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. 2025 Sep 26;15:33159. doi: 10.1038/s41598-025-91062-4

Pedunculoside alleviates lipopolysaccharide-induced acute lung injury/acute respiratory distress syndrome by inhibiting NF-κB pathway

Yu Yang 1, Zhi Xu 1, Qi Li 1, Guansong Wang 1, Jiancheng Xu 1,✉
PMCID: PMC12475281  PMID: 41006814

Abstract

Acute lung injury (ALI) is a severe respiratory disease characterized by various clinical manifestations, including intractable hypoxemia, alveolar hypertension, parenchymal edema, and progressive respiratory distress. If left untreated, ALI can progress into acute respiratory distress syndrome (ARDS). A promising therapeutic option for ALI is Pedunculoside (PE), a major bioactive flavonoid found in Butcher’s broom (Ruscus aculeatus). PE has been shown to possess significant anti-inflammatory and antioxidant effects. This study aimed to investigate the therapeutic effects of PE on ALI in mice and to understand the underlying mechanism of action. Initially, we established a mouse model of acute lung epithelial cell (MLE-12) injury induced by lipopolysaccharide (LPS). Subsequently, we treated the MLE-12 cells with PE and observed a significant improvement in cell viability and a reduction in apoptosis. Moreover, when ALI mice were treated with PE, we observed an enhancement in lung histopathological structure, a decrease in lung tissue wet-to-dry (W/D) ratio, and reduced total protein concentrations in bronchoalveolar lavage fluid (BALF). Additionally, there was a decrease in apoptotic epithelial cells in lung tissue and an increase in proliferating cells after PE intervention. PE treatment also led to reduced levels of intracellular interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and malondialdehyde (MDA), while increasing glutathione (GSH) levels and superoxide dismutase (SOD) activity in both MLE-12 cell supernatants and BALF of ALI mice. Our mechanistic studies demonstrated that PE effectively downregulated the expression of p-p65 and p-IκBα proteins. However, the induced activation of the transcription factor p65 reversed the regulatory effects of PE, partially counteracting its anti-apoptotic, anti-inflammatory, and antioxidant activities in MLE-12 cells. These findings demonstrate that PE treatment has the potential to mitigate LPS-induced ALI by inhibiting NF-κB signaling-mediated oxidative stress and inflammatory responses.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-91062-4.

Keywords: Acute lung injury, Long-stem wintergreen glycosides, Inflammation, Oxidative stress, NF-κB pathway

Subject terms: Drug delivery, Medical research

Introduction

Acute lung injury (ALI) is a respiratory disorder characterized by acute and progressive respiratory insufficiency. This condition carries a high mortality rate of 36–44% in ALI patients1. The disease is clinically manifested by various pathophysiological conditions, including intractable hypoxemia, alveolar hypertension, parenchymal edema, and progressive respiratory distress. If left untreated, it can progress to acute respiratory distress syndrome (ARDS)2. Multiple factors have been identified as potential contributors to ALI, including pulmonary inflammation, toxic inhalation, pulmonary infections, sepsis, acute pancreatitis, severe traumatic shock, and drug overdosage3. Recent decades have seen efforts focusing on developing therapeutic modalities to manage ALI, including mechanical ventilation, extracorporeal membrane pulmonary oxygenation (ECMO), and pharmacological treatment. Mechanical ventilation and ECMO primarily serve as adjuvant therapies to create a time window for subsequent pharmacological treatment. Several drugs have been employed in ALI management, such as β2 agonists (salbutamol), steroid hormones (dexamethasone), and antifungal drugs (ketoconazole)4,5. However, while these current therapeutic agents have shown some benefit in mild cases of ALI, they have failed to demonstrate therapeutic efficacy in moderate and severe cases4. Therefore, there is an urgent need to develop effective medication for ALI treatment.

In the context of pathology, ALI is an inflammatory condition characterized by extensive damage to lung epithelial and endothelial cells, activation of macrophages, and infiltration of neutrophils. Lipopolysaccharide (LPS), an endotoxin component found in the outer wall of Gram-negative bacteria, is established to trigger a rapid and intense inflammatory response. LPS potently provokes the activation of innate immunity and recruits inflammatory cells to the lungs, ultimately leading to the development of Acute Lung Injury (ALI)6. While the specific mechanisms of lung injury may vary depending on the primary cause, damage to epithelial and endothelial cells remains a crucial factor in the progression of ALI7. Pulmonary edema emerges as another key feature in ALI pathophysiology due to impaired alveolar fluid clearance8. Furthermore, severe and chronic inflammation can trigger oxidative stress to exacerbate lung tissue damage9. At the molecular level, several signaling cascades are implicated in the chronic inflammation of ALI, including Toll-like receptors (TLRs), Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), as well as different cytokines/chemokines and adhesion molecules10–12. The activation of upstream signaling pathways converges at the nuclear factor kappa-B (NF-κB) pathway, which serves as a canonical molecular cascade for mounting inflammatory response13. Therefore, targeting the NF-κB pathway and inflammatory response has emerged as a promising therapeutic strategy for ALI alleviation.

Pedunculoside (PE), a flavonoid derived from the holly (Aquifoliaceae) family, represents a major bioactive constituent with diverse biological activities14. Recent years have seen significant progress in harnessing the health benefits of PE as a flavonoid15. Extensive research has shown that PE can effectively alleviate synovial joint inflammation, prevent liver injury, improve intestinal flora, and reduce hyperlipidemia caused by a high-fat diet16–18. Furthermore, PE has exhibited promising activities in ameliorating ulcerative colitis and LPS-induced mastitis in mice by inhibiting inflammatory responses and oxidative stress19,20. However, the potential therapeutic effect of PE in treating LPS-induced ALI/ARDS remains unexplored.

This study aimed to evaluate the ameliorative effects of PE on lipopolysaccharide (LPS)-induced lung epithelial cell injury and acute lung injury (ALI) in a mouse model. We performed molecular investigations to elucidate the mechanism of action of PE on LPS-induced MLE-12 cells. Additionally, a mouse model of ALI was established by LPS induction, and the beneficial effects of PE treatment were evaluated by assessing the histopathological changes of alveolar structure, lung tissue wet-to-dry (W/D) ratio, and the levels of inflammatory and oxidative stress markers in bronchoalveolar lavage fluid (BALF).

Materials and methods

Cell culture, treatment, and grouping

Mouse lung epithelial cells (MLE-12 cell line) were obtained from the cell bank of the Chinese Academy of Sciences in Shanghai, China. The cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Sigma-Aldrich) under 5% CO2 in a humidified environment. The culture medium was supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (streptomycin and penicillin, Gibco). For the in vitro potency analysis, the cells were divided into different experimental groups, including the control, LPS, LPS + PE (5 μM, Cat#HY-N0458, MedChemExpress), LPS + PE (10 μM), and LPS + PE (15 μM) groups. For mechanistic investigations, four experimental conditions were established: control, LPS, LPS + PE (15 μM), and LPS + PE (15 μM) + mouse recombinant RANKL (100 ng/mL, Cat#315-11, PeproTech) groups. Upon reaching 80–90% confluence in the plates during the logarithmic growth phase, the cells were exposed to different concentrations of PE, 500 ng/mL of LPS (from Escherichia coli O111:B4, Cat#L4391, Sigma-Aldrich), or a combination of 100 ng/mL of mouse recombinant RANKL for 24 h.

Cell viability assay

Cell viability was assessed using the 4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MLE-12 cells in the logarithmic phase were seeded into 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h to ensure adherence. The cells were then exposed to various treatments for the designated time period. Following treatment, 20 μL of MTT (5 mg/mL, Cat#M2128, Sigma-Aldrich) was added to each well and incubated for 4 h. The supernatant was then aspirated, and the formazan crystals were dissolved in 150 μL of dimethyl sulfoxide. Absorbance was measured at 570 nm using a microplate reader (Perkin Elmer Analytical and Biochemical Instruments (Beijing) Co., Ltd).

Apoptosis determination

MLE-12 cells were seeded and treated according to the groups described in "Cell culture, treatment, and grouping" section. Apoptosis detection was conducted using Annexin V FITC/PI Dead Cell Apoptosis Kit (Cat#V13242, Thermo Fisher Scientific). After treatment, the cells (1 × 105 cells) were washed twice with pre-cooled phosphate-buffered saline (PBS) and resuspended in a reagent mixture containing 200 μL of binding buffer, 4 μL of 0.5 mg/mL propidium iodide (PI), and 2 μL of Annexin V-fluorescein isothiocyanate (FITC) solution. Following 15 min of incubation at room temperature, apoptosis was analyzed using BD Accuri C6 flow cytometry (BD Biosciences FlowMetric, Inc., Franklin Lakes, USA).

Animals

BALB/c mice (male, aged 6–8 weeks, weighing 32–35 g) were obtained from the Shanghai laboratory animal center (SLAC, Shanghai, China). The mice were housed in a specific-pathogen-free (SPF) animal facility with a standard diet and ad libitum access to drinking water under a 12-h light/dark cycle. The facility was maintained at a temperature of 20–25 °C and a relative humidity of 50–60%. All animal protocols were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Army Medical University. All experiments were performed in accordance with the guidelines and regulations outlined in the Basel Declaration. This research is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org), which ensure transparent and comprehensive reporting of animal research.

Animal grouping, drug administration, and sample collection

Male BALB/c mice (8-week-old) underwent a one-week acclimatization period prior to experiments. Mice were then randomly divided into four experimental groups (n = 6 per group): control (sham), model (LPS 12 h or LPS 24 h induction), and two PE intervention groups (PE + LPS 12 h or PE + LPS 24 h). Starting from Day 1, mice in the intervention groups received daily PE (10 mg/kg) by gavage for 7 consecutive days20, while sham and model groups received an equivalent volume of vehicle solution daily. On Day 7, mice received their final PE or vehicle gavage. One hour later, mice in the model and PE treatment groups received an intraperitoneal injection of LPS (5 mg/kg) to induce ALI21, while the sham group received an equivalent volume of saline. Mice were then euthanized at two different timepoints: 12 h (Day 7 + 12 h) or 24 h (Day 8) post-LPS administration via pentobarbital injection, and lung tissue was collected. The upper lobe of the left lung was carefully dissected and its wet weight (W) was determined using an analytical balance. The tissue was then dried at 80 °C to constant weight and reweighed to obtain the dry weight (D). The wet weight/dry weight (W/D) ratio was calculated to assess pulmonary edema.

Pathological examinations

The lung tissues were prepared for pathological examination using the following procedure. Initially, the collected lung tissues were fixed in 4% paraformaldehyde. Then, the paraffin-embedded tissues were sectioned with a thickness of 5 μm. Subsequently, the tissue sections were hydrated in a graded alcohol series (100%, 95%, 90%, and 70%) followed by immersion in distilled water. The sections were stained with hematoxylin and eosin (H&E) staining kit (Abcam). The H&E-stained sections were evaluated by two independent pathologists who were blinded to the experimental groups. The histological changes were scored on a scale of 0–2 for the following parameters: (1) alveolar congestion (0 = absent, 1 = moderate, 2 = severe); (2) hemorrhage (0 = absent, 1 = moderate, 2 = severe); (3) neutrophil infiltration or aggregation in alveolar space or vessel wall (0 = absent, 1 = moderate, 2 = severe); (4) alveolar wall thickening/hyaline membrane formation (0 = mild, < 2 × normal thickness; 1 = moderate, 2–4 × normal thickness; 2 = severe, > 4 × normal thickness); and (5) inflammatory cell infiltration (0 = absent, 1 = moderate, 2 = severe). The total score (0–10, lung injury score) was calculated by summing the individual scores, with 0 indicating normal, 1–4 indicating mild injury, 5–7 indicating moderate injury, and 8–10 indicating severe injury.

For Ki67 immunofluorescence (IF) staining, the tissue sections were exfoliated and hydrated using the same method as mentioned before. After permeabilization using TBST buffer, the sections were blocked with normal goat serum (Cat#G9023, Sigma-Aldrich) for 1 h at ambient temperature, followed by overnight incubation with anti-Ki67 antibody (Cat#ab16667, Abcam; 1:100 dilution) at 4 °C. Alexa Fluor™568-conjugated goat anti-rabbit IgG secondary antibody (Cat#A-11011, Thermo Fisher Scientific) was applied to detect the primary antibody. After TBST washing, the tissue sections were counterstained with 10 μM DAPI (Cat#D9542, Sigma-Aldrich) for 5 min. The proportion of Ki67-positive cells was quantified under an inverted fluorescence microscope (IX73, Olympus), and five randomly selected high-magnification fields (400 ×) were included for quantification in each sample.

TUNEL staining

Paraffin-embedded lung sections were deparaffinized at 70 °C (10 min) and rehydrated through a graded ethanol series (96–50%) and water (3 min each). After fixation in 4% formaldehyde and PBS washes, sections were treated with proteinase K (2 μg/mL in Tris–HCl, pH 8.0) for 10 min. Endogenous peroxidase was blocked with 0.5% H2O2 (20 min). Sections were incubated in TdT buffer containing 150 mM NaCl and 0.05% BSA (Cat#A2153, Sigma-Aldrich), followed by TUNEL staining using TdT enzyme (200 U/mL) and digoxin-dUTP (4 μM) for 1 h at 37 °C (In Situ Cell Death Detection Kit, Cat#11684795910, Roche). After PBS washes, sections were incubated with Alexa Fluor 647-anti-digoxin antibody (5 U/mL, 30 min, 37 °C) and counterstained with DAPI (10 μM, Cat#D9542, Sigma-Aldrich). TUNEL-positive cells were quantified in five random high-magnification fields per sample.

Total protein assessment in BALF

To obtain the BALF samples, lung tissues from the mice were washed with 0.5 mL of PBS six times. Subsequently, the BALF samples were centrifuged (3000 g, 20 min, 4 °C) and supernatants were transferred to low-silica binding tubes. Total protein concentration was measured using BCA protein assay kit (Cat#BCA1, Sigma-Aldrich).

Detection of inflammatory factors

The levels of inflammatory factors, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, were measured in the cell supernatant and bronchoalveolar lavage fluid (BALF) obtained from extracted lung tissues of mice. In vitro samples were collected by separating the supernatant after treating MLE-12 cells with different conditions. BALF samples were collected following the specified procedures in "Total protein assessment in BALF" section. The relative levels of cytokines were measured using commercial ELISA kits (IL-1β: Cat#RAB0275, IL-6: Cat#RAB0308, TNF-α: Cat#RAB0477, Sigma-Aldrich) according to manufacturer’s instructions.

Oxidative stress indices

Oxidative stress markers were analyzed in cell lysates and lung tissue homogenates. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Cat#R0278, Sigma-Aldrich) containing protease inhibitor cocktail (Cat#P8340, Sigma-Aldrich) for 30 min at 4 °C, then centrifuged (5000 g, 10 min). Lung tissues were homogenized in the same buffer at 4 °C and centrifuged similarly. Total protein was measured using bicinchoninic acid (BCA) kit (Cat#BCA1, Sigma-Aldrich). Malondialdehyde (MDA) (Cat#MAK085), glutathione (GSH) (Cat#CS0260), and superoxide dismutase (SOD) activity (Cat#19160) were measured using Sigma-Aldrich assay kits according to manufacturer’s protocols.

Western blot analysis

Total protein was extracted from MLE-12 cells or mouse lung tissues using RIPA lysis buffer containing phosphatase inhibitor cocktail (Cat#P5726, 1:100, Sigma-Aldrich) and protease inhibitor cocktail (Cat#P8340, 1:100, Sigma-Aldrich). Protein concentration was determined using BCA kit (Cat#BCA1, Sigma-Aldrich). Protein samples (15 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Cat#IPVH00010, Millipore) at 200 mA. Membranes were blocked in TBST containing 5% non-fat milk for 1.5 h at room temperature, then incubated overnight at 4 °C with primary antibodies: anti-p65 (Cat#ab16502, 1:1000, Abcam), anti-phospho-p65 (Cat#ab76302, 1:1000, Abcam), anti-IκBα (Cat#ab32518, 1:1000, Abcam), anti-phospho-IκBα (Cat#ab133462, 1:1000, Abcam), and anti-GAPDH (Cat#ab181602, 1:5000, Abcam). After washing, membranes were incubated with HRP-conjugated anti-rabbit IgG (Cat#ab205718, 1:5000, Abcam) for 1 h at room temperature. Protein bands were visualized using ECL substrate (Cat#32109, Pierce) on an Amersham Imager 600 system and quantified using ImageJ software (NIH, USA).

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS software (version 19.0, IBM, Chicago, IL, USA). Multiple group comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Two-group comparisons were performed using unpaired Student’s t-test. Statistical significance was set at P < 0.05. All experiments were performed at least three times independently.

Results

PE inhibits LPS-induced apoptosis in MLE-12 cells

To investigate the potential protective effects of PE on LPS-induced lung epithelial cell injury, we first examined MLE-12 cell viability using MTT assay. As depicted in Fig. 1A, LPS treatment significantly decreased MLE-12 cell viability compared to control cells, while PE treatment restored cell viability in a dose-dependent manner. We next assessed cell apoptosis using flow cytometry. LPS exposure for 24 h significantly increased the proportion of apoptotic cells, whereas PE treatment attenuated LPS-induced apoptosis in a dose-dependent manner (Fig. 1B and C). These results indicate that PE effectively protects MLE-12 cells from LPS-induced cytotoxicity and apoptosis.

Fig. 1.

Fig. 1

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PE inhibits LPS-induced apoptosis of MLE-12 cells. (A) Cell viability was assessed by MTT assay in MLE-12 cells. (B) Representative flow cytometry plots showing cell apoptosis and (C) quantification of apoptotic rates in MLE-12 cells treated with DMSO (vehicle) or PE (5, 10, and 15 μM) in the presence of LPS (500 ng/mL) for 24 h. The values are expressed as the mean ± SD from three independent experiments. ###Represents P < 0.001 versus control group; **Indicates P < 0.01, ***Signifies P < 0.001 versus LPS group.

PE attenuates LPS-induced inflammation in MLE-12 cells

To evaluate the impact of PE on LPS-induced inflammatory response in MLE-12 cells, inflammatory cytokine levels were measured in cell culture supernatants using ELISA. Following 24 h of LPS stimulation, the levels of IL-1β, IL-6, and TNF-α were significantly elevated compared to control cells. PE co-treatment significantly suppressed the LPS-induced secretion of IL-1β, IL-6, and TNF-α in a concentration-dependent manner (Fig. 2A–C).

Fig. 2.

Fig. 2

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PE reduces the production of inflammatory factors in MLE-12 cells induced by LPS. ELISA analysis showing the levels of (A) IL-6, (B) IL-1β, and (C) TNF-α in MLE-12 cells treated with DMSO (vehicle) or PE (5, 10, and 15 μM) in the presence of LPS (500 ng/mL) for 24 h. The values are expressed as the mean ± SD from three independent experiments. ###Signifies P < 0.001 versus control group; **Represents P < 0.01, ***Indicates P < 0.001 versus LPS group.

PE mitigates the oxidative stress induced by LPS in MLE-12 cells

To assess the antioxidant effects of PE, different oxidative stress markers were measured in MLE-12 cells after LPS stimulation and PE co-treatment. LPS treatment for 24 h significantly increased MDA levels while decreasing GSH levels and SOD activity in MLE-12 cells. Notably, PE co-treatment reversed these changes, showing a concentration-dependent decrease in MDA levels and increase in both GSH levels and SOD activity (Fig. 3A–C).

Fig. 3.

Fig. 3

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PE alleviates oxidative stress in LPS-induced MLE-12 cells. Quantification of (A) MDA content, (B) SOD activity, and (C) GSH levels in MLE-12 cells treated with DMSO (vehicle) or PE (5, 10, and 15 μM) in the presence of LPS (500 ng/mL) for 24 h. The values are expressed as the mean ± SD from three independent experiments. ###Represents P < 0.001 versus control group; **Signifies P < 0.01, ***Indicates P < 0.001 versus LPS group.

These results demonstrate that PE effectively protects MLE-12 cells against LPS-induced oxidative stress, which may be attributed to its anti-inflammatory properties.

PE decreases LPS-induced damage in MLE-12 cells

To elucidate the molecular mechanism underlying PE’s protective effects against LPS-induced damage in MLE-12 cells, we investigated its impact on the NF-κB pathway, a key regulator of inflammatory responses13,22. Western blot analysis revealed that LPS stimulation significantly increased phosphorylation of p65 and IκBα, while PE treatment attenuated these phosphorylation events in a dose-dependent manner (Fig. 4A), indicating suppression of NF-κB signaling activation. To confirm the role of NF-κB inhibition in PE-mediated protection, cells were co-treated with the NF-κB activator RANKL and PE. RANKL treatment partially reversed PE’s protective effects against LPS-induced cell injury (Fig. 4B and C). Additionally, RANKL administration abolished PE’s inhibitory effects on LPS-induced inflammatory cytokine production (Fig. 4D–F) and oxidative stress (Fig. 4G–I). Collectively, these data strongly suggest that PE protects MLE-12 cells from LPS-induced injury primarily through suppression of NF-κB signaling.

Fig. 4.

Fig. 4

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PE attenuates LPS-induced damage to MLE-12 cells by inhibiting the NF-κB pathway. (A) Representative immunoblots showing expression levels of p-p65, p65, p-IκBα, and IκBα proteins with β-actin as loading control. (B) Cell viability assessed by MTT assay. (C) Representative flow cytometry plots and quantification of apoptotic rates. ELISA analysis of inflammatory mediators (D) IL-6, (E) IL-1β, and (F) TNF-α. Quantification of oxidative stress markers (G) MDA content, (H) SOD activity and (I) GSH levels. MLE-12 cells were treated with DMSO (vehicle) or PE (5, 10, and 15 μM) in the presence of LPS (500 ng/mL) for 24 h. The values are expressed as the mean ± SD from three independent experiments. ###Presents P < 0.001 versus control group; ***Signifies P < 0.001 versus LPS group; ^^Indicates P < 0.01, ^^^Represents P < 0.001 versus LPS + PE group.

PE attenuates LPS-induced lung injury in ALI/ARDS mice

Following our in vitro findings, we investigated the therapeutic potential of PE in an LPS-induced ALI mouse model. Initially, we examined the histopathological changes in the lung tissues, the W/D ratio, and the protein concentration in BALF in LPS-induced ALI mouse model, with or without PE treatment. Histopathological analysis revealed that LPS administration caused severe lung injury at both 12 and 24 h, characterized by disrupted alveolar structure, diffuse interstitial edema and congestion, extensive neutrophil infiltration, and thickened alveolar walls (Fig. 5A). Furthermore, LPS-treated mice exhibited elevated lung wet/dry (W/D) weight ratio and increased BALF protein concentration (Fig. 5B and C). Treatment with PE (10 mg/kg) significantly attenuated these pathological changes, as evidenced by improved lung tissue architecture and decreased lung injury scores (Fig. 5A), reduced W/D ratio (Fig. 5B), and decreased BALF protein levels (Fig. 5C) compared to the LPS group. These results demonstrate that PE effectively ameliorates LPS-induced acute lung injury in vivo.

Fig. 5.

Fig. 5

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PE mitigates LPS-induced lung injury in ALI/ARDS mice. Mice were pretreated with PE (10 mg/kg) or vehicle by gavage for 7 days before LPS (5 mg/kg, i.p.) or saline administration. Lung tissues and BALF were collected at 12 and 24 h post-LPS challenge. (A) Representative H&E staining of lung sections showing histopathological changes (magnification ×400) and quantification of lung injury scores, scale bar: 100 μm. Quantification of (B) lung W/D ratio and (C) total protein concentration in BALF. (D) Representative immunofluorescence images and quantification of Ki67-positive cells in lung sections, scale bar: 50 μm. (E) TUNEL staining and quantification of apoptotic cells in lung sections, scale bar: 50 μm. (F) Pro-inflammatory cytokine levels (IL-1β, IL-6, and TNF-α) in BALF. (G) MDA content in lung tissues. (H) Representative immunoblots showing expression of p-p65, p65, p-IκBα, and IκBα in lung tissues, with β-actin as loading control. The values are expressed as the mean ± SD (n = 6). ###Indicates P < 0.001 versus sham group; ***Signifies P < 0.001 versus LPS 12 h group. ^^Represents P < 0.01 and ^^^Signifies P < 0.001 versus LPS 24 h group.

Subsequently, the protective effect of PE on alveolar epithelial cells in ALI mice was assessed using the immunofluorescence staining of Ki67 and TUNEL staining. LPS administration reduced alveolar epithelial cell proliferation (Ki67-positive cells) and increased apoptosis at both 12 and 24 h compared to sham group. Importantly, PE treatment significantly enhanced epithelial cell proliferation (Fig. 5D) and inhibited apoptosis (Fig. 5E) at both time points. We next assessed the anti-inflammatory and antioxidant effects of PE in vivo. LPS administration markedly elevated inflammatory cytokines (IL-1β, IL-6, and TNF-α) in BALF, increased lung tissue MDA content, and enhanced phosphorylation of p65 and IκBα. PE treatment significantly attenuated these changes, as evidenced by reduced BALF inflammatory cytokines (Fig. 5F), decreased MDA levels (Fig. 5G), and diminished p-p65 and p-IκBα expression (Fig. 5H) at both time points. These findings establish that PE effectively protects against LPS-induced lung injury in the ALI/ARDS mouse model, likely through suppression of inflammation and oxidative stress.

Discussion

Acute lung injury (ALI) is characterized by enhanced pulmonary vascular permeability, pulmonary edema, neutrophil infiltration, and inflammatory mediator release23. A major trigger of ALI is severe lung infection, particularly involving lipopolysaccharide (LPS) from Gram-negative bacterial cell walls24. LPS triggers macrophage activation, leading to pro-inflammatory cytokine production and ROS generation, ultimately initiating various inflammatory and immune responses. The hallmarks of LPS-induced ALI include increased pulmonary capillary permeability, edema, neutrophil infiltration, and inflammatory mediator release25,26. Our study demonstrates that PE treatment effectively attenuates multiple aspects of LPS-induced lung injury, including pulmonary edema, neutrophil infiltration, inflammatory cytokine production, and oxidative stress. PE also protected pulmonary epithelial cells from LPS-induced damage. These therapeutic effects align with previously documented anti-inflammatory properties of PE in various conditions, including collagen-induced arthritis, ulcerative colitis, and LPS-induced mastitis16,19,20, supporting its potential as a therapeutic agent for ALI.

During pathogenic invasion, LPS from bacterial cell wall can trigger the production of inflammatory cytokines from lung epithelial cells27–30. These mediators perpetuate the inflammatory response by stimulating macrophages and neutrophils, resulting in chronic inflammation. In lung tissue, chronic inflammation leads to alveolar wall thickening, pulmonary edema, and subsequent respiratory dysfunction31,32. Our results show that PE significantly reduced IL-6, IL-1β, and TNF-α levels in both LPS-treated MLE-12 cells and BALF from ALI mice, demonstrating its potent anti-inflammatory effects. Additionally, PE enhanced the antioxidant defense system by upregulating GSH levels and SOD activity, key components in cellular protection against oxidative stress33–35. These findings are consistent with previous studies showing that oxidative stress reduction can improve progressive lung injury36,37, suggesting that PE’s therapeutic effects are mediated through both anti-inflammatory and antioxidant mechanisms.

The inflammatory cascade begins when bacterial LPS binds to LPS-binding protein (LBP), which facilitates its transfer to TLR4/MD-2 polymers via CD14 receptors. This interaction triggers downstream signaling, leading to the recruitment and assembly of TIRAP and MyD8838,39. A key downstream effector of TLR/MyD88 signaling is NF-κB, a pivotal pro-inflammatory transcription factor complex composed of RelA (p65), NF-κB2 (p52), NF-κB1 (p50), RelB, and c-Rel proteins40,41. Under basal conditions, NF-κB remains inactive through its association with Iκ-Bα. However, upstream inflammatory signals promote Iκ-Bα dissociation, enabling NF-κB phosphorylation and nuclear translocation, ultimately inducing inflammatory cytokine expression42,43.

Our results demonstrated increased p65 and IκBα phosphorylation in LPS-treated MLE-12 cells, indicating NF-κB pathway activation. This activation correlated with enhanced inflammatory mediator production and oxidative stress44,45. PE treatment significantly reduced p-p65 and p-IκBα levels, demonstrating its inhibitory effect on NF-κB signaling. Notably, co-treatment with RANKL partially reversed PE’s protective effects and diminished its anti-inflammatory and antioxidant properties in MLE-12 cells. We further confirmed PE’s inhibitory effect on NF-κB signaling in the ALI mouse model. Taken together, these findings establish that PE ameliorates ALI through suppression of NF-κB signaling, thereby reducing inflammation and oxidative stress. This mechanism aligns with previous studies showing that NF-κB pathway inhibition provides protection against ALI progression46–48.

There are several limitations in our study that warrant further investigation. First, the mechanism by which PE represses LPS-induced NF-κB signaling activation remains to be elucidated. Second, our study design involves one week pre-treatment of PE before LPS induction in the mouse model. It is worthy further investigating whether PE intervention after LPS induction could alleviate pulmonary damages. Furthermore, whether PE treatment could protect against ALI-related death in a model of multiple LPS administrations warrants future evaluation. Another limitation is a lack of dose-dependent effcts of PE in the animal study, which could provide valuable insights into the optimal dosing regimen for therapeutic applications. Additionally, our study only included male mice; given the known sex differences in inflammatory responses and disease progression, future studies should include both male and female mice to better understand potential sex-specific effects of PE treatment in ALI.

Conclusion

In summary, this study demonstrates that PE significantly attenuates LPS-induced ALI in both cellular and mouse models. PE’s therapeutic effects stem from its dual antioxidant and anti-inflammatory properties, with the latter mediated through NF-κB pathway inhibition (Fig. 6). These findings establish PE as a promising therapeutic agent for ALI/ARDS and provide mechanistic insights that may guide the development of new treatment strategies for these conditions.

Fig. 6.

Fig. 6

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PE inhibits NF-κB pathway activation in acute lung injury. LPS triggers acute lung injury through activation of the NF-κB pathway (phosphorylation of p65 and IκB), resulting in inflammation, ROS production, and cell death, while Pedunculoside treatment prevents NF-κB pathway activation and maintains normal cellular homeostasis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (180.8KB, pdf)

Author contributions

Conception and design: Jiancheng Xu, Yu Yang, Zhi Xu Data analysis and interpretation: Yu Yang, Qi Li, Guansong Wang, Zhi Xu Manuscript writing: Jiancheng Xu, Yu Yang, Zhi Xu Final approval of manuscript: All authors.

Funding

Study on the Pathogenesis and Prevention and Treatment of Acute Respiratory Distress Syndrome.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval

All animal experimental procedures gained approval from Animal Ethics Committee of Second Affiliated Hospital of Army Medical University. The experimental protocol was performed in accordance with the relevant guidelines and regulations of the Basel Declaration. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (180.8KB, pdf)

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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