS-mediated influence on proliferation was attenuated (Figure 3B). As a marker of cellular damage, the release of LDH was further measured. LPS stimulation raised LDH activity in culture supernatants of HLMVECs, but the addition of SS-31 undermined the improvement in LDH activity (Figure 3C). Consistently, LPS stimulation evoked the apoptosis of HLMVECs, whereas SS-31 treatment lessened the apoptosis of HLMVECs in the presence of LPS stimulation (Figure 3D). As expected, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and cleaved caspase-3 protein levels were up-regulated in LPS-stimulated HLMVECs, accompanied by a decrease in Bcl-2 protein levels. However, these alterations were reversed after SS-31 administration (Figure 3E). Notably, SS-31 did have a marked effect on the above parameters in HLMVECs (Figure 3A-3E). All results manifested that SS-31 administration may mitigate LPS-induced HLMVEC injury by repressing the TXNIP/NLRP3 pathway.

Figure 3 SS-31 treatment may ease LPS-induced HLMVEC injury and apoptosis via mediating the TXNIP/NLRP3 pathway. (A-E) HLMVECs were treated as follows: control, SS-31, LPS, LPS + SS-31. (A) Detection of TXNIP and NLRP3 protein levels in HLMVECs was carried out with western blot. (B) The proliferative ability of HLMVECs was evaluated by EDU assays. (C) Cell injury was determined by assessing the release of LDH. (D) The apoptosis of HLMVECs was detected with flow cytometry. (E) Protein levels of Bax, Bcl-2, and cleaved caspase-3 were detected by western blot. Data are represented as mean ± SEM. Statistical analysis was performed using one-way ANOVA (B-E). *, P<0.05; ***, P<0.001. ANOVA, analysis of variance; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; DAPI, 4',6-diamidino-2-phenylindole; EDU, 5-ethynyl-2'-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLMVEC, human lung microvascular epithelial cell; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; SEM, standard error of the mean; SS-31, Szeto-Schiller 31; TXNIP, thioredoxin-interacting protein.

SS-31 impairs OxS, permeability, and inflammatory response in HLMVECs under LPS stimulation

Oxidative damage is an important component in lung injury, so we further investigated the effect of SS-31 on HLMVECs stimulated with LPS. The results displayed that LPS stimulation elevated ROS production and MDA levels but decreased SOD activity in HLMVECs, yet SS-31 administration undercut changes in these parameters (Figure 4A-4C). Changes in vascular permeability can be used as a biomarker for ARDS diagnosis, so the effect of SS-31 treatment on the permeability of HLMVECs urged by LPS was further analyzed. We observed that SS-31 administration diluted LPS stimulation-mediated up-regulation of intercellular adhesion molecule-1 (ICAM-1), poly (ADP-ribose) polymerase (PARP), and vascular cell adhesion molecule-1 (VCAM-1) protein levels in HLMVECs, implying that SS-31 impairs LPS-induced enhancement in the permeability of HLMVECs (Figure 4D). As for the inflammatory response, LPS stimulation-compelled the release of TNF-α, IL-1β, and IL-6 from HLMVECs was attenuated following administration of SS-31 (Figure 4E-4G). Notably, SS-31 treatment increased SOD activities in HLMVECs, but had no significant effect on other parameters (Figure 4A-4G). All results manifested that SS-31 attenuates LPS-induced OxS, permeability, and inflammatory response for HLMVECs.

Figure 4 SS-31 lightens LPS-induced OxS, permeability, and inflammation in HLMVECs. (A-G) HLMVECs were treated as follows: control, SS-31, LPS, LPS + SS-31. (A-C) Production of ROS, MDA, and SOD in HLMVECs was measured using the corresponding kit. (D) Protein levels of ICAM-1, PARP, and VCAM-1 in HLMVECs were detected by western blot. (E-G) Release of TNF-α, IL-1β, and IL-6 from HLMVECs was analyzed by ELISA. Data represent the mean ± SEM. Statistical analysis was performed using one-way ANOVA (A-G). *, P<0.05; ***, P<0.001. ANOVA, analysis of variance; DHE, dihydroergotamine; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLMVEC, human lung microvascular epithelial cell; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; LPS, lipopolysaccharide; MDA, malondialdehyde; OxS, oxidative stress; PARP, poly (ADP-ribose) polymerase; PE-A, peripheral access; ROS, reactive oxygen species; SEM, standard error of the mean; SOD, superoxide dismutase; SS-31, Szeto-Schiller 31; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.

SS-31 may ameliorate histological lesions and edema in the pulmonary of neonatal mice with ARDS via repressing the TXNIP/NLRP3 pathway

To further validate the efficacy of SS-31 on ARDS, neonatal mice with ARDS were constructed by intraperitoneal injection of LPS and then treated with SS-31 for 12 hours. HE staining showed that lung tissues in the LPS group showed significant injury, as evidenced by disruption of alveolar walls, incompleteness of cellular contours, and infiltration of massive inflammatory cells. However, administration of SS-31 relieved the above pathological changes, with a better efficacy at a higher dose (Figure 5A). LPS-induced ARDS mice showed a significant increase in wet/dry values, but administration of high doses of SS-31 reduced wet/dry values, suggesting that SS-31 administration can reduce pulmonary edema (Figure 5B). Interestingly, TXNIP, NLRP3, ASC, and caspase-1 protein levels were up-regulated markedly in LPS-induced ARDS mice-derived lung tissues, but SS-31 treatment decreased TXNIP, NLRP3, ASC, and caspase-1 protein levels, suggesting that SS-31 may work by targeting TXNIP-mediated activation of NLRP3 inflammasomes (Figure 5C). Overall, SS-31 may improve lung injury and edema in neonatal mice with ARDS by repressing the TXNIP/NLRP3 pathway.

Figure 5 SS-31 may ease lung injury and edema in neonatal mice with ARDS by repressing the TXNIP/NLRP3 pathway. (A-C) Neonatal mice were treated as follows: control, LPS, SS-31-5 mg/kg, and SS-31-10 mg/kg. (A) Histopathological alterations in the lungs of neonatal mice were assessed by HE staining. (B) Wet-to-dry weight ratio of lung tissues was assessed. (C) Western blot detection of TXNIP, NLRP3, ASC, and caspase-1 protein levels in lung tissues. Data represent the mean ± SEM. Statistical analysis was performed using one-way ANOVA (A-C). *, P<0.05; **, P<0.01; ***, P<0.001. SS-31-5 mg/kg: LPS + SS-31 5 mg/kg; SS-31-10 mg/kg: LPS + SS-31 10 mg/kg. ANOVA, analysis of variance; ARDS, acute respiratory distress syndrome; ASC, apoptosis-associated speck-like protein; HE, hematoxylin and eosin; LPS, lipopolysaccharide; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; SEM, standard error of the mean; SS-31, Szeto-Schiller 31; TXNIP, thioredoxin-interacting protein.

SS-31 ameliorates cell apoptosis, inflammatory response, and OxS in neonatal mice with ARDS by targeting TXNIP

Subsequently, apoptosis of lung tissue cells in mice was analyzed. TUNEL staining displayed an overt elevation in the proportion of apoptotic cells in lung tissues derived from LPS-induced ARDS mice, however, SS-31 treatment ameliorated cell apoptosis in lung tissues (Figure 6A). Furthermore, serum IL-6, IL-1β, and TNF-α levels were increased obviously in ARDS mice, whereas SS-31 administration decreased these changes in ARDS mice, suggesting that SS-31 treatment attenuates inflammation in ARDS mice (Figure 6B-6D). Subsequently, OxS was assessed by measuring SOD activity, MDA levels, and ROS production in lung tissues. LPS-induced ARDS mice exhibited higher SOD activity, MDA levels, and ROS production in lung tissues, but the abnormal alterations in these parameters were alleviated following SS-31 treatment (Figure 6E-6G). Collectively, SS-31 may improve ARDS in neonatal mice by attenuating apoptosis, inflammatory response, and OxS.

Figure 6 SS-31 may attenuate apoptosis, inflammatory response, and OxS in the lungs of ARDS neonatal mice. (A-G) Neonatal mice were treated as follows: control, LPS, SS-31-5 mg/kg, and SS-31-10 mg/kg. (A) Cell apoptosis in lung tissues was determined by TUNEL staining. (B-D) Serum IL-6, IL-1β, and TNF-α levels were detected by ELISA. (E,F) OxS was analyzed by analyzing SOD activities and MDA levels in lung tissues. (G) ROS levels were measured in lung tissues by staining with BBoxiProbe^® O06 ROS fluorescent probe. Data represent the mean ± SEM. Statistical analysis was performed using one-way ANOVA (A-C). *, P<0.05; **, P<0.01; ***, P<0.001. SS-31-5 mg/kg: LPS + SS-31 5 mg/kg; SS-31-10 mg/kg: LPS + SS-31 10 mg/kg. ANOVA, analysis of variance; ARDS, acute respiratory distress syndrome; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; MDA, malondialdehyde; OxS, oxidative stress; ROS, reactive oxygen species; SEM, standard error of the mean; SOD, superoxide dismutase; SS-31, Szeto-Schiller 31; TNF-α, tumor necrosis factor-α; TUNEL, TdT-mediated dUTP nick end labeling.

Discussion

In this study, the GSE5883 dataset combined with enrichment analysis of GO-BP, GO-CC, GO-MF, and KEGG discovered that C3, TXNIP, and IL-6 were hub genes in ARDS, particularly the TXNIP gene that is associated with NLRP3 inflammasomes. Using the STITCH database, SS-31 was identified as a potential therapeutic agent in mediating the TXNIP gene. Therefore, the study focused on investigating whether SS-31 affects ALI by mediating the TXNIP/NLRP3 pathway in neonatal ARDS.

The key mechanism by which SS-31 protects cells from OxS damage lies in the reduction of ROS production in mitochondria (18). SS-31 also promotes the production of adenosine triphosphate which is a key molecule in cell energy metabolism (19). Moreover, SS-31 can inhibit the formation of the mitochondrial permeability transition pore, which is an important protective mechanism against apoptosis and necrosis (20). Through its anti-oxidant and mitochondrial protective effects, SS-31 may assist in lessening the pathological process and improving clinical outcomes in serious diseases, such as ARDS (15,21).

An increase in Bax may be due to activation of inflammatory factors in neonatal ARDS, resulting in damage to alveolar epithelial cells and endothelial cells (22). Nhu et al. found that SS-31 may inhibit caspase-3 activation indirectly by decreasing Bax expression and attenuating mitochondrial damage, thereby lessening apoptosis (23). Vascular permeability holds a key role in developing ARDS in newborns, and the vascular permeability markers ICAM-1, PARP, and VCAM-1 are significantly up-regulated in neonatal ARDS (24,25). Inflammatory factors play a crucial role in the pathological process of neonatal ARDS, among which TNF-α, IL-1β, and IL-6 are involved in modulating the immune response and inflammatory process and exert a significant influence on ARDS pathogenesis and progression (26). Here, we discovered that SS-31 ameliorated LPS-induced decreases in viability and proliferation as well as increases in apoptosis, vascular permeability, OxS, and inflammation in HLMVECs. The efficacy of SS-31 was verified by constructing LPS-induced ARDS animal models with neonatal mice, and the results demonstrated that SS-31 attenuated histopathological changes notably in the lungs of neonatal ARDS mice, accompanied by reduced lung oedema, improved alveolar capillary barrier integrity, and reduced OxS, inflammation, and apoptosis. These results manifested the pleiotropic role of SS-31 in the treatment of neonatal ARDS, including anti-OxS, anti-inflammation, anti-apoptotic, and improvement of increased vascular permeability, which provides a new perspective on the comprehensive treatment of ARDS.

TXNIP is not only a key molecule that links endoplasmic reticulum stress and programmed cell death, but also an important mediator in maintaining the homeostasis of energy metabolism and OxS (7). TXNIP regulates OxS by binding to TRX and inhibiting TRX activity. Thus, dis-regulation of TXNIP may lead to enhanced apoptosis and inflammation (27). TXNIP, an early inflammatory protein, can activate NLRP3 inflammasomes, resulting in prompting the secretion of IL-1β and IL-18 (28). Zhai et al. showed that NLRP3 inflammasome activation can be significantly inhibited by interfering with TXNIP expression, thereby reducing hepatic inflammation and lipid-induced hepatocyte injury (29). Yang et al. unveiled that inflammatory cytokines are secreted in large quantities through the TXNIP/NLRP3 pathway in animal models with global cerebral ischemic-reperfusion injury (30). Furthermore, LPS induces ALI via the TXNIP/NLRP3 pathway-mediated pyroptosis of alveolar epithelial cells (11,31). In the current study, we discovered that serum TXNIP, NLRP3, ASC, and caspase-1 levels were notably higher in newborns with ARDS, and a positive correlation was observed between TXNIP and NLRP3, manifesting that TXNIP may exert a vital role in the NLRP3 inflammasome activation in neonatal ARDS. Interestingly, SS-31 treatment repressed TXNIP and NLRP3 protein levels in LPS-stimulated HLMVECs and LPS-induced neonatal ARDS mouse models. All these outcomes compel us to conclude that SS-31 may improve ALI by targeting the TXNIP/NLRP3 pathway in neonatal ARDS by inhibiting OxS, inflammation, apoptosis, and vascular permeability. Unfortunately, we did not identify the efficacy of SS-31 in LPS-induced ARDS cells and animal models by knocking down TXNIP, which is a direction for our future work.

Conclusions

Our findings revealed the potential of SS-31 in managing neonatal ARDS. SS-31 eased LPS-induced injury in HLMVECs and improved ALI in LPS-induced ARDS neonatal mice via reducing OxS, inflammation, and apoptosis, as well as vascular permeability. These positive effects may be related to the inhibitory effect of SS-31 on the TXNIP/NLRP3 pathway, providing a direction for developing new therapeutic strategies for neonatal ARDS.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-165/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-165/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-165/prf

Funding: This study was supported by Nantong Municipal Health Commission Research Project in 2023 (No. QNZ2023030), Nantong Municipal Health Commission Research Project (No. MSZ2022011), and Nantong Science and Technology Bureau Research Project in 2022 (No. MSZ2022020).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-165/coif). All authors report the funding from Nantong Municipal Health Commission Research Project in 2023 (No. QNZ2023030), Nantong Municipal Health Commission Research Project (No. MSZ2022011), and Nantong Science and Technology Bureau Research Project in 2022 (No. MSZ2022020). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was reviewed and approved by the ethics committee of Nantong First People’s Hospital (No. 2021KT112). Parents of all participants had provided informed written consent. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Animal care and experimental procedures were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committees of Nantong University (No. P20230218-024; date: March 8, 2023).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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