Astragaloside IV Alleviates Trueperella pyogenes-Induced Endometritis via the Nrf2/HO-1 Signaling Pathway

TL;DR

Astragaloside IV, a compound from traditional Chinese medicine, protects against endometritis caused by T. pyogenes by reducing inflammation and oxidative stress through the Nrf2/HO-1 pathway.

Contribution

This study is the first to demonstrate that Astragaloside IV alleviates T. pyogenes-induced endometritis via the Nrf2/HO-1 signaling pathway.

Findings

AS-IV reduces T. pyogenes-induced endometrial damage by suppressing inflammation, apoptosis, and oxidative stress.

Transcriptomic analysis shows AS-IV's effects are linked to inflammation, apoptosis, and oxidative stress pathways.

Nrf2 and HO-1 mediate the protective effects of AS-IV, confirmed by Nrf2 inhibition experiments.

Abstract

The increasing antimicrobial resistance of T. pyogenes, one of the principal pathogens associated with endometritis, presents a formidable challenge in veterinary medicine. Astragaloside IV (AS-IV) is a triterpene saponin compound isolated from the traditional Chinese medicine Astragalus membranaceus. While recognized as the primary bioactive constituent of Astragalus membranaceus with diverse pharmacological properties, its potential to counteract T. pyogenes-induced endometritis has yet to be elucidated. In the current study, T. pyogenes infection models were successfully established in both mouse uteri and cultured goat endometrial epithelial cells (gEECs). Integrating histopathology, molecular biology and transcriptomic technology, this study characterized the multifaceted biological effects of AS-IV. Transcriptomic analysis indicates that the regulatory effects of AS-IV on T. pyogenes-induced infection are primarily associated with the enrichment of signaling pathways related to inflammation, apoptosis, and oxidative stress. Subsequent validation demonstrated that AS-IV treatment effectively alleviated T. pyogenes-induced endometrial damage by suppressing inflammation, apoptosis, and oxidative stress. These effects were mediated through Nrf2 and its downstream target HO-1, a mechanism further confirmed by the loss of protection upon Nrf2 inhibition. In summary, AS-IV protects the endometrium against T. pyogenes-induced inflammatory and oxidative damage by activating the Nrf2/HO-1 signaling pathway.

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Full text

1. Introduction

Endometritis is a prevalent inflammatory disorder in domestic animals following parturition or abortion, the occurrence of which is closely linked to the dynamic shifts in microbial communities during uterine involution [1]. During this transition, the pathological invasion or proliferation of opportunistic pathogens, such as Escherichia coli, Trueperella pyogenes (T. pyogenes), and Staphylococcus species, can disrupt microecological homeostasis and the local endometrial immune environment, thereby precipitating infectious endometritis [2,3]. As a versatile opportunistic pathogen, T. pyogenes exhibits an extensive host range, contributing to a diverse array of infectious conditions in both livestock and wild animals [4]. T. pyogenes primarily mediates purulent inflammatory processes in affected animals [5]. Pyolysin (PLO), a primary virulence factor of T. pyogenes, is a cholesterol-dependent pore-forming cytolysin. It targets cholesterol-rich lipid rafts on cell membranes to form transmembrane pores, thereby leading to direct cytolysis [6]. Furthermore, PLO triggers macrophage pyroptosis, which subsequently liberates a cascade of pro-inflammatory cytokines [7]. T. pyogenes expresses various secondary virulence factors, including neuraminidase, extracellular matrix-binding proteins and fimbriae, all of which are pivotal in mediating adhesion and colonization within host tissues [8]. Currently, clinical isolates of T. pyogenes exhibit varying resistance profiles to various antimicrobial agents [9], with documented multidrug resistance against several classes, including macrolides and lincosamides [10].

The indiscriminate use of antibiotics has intensified the global crisis of antimicrobial resistance, compelling a strategic shift toward natural products and traditional medicine in the search for novel therapeutic agents. AS-IV the principal bioactive constituent of Astragalus membranaceus, exhibits a broad pharmacological profile underpinned by potent antibacterial, anti-inflammatory, and immunomodulatory activities [11,12,13,14,15]. The Nrf2/HO-1 pathway serves as a pivotal defensive system in response to oxidative stress, inflammation, and exogenous stimuli. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1 via its Nrf2 domain and undergoes ubiquitin-mediated proteasomal degradation [16]. Upon exposure to oxidative stress, specific cysteine residues of Keap1 undergo covalent modification, inducing conformational changes that abrogate its ability to mediate Nrf2 degradation. Consequently, Nrf2 accumulates in the cytoplasm and subsequently translocates into the nucleus, where it initiates the transcription of HO-1. This, in turn, orchestrates a series of cytoprotective responses, including anti-inflammation, scavenging of reactive oxygen species (ROS), and inhibition of lipid peroxidation [17]. Activation of the Nrf2/HO-1 signaling pathway represents a primary molecular mechanism through which AS-IV exerts its diverse pharmacological effects. In various models of organ injury, such as hepatic, renal, and cardiac diseases [18,19,20], as well as heavy metal toxicity [21], AS-IV manifests therapeutic efficacy by attenuating ferroptosis and alleviating oxidative stress. By engaging multi-targeted mechanisms, it stabilizes Nrf2 and subsequently upregulates HO-1 alongside other antioxidant genes. This integration fosters a synergistic network of antioxidant, anti-inflammatory, and anti-apoptotic actions [22], ultimately providing comprehensive protection across multiple organ systems.

Endometritis pathogenesis is characterized by an interplay among endocrine, immune, and cytokine systems, where endometrial defense mechanisms serve as the primary determinant of the clinical outcome following bacterial challenge [23]. To date, the potential of AS-IV to mitigate T. pyogenes-induced endometrial injury by targeting the Nrf2/HO-1 signaling pathway has yet to be fully elucidated. However, based on previous research, we hypothesize that AS-IV may alleviate endometritis by modulating the Nrf2/HO-1 signaling pathway, thereby identifying this pathway as a potential therapeutic target. In the present study, employing both in vivo and in vitro models, we elucidated the effects of AS-IV on inflammation, apoptosis, oxidative stress, and the Nrf2/HO-1 pathway in uterine tissues and endometrial epithelial cells. Furthermore, by harnessing transcriptomic sequencing data, we conducted a comprehensive analysis of its therapeutic mechanisms. This research seeks to establish a novel theoretical framework and provide innovative therapeutic strategies for the clinical management of endometritis.

2. Materials and Methods

2.1. Mice and Treatments

A total of thirty-six 8-week-old female specific-pathogen-free (SPF) Kunming (KM) mice, weighing 30–40 g, were obtained from the Second Affiliated Hospital of Harbin Medical University. Mice were housed in individual cages with free access to food and water under standardized conditions. Throughout the study, the non-experimental mortality rate remained below 10% across all groups.

AS-IV (≥98%) was purchased from Beyotime Biotechnology (Shanghai, China). The T. pyogenes strain was provided by Northwest A&F University. Mice were randomly assigned to three groups (n = 12 per group): a control group (Con), a T. pyogenes-infected group (TP), and an AS-IV–pretreated T. pyogenes group (AS-IV + TP). Mice in the prevention group were orally administered AS-IV (60 mg/kg/day), while mice in the Con and TP groups received an equivalent volume of physiological saline. All administrations were conducted daily for 14 consecutive days. On the final day of the 14-day administration period, endometritis was induced by intrauterine injection of 20 μL of a T. pyogenes suspension (1 × 10^10^ CFU/mL) using a microsyringe. After successful establishment of the model, the mice were observed for 3 days and then euthanized. Uterine and ovarian tissues were collected for subsequent analyses. Meanwhile, body weights, as well as the weights of the ovaries and uteri, were recorded to calculate the corresponding organ coefficients.

2.2. Cell Culture and Treatments

Goat endometrial epithelial cells (gEECs) used in this study were an immortalized cell line established by transfection with human telomerase reverse transcriptase (hTERT), as previously described. The gEECs were obtained through an academic collaboration with Professor Yaping Jin (College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China) and were used exclusively for scientific research in accordance with relevant institutional and ethical guidelines. The gEECs were cultured in DMEM/F12 (Gibco, Thermo Fisher Scientific, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum (FBS, ZETA life, Menlo Park, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Beyotime, Shanghai, China). ML385, a specific Nrf2 inhibitor, was purchased from Solarbio Life Sciences (Beijing, China). The cells were maintained in a humidified incubator at 37 °C with 5% CO_2_. For the experiments, cells were randomly assigned into four groups: (1) a control group (Con, without treatment), (2) a TP treatment group (TP), (3) an AS-IV with TP group (AS-IV + TP) and (4) a ML385 treatment group (AS-IV + TP + ML385). The TP group served as the intracellular infection model. Cells were inoculated with Mycoplasma hominis (MOI = 100) for 2 h. Following incubation, the culture medium was replaced with fresh medium supplemented with 50 μg/mL gentamicin (Solarbio, Beijing, China). The cells were then incubated for an additional 2 h. Following a 20-h pretreatment with 20 μg/mL of the AS-IV, cells in the AS-IV and ML385 (Solarbio, Beijing, China) groups were subjected to the same experimental protocol as described for TP group. For the ML385 group, following the establishment of the intracellular infection model, cells were treated with 10 μg/mL ML385 for an additional 24 h.

2.3. Histological Observation

Uterine tissues were fixed in 4% paraformaldehyde and embedded in paraffin to yield 4-μm-thick sections. These sections were then stained with Hematoxylin and Eosin (H&E) before being examined via light microscopy (SWE-CX63, Servicebio, Wuhan, China) for histopathological evaluation.

2.4. Complete Blood Count and MPO Assay

Whole blood was collected in EDTA-K2 anticoagulant tubes and mixed thoroughly. Total white blood cell (WBC) and lymphocyte counts were performed within 2 h of collection using an automated hematology analyzer (BC-2800vet, Mindray, Shenzhen, China). Uterine tissues were homogenized at a ratio of 1:10 (weight/volume) in ice-cold reaction buffer. Subsequently, myeloperoxidase (MPO) activity was determined using an MPO assay kit ( Solarbio, Beijing, China) according to the manufacturer’s instructions, with absorbance measured at 460 nm using a microplate reader.

2.5. Oxidative Stress Detection

Uterine tissues were homogenized in ice-cold reaction buffer at a ratio of 1:10 (w/v). Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were measured using commercial assay kits (Solarbio, Beijing, China) by determining absorbance at 532/600 nm and 450 nm, respectively, with calculations performed according to the manufacturer’s instructions. Intracellular reactive oxygen species (ROS) levels were assessed using a corresponding ROS assay kit (Solarbio, Beijing, China). Briefly, 1 × 10^6^ cells were incubated in serum-free medium with a fluorescent probe (10 μmol/L) and analyzed by flow cytometry, with dichlorofluorescein (DCF) fluorescence detected in the FITC channel to quantify ROS levels.

2.6. Total RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

Total RNA was isolated from mice uterine tissues and gEECs by the TRIzol method according to the instructions of the manufacturer. Subsequently, reverse transcription and RT-qPCR were performed using the Evo M-MLV RT Kit (Accurate Biology, Changsha, China) and the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China), respectively. All reactions were conducted in triplicate, and target gene expression was normalized to the internal control β-actin. Detailed primer information is provided in Table S1 Primer sequences used for quantitative real-time PCR. Relative quantification of gene expression was determined via the 2^−ΔΔCT^ method.

2.7. Western Blot Analysis

Total protein was extracted from mice uterine tissues and gEECs by lysis in RIPA buffer (Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein samples were resolved via SDS-PAGE and subsequently transferred onto PVDF membranes. Following a 2-h blocking step with 5% skimmed milk, the membranes were incubated overnight at 4 °C with primary antibodies at optimized concentrations. The membranes were then incubated for 2 h with HRP-conjugated secondary antibodies (Thermo Fisher Scientific, Shanghai, China), and protein bands were visualized using an imaging system (SCG-W3000 Plus, Servicebio, Wuhan, China).

2.8. Transcriptome Sequencing

RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was employed as the starting material for library construction, where mRNA was first purified using poly-T oligo-attached magnetic beads and subsequently fragmented via divalent cations under high temperature. First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase, followed by second-strand synthesis mediated by DNA Polymerase I and RNase H. After end-repair, 3′ adenylation, and ligation of hairpin loop adapters, cDNA fragments with a size range of approximately 370–420 bp were selected using the AMPure XP system. Following PCR amplification and purification, library quality was validated on the Agilent Bioanalyzer 2100 system. Upon successful quality validation, the clustering of index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA) in accordance with manufacturer instructions. Finally, the libraries were sequenced on the Illumina Novaseq platform, generating 150 bp paired-end reads. The transcriptomic sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1419508.

2.9. Statistical Analysis

All experiments were independently performed at least three times with a minimum of three biological replicates per experiment. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (version 10.3). Comparisons between two groups were conducted using unpaired Student’s t-tests. For comparisons among three or more groups, one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s post hoc tests was applied, as appropriate. A p value < 0.05 was considered statistically significant.

3. Result

3.1. Transcriptomic Analysis of the Regulatory Effects of AS-IV on T. pyogenes-Induced Infection in gEECs

Transcriptomic analysis (RNA-seq) was performed on gEECs to elucidate the molecular regulatory mechanisms of AS-IV against T. pyogenes-induced endometritis. Differential expression analysis revealed that T. pyogenes infection induced 2279 differentially expressed genes (DEGs) in gEECs, consisting of 1320 significantly upregulated and 959 downregulated genes compared to the Con group. Compared to the TP group, treatment with AS-IV induced an additional 2790 DEGs, of which 1530 were significantly upregulated and 1260 were downregulated (Figure 1B).

Gene Ontology (GO) enrichment analysis (Figure 1D) indicated that T. pyogenes infection predominantly activated oxidative stress-related pathways, specifically oxidoreductase activity and monooxygenase activity, alongside key inflammatory functions such as cytokine activity and chemokine receptor binding. In contrast, AS-IV treatment effectively counteracted these changes, notably downregulating energy metabolism genes associated with nicotinamide nucleotide metabolism and ATP catabolism. Additionally, it reinstated critical homeostatic processes, including microtubule cytoskeleton organization and vesicle-mediated transport, thereby modulating the secretion of inflammatory cytokines.

Further signaling network analysis via Kyoto Encyclopedia of Genes and Genomes (KEGG) (Figure 1E) revealed that T. pyogenes infection significantly enriched DEGs in core innate immune and inflammatory modules. The infection specifically activated the NOD-like receptor, TNF, and NF-κB signaling pathways, alongside the dysregulation of critical cell fate processes such as the cell cycle, apoptosis, and ferroptosis. Conversely, AS-IV treatment effectively attenuated these infection-induced signaling perturbations. This restoration involved the marked downregulation of core metabolic processes, particularly oxidative phosphorylation, as well as essential genomic maintenance pathways such as DNA replication and base excision repair. Key DEGs were then screened for in-depth functional validation (Figure 1C). The findings indicated that AS-IV administration markedly suppressed the expression of key innate immune signaling components (TICAM1, IRF9), oxidative stress indicators (HMOX1, GDF15), and immunochemical mediators (CCL17, CXCR4). Moreover, the modulation of genes associated with cell cycle arrest and apoptosis (CDKN1A, BNIP3) underscores the potent effect of AS-IV on maintaining cellular homeostasis and regulating programmed cell death pathways.

Network pharmacology analysis (Figure 1F,G) identified potential crosstalk between AS-IV targets and endometritis-associated genes, yielding 19 overlapping targets. Subsequent protein–protein interaction (PPI) network and topological analysis designated key inflammatory mediators and hormone receptors as hub genes. These findings suggest that the therapeutic effects of AS-IV are primarily mediated through immune modulation and tissue remodeling.

3.2. AS-IV Alleviates T. pyogenes-Induced Endometrial Inflammation

The protective effects of AS-IV against T. pyogenes-induced inflammatory injury were evaluated by correlating uterine histopathological alterations with the expression levels of key pro-inflammatory cytokines. While the Con group maintained normal uterine morphology, challenge with T. pyogenes triggered severe pathological changes, including hyperemia, diffuse edema, and focal hemorrhage. Notably, these alterations were markedly attenuated by AS-IV treatment. Histopathological evaluation via H&E staining revealed that T. pyogenes challenge disrupted the inherent uterine stratification, characterized by extensive epithelial desquamation, focal necrotic loci, and a notable rarefaction of endometrial glands. Conversely, treatment with AS-IV substantially reversed these deleterious alterations, as evidenced by the restoration of mucosal integrity and a significant attenuation of stromal inflammatory cell infiltration (Figure 2A). Consistently, administration of AS-IV yielded significant reductions in both uterine indices and histopathological scores compared to the TP group (p < 0.05). These quantitative data strongly corroborate the marked attenuation of inflammatory injury (Figure 2B,C). Additionally, the downregulation of occludin in the TP group was significantly attenuated by AS-IV (p < 0.05), indicating a restoration of endometrial epithelial barrier integrity (Figure 2Q,R). In terms of systemic inflammation, AS-IV significantly suppressed T. pyogenes-induced elevations in peripheral leukocyte and lymphocyte counts (p < 0.05). Additionally, uterine myeloperoxidase (MPO) activity was markedly lower in the AS-IV group compared to the TP group (p < 0.05). These results indicate that AS-IV alleviates the systemic inflammatory burden and limits localized tissue injury by inhibiting neutrophil infiltration into the endometrium (Figure 2D–F).

The optimal concentration of AS-IV was initially established using a CCK-8 assay to investigate the molecular mechanisms underlying the modulation of T. pyogenes-induced endometritis (Figure 2L). Parallel investigations across in vitro and in vivo models confirmed the anti-inflammatory potency of the treatment. In alignment with the in vivo observations, T. pyogenes infection triggered a marked elevation in the mRNA expression of inflammatory mediators, specifically TNF-α, IL-1β, IL-6, IL-8, and COX-2 (p < 0.05). RT-qPCR analysis (Figure 2G–P) further showed that the administration of AS-IV significantly inhibited this infection-induced transcriptional surge, effectively dampening the inflammatory cascade (p < 0.05).

Overall, findings at both the in vitro and in vivo levels indicate that AS-IV effectively inhibits the transcriptional upregulation of key pro-inflammatory mediators. Such suppression represents a fundamental molecular mechanism through which the treatment alleviates the severe inflammatory response triggered by T. pyogenes infection.

3.3. Modulation of T. pyogenes-Induced Apoptosis by AS-IV in Endometritis

The regulatory impact of AS-IV on T. pyogenes-induced apoptosis was assessed by quantifying the expression of key apoptotic genes and proteins, alongside phenotypic characterization using RT-qPCR, Western blotting, and flow cytometry.

At the transcriptional level (Figure 3A–E), mRNA expression of Caspase-8 and Caspase-9 was significantly elevated in the TP group compared with the Con group (p < 0.05). Concurrently, the BAX/Bcl2 ratio, a pivotal indicator of the mitochondria-dependent apoptotic pathway, exhibited a significant increase (p < 0.05), suggesting the activation of mitochondrial-mediated programmed cell death and AS-IV effectively suppressed the aberrant increase in apoptotic gene expression (p < 0.05). Moreover, Western blotting (Figure 3F–K) demonstrated that AS-IV administration counteracted the infection-induced upregulation of the core endoplasmic reticulum (ER) stress transcription factor ATF4. This regulatory effect involved a reduction in the pro-apoptotic protein BAX, an increase in the anti-apoptotic protein Bcl-2, and a marked reversal of cleaved caspase-9 accumulation (p < 0.05). At the cellular phenotypic level, flow cytometric analysis (Figure 3L) confirmed a significant increase in cell death following T. pyogenes infection (p < 0.05). Conversely, intervention with AS-IV markedly reduced the apoptotic cell population (p < 0.05), providing functional validation for the strong anti-apoptotic potential of this treatment. Collectively, these results suggest that AS-IV may attenuate T. pyogenes-induced apoptosis through regulation of apoptotic signaling pathways, which is associated with reduced uterine tissue damage.

3.4. AS-IV Alleviates T. pyogenes-Induced Oxidative Stress in Endometritis

This study utilized oxidative stress assay kits, RT-qPCR, and Western blotting to quantify specific markers and systematically evaluate alterations in oxidative damage phenotypes and related signaling pathways. The administration of AS-IV effectively attenuated T. pyogenes-induced oxidative injury, characterized by significantly decreased MDA levels, enhanced SOD activity, and reduced intracellular reactive ROS accumulation (p < 0.05). This suggests that AS-IV provides protection by dampening oxidative stress via diverse regulatory pathways (Figure 4A,B,E). Further analysis showed that the antioxidant properties of AS-IV are linked to the activation of endogenous defense systems. AS-IV administration significantly enhanced the mRNA levels of Nrf2 and the downstream target HO-1 (p < 0.05), counteracting the inhibitory effects of T. pyogenes infection (Figure 4C–G). These findings were confirmed by Western blotting (Figure 4H–J), which showed that AS-IV significantly increased the protein expression of Nrf2 and HO-1 (p < 0.05), establishing a consistent activation profile from transcription to translation. In summary, AS-IV attenuated T. pyogenes-induced oxidative injury by activating the Nrf2/HO-1 signaling pathway, thereby reducing oxidative damage and enhancing antioxidant capacity.

3.5. Dependence of AS-IV Mediated Protection on Nrf2 Signaling in T. pyogenes-Induced Endometrial Damage

To elucidate the pivotal role of the Nrf2/HO-1 signaling pathway in AS-IV mediated protection against T. pyogenes-induced uterine damage, the specific inhibitor ML385 was employed to suppress Nrf2 activity. The protective mechanisms of AS-IV were validated using RT-qPCR, Western blotting, and flow cytometry. Regarding the inflammatory response (Figure 5A–D), pharmacological inhibition of Nrf2 with ML385 significantly elevated the mRNA expression of IL-6, IL-8 and COX-2 compared to the group without the inhibitor (p < 0.05). These results demonstrate that the anti-inflammatory efficacy of AS-IV is mediated via the Nrf2 signaling pathway, as its protective effects were effectively antagonized by the inhibitor. In terms of apoptosis (Figure 5E–K), the anti-apoptotic efficacy of AS-IV was significantly neutralized by the inhibitor ML385. Treatment with the inhibitor led to a marked increase in the mRNA expression of Caspase-8 and Caspase-9 (p < 0.05). In agreement, Western blotting showed that ML385 induced pro-apoptotic BAX expression and elevated the BAX/Bcl-2 ratio, alongside a significant upregulation of the effector protein cleaved caspase-9 (p < 0.05). Phenotypic validation through flow cytometry confirmed a significant rise in the apoptotic cell population (p < 0.05). These findings collectively demonstrate that the anti-apoptotic property of AS-IV depends on the activation of the Nrf2/HO-1 signaling pathway. In terms of oxidative stress (Figure 5L–Q), inhibiting Nrf2 impaired the cellular antioxidant defense. RT-qPCR and Western blotting demonstrated that the administration of ML385 suppressed Nrf2 expression and downregulated the downstream mediator HO-1 (p < 0.05). These alterations led to a significant increase in intracellular ROS levels, confirming that the protective capacity of AS-IV to neutralize oxidative stress through this specific signaling pathway was effectively abolished.

In summary, the pharmacological blockade of the Nrf2 pathway markedly diminished the ability of AS-IV to alleviate T. pyogenes-induced inflammatory response, apoptosis, and oxidative damage. The results indicate that the therapeutic efficacy of AS-IV in uterine tissue is predominantly mediated through the activation of the Nrf2/HO-1 pathway, which orchestrates the suppression of pro-inflammatory mediators, the preservation of apoptotic homeostasis, and the optimization of ROS clearance.

4. Discussion

Current pathological models of endometritis predominantly focus on non-specific inflammatory processes; however, systematic investigations into the specific pathogenic mechanisms driven by T. pyogenes remain scarce [24]. As an opportunistic pathogen, T. pyogenes can cause a variety of infections, including endometritis, mastitis, and pneumonia [25]. At present, antibiotic therapy remains the cornerstone of clinical treatment following infection. However, T. pyogenes strains isolated from cows with endometritis have been found to harbor tetracycline resistance genes [26]. Consequently, the use of active constituents derived from traditional Chinese medicine, notably alkaloids, flavonoids, glycosides, and phenolic compounds [27,28,29,30], as alternative or adjunctive therapeutic strategies has emerged as a significant research focus [31]. Following infection, the endometrium orchestrates the activation of various cytokines and chemokines, alongside diverse immune signaling pathways, which in turn trigger pathological events including inflammatory infiltration, apoptosis, and oxidative stress [32]. Building upon these premises, the present study employed T. pyogenes to establish an endometritis infection model. We systematically investigated the multi-target therapeutic mechanisms of AS-IV in alleviating inflammation and oxidative stress, as well as inhibiting apoptosis, through the modulation of the Nrf2/HO-1 signaling pathway (Figure 6).

To further elucidate the aforementioned molecular mechanisms, we performed RNA-seq-based transcriptomic analysis on gEECs. As indicated by transcriptomic findings, T. pyogenes infection initiates a highly synergistic stress response [33], characterized by the sustained activation of inflammatory pathways and the disruption of redox homeostasis. These processes ultimately converge on key signaling networks that regulate cell survival and death [34]. Differential gene expression analysis indicates that T. pyogenes infection significantly disrupts uterine immune homeostasis during inflammatory processes. This disruption may be mediated by the modulation of cell adhesion and cytoskeletal remodeling, which in turn facilitates the migration and infiltration of inflammatory cells, thereby exacerbating the host immune response [35]. Regarding apoptosis, T. pyogenes elicits a potent stress response that disrupts cell cycle progression via cumulative DNA damage, ultimately triggering programmed cell death [36]. Regarding oxidative stress, the enrichment of peroxisome-related genes in the GO analysis is directly associated with the regulation of reactive ROS metabolism and redox balance, coupled with alterations in energy metabolism pathways. These findings suggest that T. pyogenes infection may disrupt cellular redox equilibrium by interfering with ROS metabolic homeostasis and energy utilization processes [37]. In contrast, AS-IV treatment modulates cellular metabolic homeostasis and regulates pivotal genes and pathways essential for sustaining fundamental physiological functions [38]. By targeting T. pyogenes-induced insults, AS-IV alleviates oxidative stress within host cells, thereby reshaping the immune microenvironment and restoring cellular homeostasis during endometritis [39], which ultimately underpins its therapeutic efficacy.

At the histopathological level, T. pyogenes infection of the uterus triggers cellular swelling, vacuolization, and diffuse necrosis in gEECs [40], thereby impairing their structural barrier integrity. This disruption facilitates the stimulation of Toll-like receptors on underlying stromal cells, driving the aberrant expression of pro-inflammatory mediators [41]. The histological hallmarks of endometritis, including inflammatory cell infiltration, epithelial disruption, and stromal reaction, serve as key criteria for assessing disease progression [42]. Mechanistically, T. pyogenes infection and its virulence factors are closely linked to cytolytic and membrane-perforating activities that compromise cellular integrity [43]. Uterine indices and histopathological scores provide evidence that AS-IV treatment effectively reverses the progression of T. pyogenes-induced damage, thereby facilitating the resolution of tissue inflammation and promoting structural restoration [44]. The recovery of occludin expression, as a key indicator of endometrial barrier function [45], further demonstrates the efficacy of AS-IV in mitigating T. pyogenes-induced barrier impairment, thereby shielding the tissue from the dual insult of inflammation and programmed cell death [46]. Disruption of immune homeostasis affects the uterine inflammatory microenvironment and potentially interferes with successful embryo implantation [47]. In the context of T. pyogenes infection, the mass infiltration of neutrophils and macrophages exacerbates the inflammatory response by liberating large quantities of pro-inflammatory factors [48]. Thus, maintaining immune homeostasis remains a critical therapeutic strategy for alleviating endometritis. The therapeutic administration of AS-IV provides a potent intervention against the immune dysregulation induced by T. pyogenes infection. Such efficacy suggests that AS-IV mitigates the progression of endometritis through the systemic regulation of host homeostasis [49]. It is noteworthy that COX-2, a primary driver of inflammatory responses, is significantly induced during bacterial infection [50]. This factor plays a pivotal role in both the establishment and persistence of the inflammatory microenvironment.

In the context of apoptotic mechanisms, caspase-9 serves as the pivotal executor molecule within the mitochondria-dependent intrinsic apoptotic pathway. Its activation triggers a downstream cascade of caspase proteolytic cleavage events, ultimately culminating in programmed cell death [51]. As a key component of the Bcl-2 protein family, BAX functions as a mitochondrial pro-apoptotic effector [52]. By mediating the formation of transmembrane pores in the mitochondrial outer membrane, BAX triggers the release of cytochrome c, a crucial step in the initiation of the intrinsic apoptotic cascade [53]. The results demonstrate that T. pyogenes infection triggers a robust activation of the intrinsic mitochondrial apoptotic cascade. Crucially, this transition does not occur in isolation but is mechanistically intertwined with excessive inflammatory signaling and impaired redox homeostasis [54]. AS-IV treatment significantly downregulated the aberrant expression of key pro-apoptotic molecules, including Caspase-9 and BAX, while simultaneously preserving the relative stability of Bcl-2 levels. The results suggest that AS-IV exerts multi-target effects on gene expression, effectively counteracting T. pyogenes-induced apoptotic responses by restoring the equilibrium between pro-apoptotic and anti-apoptotic regulators [55]. This intervention specifically targets the intrinsic apoptotic cascade, as evidenced by the synchronized downregulation of key mediators at both the mRNA and protein levels. Such consistency across transcriptional and translational dimensions underscores the robust synergistic mechanism by which AS-IV alleviates programmed cell death in uterine tissue [56].

To further elucidate the underlying mechanism, we examined whether AS-IV mitigates T. pyogenes-induced injury by modulating oxidative stress through the Nrf2 pathway. Intracellular ROS levels serve as a critical index for assessing oxidative stress [57]. Infection with T. pyogenes severely compromises redox equilibrium, where the resulting overproduction of ROS triggers the oxidative degradation of essential macromolecules, including proteins, lipids, and nucleic acids [58]. This process facilitates both necrotic and apoptotic cell death. Consequently, the attenuation of ROS production indicates that AS-IV achieves its therapeutic potential by neutralizing pro-oxidant species produced during the inflammatory response [59]. Uterine tissue analysis substantiated the Nrf2/HO-1 signaling findings, revealing that T. pyogenes challenge significantly intensified oxidative stress [60]. Pathogen entry compromises the redox equilibrium of the host [61], while the impairment of endogenous defense mechanisms suppresses both the enzymatic activity and protein expression of antioxidant systems. This cascade promotes the accumulation of lipid peroxides, thereby driving extensive tissue damage. Triggered by AS-IV treatment [62], Nrf2 translocates from the cytoplasm to the nucleus, where it facilitates the transcription of ARE-related genes to induce HO-1 expression. This regulatory pathway effectively alleviates oxidative stress while simultaneously suppressing the activation of apoptotic signaling pathways [63,64,65]. The specific inhibitor ML385 is widely employed to blockade the Nrf2 signaling pathway in diverse biological models [66]. This is exemplified by its ability to abrogate the therapeutic benefits of Lentinan (LNT), effectively counteracting its anti-inflammatory and cytoprotective properties in LPS-challenged bovine mammary epithelial cells [67]. To validate the involvement of this pathway, the Nrf2 signaling cascade was pharmacologically inhibited in the presence of AS-IV. The subsequent loss of AS-IV-mediated protection against inflammatory responses, apoptosis, and oxidative damage suggests that the therapeutic efficacy of AS-IV in T. pyogenes-induced endometritis is contingent upon the activation of the Nrf2/HO-1 signaling pathway. In summary, our results suggest that AS-IV functions as a pleiotropic agent against endometritis via a sophisticated, multi-level regulatory network. By mobilizing the central Nrf2/HO-1 pathway, AS-IV orchestrates a synergistic modulation of three interconnected pathological axes, namely oxidative stress, inflammation, and programmed cell death. Collectively, these findings highlight oxidative stress as the primary cellular response to T. pyogenes, while inflammation and apoptosis serve as complementary mechanisms, as indicated by exploratory RNA-seq analysis and targeted experimental validation.

5. Conclusions

Through activation of the Nrf2/HO-1 signaling pathway, AS-IV promotes the restoration of redox and inflammatory homeostasis in both in vivo murine models and in vitro cellular systems. These findings demonstrate that AS-IV exerts a protective effect against endometritis-associated pathological alterations by modulating Nrf2-dependent antioxidant responses.