Preterm birth accounts for nearly 11% of all live births and remains a leading contributor to neonatal morbidity and mortality worldwide [1, 2]. Respiratory distress syndrome, driven by inadequate lung maturation and surfactant deficiency, is one of the most critical complications associated with early delivery [3]. For decades, antenatal corticosteroids (ACS) have represented the cornerstone of perinatal management, effectively reducing respiratory distress syndrome by accelerating the structural and functional maturation of the fetal lung [4]. Their primary mechanism involves promoting type II pneumocyte differentiation and increasing expression of key surfactant proteins (SP-A, SP-B, SP-C) as well as ABCA3, a pivotal regulator of phospholipid transport and lamellar body formation [5].

Despite their well-documented respiratory benefits, concerns regarding the long-term systemic effects of ACS have intensified. A growing body of work suggests that prenatal glucocorticoid exposure may induce persistent developmental programming in extra-pulmonary organs—including alterations in the hypothalamic–pituitary–adrenal axis [6–8] and potential impacts on neurodevelopment and cardiometabolic pathways [9]. Experimental studies further demonstrate that prenatal dexamethasone can impair testicular barrier integrity, suppress testosterone production, and disrupt ovarian development, indicating a broader spectrum of glucocorticoid-related developmental toxicity [10–12]. These findings underscore the need for alternative or adjunctive agents capable of supporting lung maturation while minimizing systemic endocrine and developmental risks.

Within this context, erdosteine has emerged as a biologically plausible candidate. A thiol-containing prodrug, erdosteine is metabolized to active free-sulfhydryl derivatives—particularly the M1 metabolite—conferring antioxidant, anti-inflammatory, and mucoregulatory properties [13]. Clinically, it is used in chronic obstructive pulmonary disease and upper respiratory infections, where it reduces mucus viscosity and mitigates oxidative stress [14, 15]. Yet, despite its favorable safety profile and lung-directed antioxidant capacity, its role in fetal or neonatal lung maturation remains unexplored.

Timed-pregnant Sprague–Dawley rats were obtained from the institutional breeding facility. Pregnancy was confirmed solely by the detection of a vaginal copulatory plug, following standard rodent reproductive assessment protocols.

Animals were housed under controlled environmental conditions (22 ± 2°C, 50–60% relative humidity, 12-hour light–dark cycle) with ad libitum access to food and water.

All husbandry procedures conformed to the Guide for the Care and Use of Laboratory Animals and adhered to AAALAC International standards.

On gestational day 16, preterm delivery was induced by cesarean section under anesthesia. Immediately thereafter, pregnant rats were euthanized under deep anesthesia using an intraperitoneal injection of sodium pentobarbital (200 mg/kg). Adequate depth of anesthesia was confirmed by the absence of corneal and pedal withdrawal reflexes prior to euthanasia. This chemical method was selected in accordance with institutional animal care guidelines to ensure rapid and humane death.

Newborn pups were randomly allocated into four groups:

To avoid litter-based clustering effects, no more than one pup per litter was assigned to each group. Group sizes ranged from 8 to 12 pups depending on litter availability.

Pups in the ERDO group received:

This dose was chosen based on established respiratory safety data and preclinical efficacy reports.

All treatments were administered once daily from postnatal day 0 to postnatal day 5.

On postnatal day 5, pups were euthanized with high-dose pentobarbital. Lungs were inflation-fixed with 10% neutral-buffered formalin, processed routinely, embedded in paraffin, and sectioned at a thickness of 5 µm for histopathological and immunohistochemical analyses.

Preterm birth–related respiratory distress syndrome remains a major cause of neonatal morbidity and mortality and is primarily driven by surfactant deficiency and structural immaturity of the lung. Antenatal corticosteroids are still the standard of care to accelerate fetal lung maturation and reduce early respiratory complications. However, concerns about possible long-term cardiometabolic or neurodevelopmental effects, especially with repeated or high-dose exposure, have renewed interest in alternative or adjunctive strategies that might support lung maturation with a more favourable safety profile [16–20].

In this preterm rat model, dexamethasone and betamethasone produced a nearly normal histological appearance, whereas erdosteine resulted in preserved alveolar structure with only mild interstitial mononuclear cell infiltration. This indicates that erdosteine does not exert harmful tissue effects but induces a modest inflammatory response. The minimal inflammation in the corticosteroid groups aligns with studies showing that antenatal betamethasone reduces inflammatory cell accumulation and stabilises alveolar structure [21, 22]. Given that glucocorticoids suppress NF-κB–dependent cytokine release and leukocyte recruitment [23], the reduced inflammatory scores in DEX and BET are not unexpected. By contrast, the mild infiltration with erdosteine may reflect a more balanced immunomodulatory profile in which oxidative stress is attenuated without complete suppression of local defence mechanisms [13, 14, 24].

Caspase-3 expression was increased in both the interstitial and bronchiolar epithelium of the corticosteroid-treated groups, consistent with previous ovine and rodent studies reporting steroid-induced epithelial apoptosis and thinning of alveolar septa [22, 25]. In contrast, caspase-3 positivity remained low in the erdosteine group, paralleling evidence that erdosteine and its active metabolite limit reactive oxygen species and mitigate mitochondrial stress–related apoptotic pathways [14, 24, 26]. These findings suggest that erdosteine may confer protection against excessive apoptosis during early lung development, in contrast to the more pronounced remodelling triggered by classical glucocorticoids.

The PCNA expression pattern further supports this divergence. Dexamethasone showed the highest proliferative activity, followed by betamethasone, whereas erdosteine and control lungs exhibited lower levels. This is compatible with reports that glucocorticoids transiently enhance epithelial proliferation and differentiation before promoting maturation of alveolar structures [27, 28]. The relatively modest PCNA positivity in the erdosteine group implies that it does not directly stimulate proliferation but may facilitate maturation through preservation of cellular integrity and redox balance—an effect that could support surfactant pathways without provoking excessive architectural remodelling.

One of the most striking findings is the ABCA3 expression profile. ABCA3, a key ATP-binding cassette lipid transporter in type II pneumocytes, is essential for phospholipid import, lamellar body maturation and surfactant homeostasis. Experimental and clinical studies demonstrate that ABCA3 dysfunction impairs lamellar body biogenesis, alters surfactant lipid composition and compromises alveolar stability [29–32]. Loss-of-function ABCA3 variants are associated with lethal neonatal respiratory distress and interstitial lung disease, highlighting its central role in lung development [31, 33, 34]. Although corticosteroids are known to increase ABCA3 expression and lamellar body maturation in experimental settings [5, 35], our data showed the highest ABCA3 staining in the erdosteine group across both interstitial and bronchiolar regions. This suggests that erdosteine may amplify surfactant-related lipid transport not only through pathways overlapping with glucocorticoid responsiveness but also via redox-sensitive mechanisms that preserve phospholipid-synthesising enzymes and membrane transporter activity. The parallel increase in interstitial and epithelial compartments supports a global enhancement of surfactant trafficking.

SFTPA1 displayed a compartment-specific profile. Interstitial staining was most pronounced in the erdosteine group, while epithelial expression was higher in the control and DEX groups and lower in BET and ERDO. SFTPA1 is a major hydrophilic surfactant protein involved in surfactant structure, surface-tension regulation, pathogen opsonisation and immunomodulation [36–38]. Its expression increases during the late canalicular and saccular stages, and contemporary explant, organoid and epithelial models confirm glucocorticoid-driven upregulation of SP-A [39–42]. The distribution observed here suggests that erdosteine may preferentially enhance interstitial or stromal surfactant-related immunoregulation, whereas corticosteroids more strongly influence epithelial differentiation and secretion patterns.

AQP5 and SP-B were immunonegative across all groups at postnatal day 5. This is consistent with developmental reports demonstrating that both markers increase closer to term and early postnatally, often remaining low at very early time points, particularly in immature lung models [43–45]. Thus, their absence likely reflects developmental stage rather than intervention-specific suppression.

Taken together, these findings indicate that erdosteine supports preterm lung maturation in a manner partially overlapping with but distinct from classical corticosteroids. Erdosteine increased ABCA3 and interstitial SFTPA1 expression while maintaining low apoptosis and modest proliferation, accompanied by preserved lung architecture. Dexamethasone and betamethasone induced stronger apoptotic and proliferative responses, leading to near-normal histology but relatively lower ABCA3 levels than erdosteine. These patterns raise the hypothesis that thiol-based antioxidant therapy may complement or, in selected circumstances, partially substitute glucocorticoids in supporting surfactant biogenesis while potentially reducing systemic programming effects. Nevertheless, such strategies require rigorous evaluation of timing, dose and interaction with standard ACS regimens, ideally in clinically relevant models and, ultimately, in human trials.

This study has several limitations. First, the sample size was modest and analyses were conducted at a single early postnatal time point, precluding assessment of longer-term structural or functional outcomes. Second, the immunohistochemical approach was semi-quantitative and targeted selected markers; molecular analyses and functional evaluations (lung mechanics, gas exchange) were not performed. Third, only one dosing regimen of each intervention was tested, and dose–response or combination protocols were not explored. Finally, systemic effects on other organs—which are pertinent to the broader safety considerations of ACS and antioxidant therapies—were beyond the study’s scope.

Despite these limitations, this study provides initial experimental evidence that erdosteine can modulate key surfactant-regulatory markers such as ABCA3 and SFTPA1 in the preterm lung, with an apoptosis–proliferation profile distinct from dexamethasone and betamethasone. These results support further investigation of erdosteine as a potential adjunct or alternative to antenatal corticosteroids, particularly in models integrating functional respiratory outcomes and long-term follow-up.

In this preterm rat model, erdosteine exhibited a maturation profile that was clearly distinct from that of dexamethasone and betamethasone. While classical antenatal corticosteroids generated robust apoptotic and proliferative activity consistent with accelerated structural maturation, erdosteine produced minimal apoptosis, preserved alveolar architecture and a comparatively modest proliferative response. Most notably, erdosteine markedly increased ABCA3 expression and enhanced interstitial SFTPA1 staining, suggesting a favourable impact on surfactant-related lipid transport and innate immune readiness. These effects emerged without the degree of tissue remodelling observed in the corticosteroid groups.

Taken together, the data indicate that thiol-based antioxidant therapy may support surfactant biogenesis and early lung maturation through mechanisms partially overlapping with, yet biologically distinct from, glucocorticoid-driven pathways. Although the present findings are limited by sample size, a single postnatal time point and the absence of functional respiratory assessments, they provide preliminary experimental evidence that erdosteine could serve as an adjunctive or, under selected conditions, a partial alternative to antenatal corticosteroids. Future studies incorporating dose–response analyses, combined regimens, long-term structural and functional outcomes, and translationally relevant models will be essential to define the therapeutic potential and safety profile of erdosteine in the context of preterm lung maturation.