Combining network pharmacology and experimental validation to demonstrate that salidroside alleviates acute lung injury by inhibiting ferroptosis via the MAPK/GPX4 pathway

Abstract

Objective

This study aimed to combine network pharmacology with in vitro experiments to identify the key targets and potential mechanisms of salidroside (Sal) in the treatment of acute lung injury (ALI).

Methods

Potential targets related to Sal and ALI were retrieved from the ChEMBL, SuperPRED, SwissTargetPrediction, GeneCards, OMIM, and CTD databases. Overlapping targets were imported into the STRING database and Cytoscape software to construct a protein-protein interaction (PPI) network and identify core targets. Functional enrichment analysis of these core genes, including GO and KEGG pathways, was performed using the DAVID database. Two genes, MAPK14 and GPX4, directly relevant to subsequent validation, were selected for molecular docking analysis. Furthermore, an in vitro model of ALI was established using LPS-induced alveolar type II epithelial cells to verify the protective mechanism of Sal.

Results

A total of 355 potential targets associated with Sal in ALI treatment were identified. In vitro experiments showed that, compared to the LPS group, the Sal group exhibited significantly reduced secretion of IL-6, ROS, p-MAPK, MDA, and Fe²⁺, along with increased GPX4 expression and attenuated lung injury.

Conclusion

Integrated network pharmacology and experimental validation suggest that Sal pretreatment alleviates inflammatory response and oxidative stress, likely through regulation of the MAPK/GPX4 signaling pathway, thereby providing protection against lung tissue injury.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-026-03529-1.

Introduction

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), are life-threatening conditions characterized by widespread diffuse damage to alveolar epithelial cells (AECII) and uncontrolled inflammation. These conditions manifest as bilateral pulmonary opacities on imaging accompanied by severe hypoxemia [1, 2]. The underlying mechanisms are highly complex, involving an imbalance in inflammatory responses, regulation of aquaporins, dysregulation of coagulation/fibrinolysis systems, apoptosis, autophagy, fever, among others [3]. Despite significant efforts directed towards the immune response against infections—exacerbated by the emergence of novel pathogens such as the global pandemic of COVID-19 [4, 5], and the ongoing rise in drug resistance, effective pharmacological therapies for ALI remain elusive [6]. Increasing evidence suggests that acute lung injury is closely associated with non-apoptotic pathways including pyroptosis [7–9] and ferroptosis [10–12]. Ferroptosis is a recently identified form of regulated cell death distinct from necrosis and apoptosis; it is characterized by iron-dependent accumulation of lipid peroxides (LPO) [13] and manifests through decreased activity of key antioxidant enzymes such as GPX4 within the glutathione system [14]. Iron deposition has been observed in the lower respiratory tract of ALI patients; this accumulation can lead to inflammatory responses, oxidative stress, and mitochondrial dysfunction which further exacerbate lung injury severity [15]. A deeper understanding of the molecular mechanisms underlying ferroptosis in ALI may provide new therapeutic avenues for treating acute lung injury.

Previous studies have indicated that traditional Chinese medicine exhibits therapeutic effects on sepsis-induced acute lung injury [16–18]. Salidroside is a bioactive component derived from Rhodiola rosea. Its antioxidative properties [19], anti-aging effects [20], anticancer activities [21], and immunomodulatory functions [22] have been widely validated. Our prior research demonstrated that salidroside could prevent sepsis-related acute lung injury while reducing pro-inflammatory cytokine production [23], highlighting its potential clinical value. A brief comparison of the therapeutic effects of salidroside and known anti iron toxicity compounds (Table 1).

Table 1.

A brief comparison of the therapeutic effects of Salidroside and known anti iron toxicity compounds

Comparative dimension	Salidroside	Deferasirox/ Deferasirox	Idebenone
Main source	Natural active ingredients derived from plants	Artificially synthesized specialized therapeutic drugs	Artificially synthesized specialized therapeutic drugs
Main mechanism of action	↓Fe²⁺, ↑GPX4, inhibition of lipid peroxidation [26, 27].	It specifically binds to free iron (especially toxic iron) in the body, forms a complex, and is excreted from the body through urine or feces, directly clearing excess iron [28].	↓MDA, ↓ROS, Protect cells [29]

↓: inhibit; ↑:activate

Network pharmacology offers insights into disease pathogenesis and progression from a systems biology perspective; it can elucidate the actions of traditional Chinese medicines [24]. Molecular docking serves as a computational method to assess binding affinities between small molecules and specific receptors through active component screening [25]. Therefore, we employed network pharmacology alongside molecular docking techniques to uncover the mechanistic pathways through which salidroside exerts its therapeutic effects on ALI. Finally, we conducted further validation at the cellular level regarding both efficacy and mechanism.

Materials and methods

Network pharmacology and molecular docking

Target identification for Salidroside

The compound Salidroside was input into the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) [30] to obtain its SMILES identifier. Subsequently, this identifier was entered into the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) [31], where targets with a Probability > 0 were selected, along with data from the CHEMBL database (https://www.ebi.ac.uk/chembl/) [32] and Super-PRED database (https://prediction.charite.de) [33]. The species filter was restricted to “Homo sapiens”. Target proteins were standardized using the UniProt database (https://www.uniprot.org). After integrating prediction results and re-moving duplicates, a final list of target genes for Salidroside was compiled [34].

Acquisition of acute lung injury targets

Targets associated with acute lung injury were primarily sourced from GeneCards (https://www.genecards.org/) [35], OMIM ( https://www.omim.org/ ) [36] and CTD databases ( https://ctdbase.org ) [37], by searching for the keyword “acute lung injury” .Following integration and removal of duplicate target genes across these three databases, a set of disease-specific target genes related to acute lung injury was obtained.

Core gene identification

Venny 2.1 online software tool (https://bioinfogp.cnb.csic.es/tools/venny/) was utilized to analyze common action targets between Salidroside and acute lung injury in order to identify shared drug-disease targets. A protein-protein interaction network diagram was constructed using the STRING online platform (https://string-db.org/) [38] with species limited to “Homo sapiens” and a confidence score set at ≥ 0.9. This network data was then imported into Cytoscape 3.8.2, where core genes were ranked based on values assigned through topological network algorithms via the cytoHubba plugin for further exploration and analysis of core gene networks.

GO and KEGG pathway enrichment analysis

To further investigate the mechanism of Salidroside in treating acute lung injury (ALI), core genes were submitted to the DAVID database ( https://david.ncifcrf.gov/ ) [39] for Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The GO enrichment analysis was visualized using bubble plots that display the top ten biological processes, cellular components, and molecular functions, while a bar chart illustrated the top twenty KEGG pathways.

Molecular docking

The structure of Salidroside was obtained from the PubChem database, converted into a three-dimensional format, and saved in *MOL format. AutoDock version 1.5.6 was used to process this structure, saving it as a *PDBQT file. The crystal structure of the target protein was retrieved from the PDB database (http://www.RLsb.org). PyMol and AutoDock software were employed to manipulate the target protein molecule, which was also saved in *PDBQT format. Using AutoDock-Vina, molecular docking between Salidroside and the target protein was performed; results were visualized using PyMol version 1.7.x.

Cell experiments

Cells and reagents

Rat alveolar type II epithelial cells (RLE-6TN) were purchased from Hunan Fenghui Biotechnology Co., Ltd.; CCK-8 assay kit (Lot: C0038) was obtained from Shanghai Biyuntian Biotechnology Co., Ltd.; Ferrostatin-1 (Lot: HY-100579) was purchased from Shanghai MCE Bioactive Molecular Master. Rat Slc7a11 (Cystine/glutamate transporter) ELISA Kit (Lot: ER1709) was purchased from Wuhan Fine Biotech Co., Ltd. ANNEXIN V-FITC/PI apoptosis detection kit (Lot: CA1020) was sourced from Beijing Solarbio Technology Co., Ltd.; H2DCFDA (Synonyms: DCFH-DA; 2’,7’-Dichlorodihydrofluorescein diacetate) (Lot: 4091990) was acquired from MedChemExpress in the USA; Fe2 + ELISA kit (Lot: BC5415) was purchased from Beijing Solarbio Technology Co., Ltd.; MDA ELISA kit (Lot: S0131S) was obtained from Shanghai Biyuntian Biotechnology Co., Ltd.; TBST wash buffer was sourced from Beijing Solarbio Technology Co., Ltd.; antibody dilution buffer and 5% BSA blocking solution were both purchased from Beijing Zhongshu Jinqiao Biotechnology Co., Ltd. The GPX4 Monoclonal antibody (Lot: 67763-1-Ig, dilution ratio 1:5000 for a molecular weight of 20 kDa) was acquired from Proteintech in the USA; Anti-CD130 (gp130) antibody [EPR24557-50] (Lot: ab283685, dilution ratio 1:1000 for a molecular weight of 130 kDa), Anti-IL-6 antibody [EPR23819-103] (Lot: ab290735, dilution ratio 1:1000 for a molecular weight of 23 kDa), and Anti-p38 phospho T180 + Y182 antibody(Lot: ab4822, dilution ratio 1:1000 for a molecular weight of 38 kDa) were all sourced from Abcam in the UK. The p38 MAPK Polyclonal antibody (Lot: 14064-1-AP, dilution ratio༚1༚5000, molecular weight༚38 kDa) was obtained from Proteintech in the USA; Beta Actin Monoclonal antibody (Lot༚29380-1-AP, dilution ratio༚1༚5000, molecular weight༚42 kDa)was also acquired from Proteintech; Goat Anti-Mouse IgG/HRP(Lot༚SE131, dilution ratio༚1༚2000༉was purchased from Beijing Solarbio Technology Co., Ltd.; Goat Anti-Rabbit IgG H&L (HRP) (Lot : ab6721, dilution rate : 1 :2000 ) came from Abcam Limited in the UK.

Cell culture

Cell Culture Rat alveolar type II epithelial cells (RLE-6TN) were cultured using DMEM complete medium, which was prepared by adding 10% fetal bovine serum and 1% antibiotics. Both cell lines were placed in an incubator containing 95% O2 and 5% CO2, maintained at a constant temperature of 37 °C. Once the cells reached approximately 90% confluence, they were digested for passage using 0.25% trypsin, with logarithmic phase cells being used for experimental procedures.

Cell proliferation detection

CCK-8 Assay for Cell Proliferation RLE-6TN cells were seeded in a 96-well plate, with a total volume of culture medium per well set to 200 µl. The groups included: culture medium group containing cells (control group), LPS group (5 µg/ml), and LPS + Sal group (20 µg/ml、Sal-L), LPS + Sal group (40 µg/ml、Sal-M), LPS + Sal group (80 µg/ml、Sal-H), LPS + Ferrostatin-1 group (2µM). After drug treatment for eight hours, CCK-8 reagent (20 µl) was added to each well, followed by incubation in the incubator for an additional period of one to four hours until the color developed into deep brown. Subsequently, the optical density (OD) value of each well was measured at a wavelength of 450 nm using a microplate reader. The percentage of cell proliferation was calculated as follows: Cell proliferation percentage = [(Experimental Group OD - Blank Group OD) / (Control Group OD - Blank Group OD)] ×100%.

Cell apoptosis detection

Flow Cytometry Detection of Apoptosis After appropriate culture stimulation, all cells along with their culture medium were transferred into centrifuge tubes and centrifuged at room temperature at 1000 rpm for five minutes to remove the supernatant. Cells were then resuspended in PBS solution (3 ml), followed by another round of centrifugation under the same conditions; this step was repeated twice more before resuspending the final pellet in binding buffer solution while removing excess supernatant after each spin cycle. For staining, each sample received a mixture consisting of 100 µl binding buffer along with Annexin V and PI staining solution (5 µl each). The samples were gently mixed and incubated at room temperature away from light for fifteen minutes before undergoing another centrifugation step at room temperature at 1000 rpm for five minutes to discard supernatants again; finally, they were resuspended in PBS solution prior to immediate analysis via flow cytometry.

ROS detection

Flow Cytometry Detection of Reactive Oxygen Species (ROS) Concentration A stock solution comprising H2DCFDA dissolved in DMSO was prepared by dissolving five milligrams into one point zero three milliliters DMSO as needed; subsequently diluted with pre-warmed serum-free cell culture media or PBS to achieve working concentrations ranging from five to ten micromolar H2DCFDA solutions according to manufacturer instructions.

ELISA detection of Fe²⁺ and MDA

ELISA Detection of Fe²⁺ and Malondialdehyde (MDA) Concentrations Cell supernatants were collected, and the concentrations of Fe²⁺ and MDA were measured according to the instructions provided in the Fe²⁺ and MDA ELISA kit. The optical density values for each well were determined using a microplate reader at a wavelength of 450 nm.

Immunofluorescence detection of GPX4

Immunofluorescence Detection of Glutathione Peroxidase 4 (GPX4) Concentration To assess the protein levels of glutathione peroxidase 4 (GPX4) in cells, treated RLE-6TN cells were seeded onto glass-bottom culture dishes. After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked in PBS containing 5% bovine serum albumin at room temperature for one hour. Cells were then incubated overnight at 4 °C with a primary antibody specific to GPX4. Following PBS washes, cells were incubated in the dark at room temperature for one hour with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. The cell nuclei were counterstained with DAPI (1 µg/mL) for five minutes. Finally, samples were mounted using an anti-fade mounting medium. All images from samples were acquired using a microscope model such as Nikon A1R + laser confocal microscope under a ×60 oil immersion objective lens to ensure reliable comparisons of fluorescence intensity; all experimental groups and control groups underwent staining within the same batch while utilizing identical microscopy parameters during image acquisition.

Western blot

Protein Immunoblotting Analysis Cell samples from four different groups were collected and lysed using RIPA buffer supplemented with PMSF (100 mM) in a ratio of 100 µL RIPA + 1 µL PMSF by pipetting gently before placing on ice for thirty minutes post-lysis; subsequently centrifuged at 12,000 rpm for thirty minutes at 4 °C after lysis completion. Protein concentration was quantified using BCA assay kits following their operational protocols. For protein blotting analysis, BeyoECL Plus reagent kit (P0018S; Beyotime, China) was employed along with imaging performed via Tanon Imaging System (Tanon, China).

Statistical analysis

The results are expressed as mean ± SD. Use unpaired two-sided student t-tests for two group comparisons or one-way ANOVA tests, and then use Bonferroni post hoc tests for multiple comparisons to analyze differences. P < 0.05 is considered to indicate significant statistical differences.

Results

Network Pharmacology and molecular docking results

The results of network pharmacology and molecular docking indicate that Salidroside exerts its therapeutic effects on acute lung injury (ALI) through a synergistic action involving multiple targets and pathways. The flowchart is illustrated in Fig. 1.

Fig. 1.

Flow chart of network pharmacology and molecular docking

Intersection genes between Salidroside and acute lung injury

In this study, we identified 382 targets associated with Salidroside from the ChEMBL, Swiss TargetPrediction, and Super-PRED databases. Subsequently, we determined 11,036 targets closely related to ALI using the GeneCards, OMIM, and CTD databases. Through Venn diagram analysis, we obtained a set of 355 overlapping targets as potential candidates for Salidroside treatment of ALI (Fig. 2).

Fig. 2.

The Venn diagram of related targets between Sal and ALI

Protein interaction network diagram and core genes acquisition

Using the STRING database along with Cytoscape software, we constructed a protein-protein interaction network diagram for detailed analysis to gain insights into the topological features of these nodes. This visualization effectively conveys the significance of each node through size and color; it includes 213 nodes and 470 edges where larger nodes with darker colors represent higher degree values within the network (Fig. 3A). Further analysis utilizing the cytoHubba plugin in Cytoscape allowed us to identify 20 key targets involved in Salidroside’s treatment of ALI (Fig. 3B).

Fig. 3.

Protein-protein interaction network diagram and core genes acquisition. A: Protein-protein interaction network diagram; B: Core genes

GO and KEGG pathway analysis for Sal treatment in ALI

The top twenty key genes were imported into the DAVID database for Gene Ontology (GO) enrichment analysis as well as KEGG pathway enrichment analysis. The GO enrichment results indicated involvement in 190 biological processes, 28 cellular components, and 44 molecular functions. We selected the top ten entries from each category which are visually represented in an enrichment chart (Fig. 4), providing a clear overview related to Salidroside’s therapeutic effects on ALI—primarily concerning signal transduction, cytosol localization, protein binding among others. The KEGG pathway enrichment analysis revealed associations with151 signaling pathways predominantly linked to MAPK signaling pathway and VEGF signaling pathway; details regarding the top twenty results are shown in Fig. 5.

Fig. 4.

GO enrichment analysis

Fig. 5.

KEGG enrichment analysis

Molecular docking

Molecular docking analysis was conducted between Sal and two genes related to the study, MAPK14 and GPX4. The results highlighted the strong binding affinity of Sal to these key proteins (Table 2). To further elucidate the complex binding configurations, we utilized PyMOL for visualization, displaying the lowest energy binding conformation (Fig. 6). This visualization vividly illustrates the close relationship between Sal and its critical targets, providing valuable insights into their interaction’s structural basis and reinforcing the significance of Sal in treating ALI at a molecular level.

Table 2.

Verification results of molecular Docking

Ingredients	targets	Affifinity (kcal/mol)	Distance from best mode
			rmsd l.b.	rmsd u.b.
Salidroside	MAPK14	− 6.7	0	0
	GPX4	− 6.4	0	0

Fig. 6.

Molecular docking. A: Salidroside with MAPK14; B: Salidroside with GPX4

Cellular experimental results

Acute lung injury caused by septic infection leads to widespread diffuse damage and uncontrolled inflammation in alveolar epithelial cells. Therefore, this study selected rat alveolar type II epithelial cells for subsequent research; the flowchart is shown in Fig. 7.

Fig. 7.

Cell experiment flowchart

Cell proliferation assay

The proliferation of cells from different groups was assessed using a CCK-8 kit, as illustrated in Fig. 8. The cell proliferation rate in the LPS group was significantly lower than that of the control group (P < 0.01); However, compared with the LPS group, the addition of Sal 40 µg/ml significantly increased the cell proliferation rate (P < 0.01). This indicates that Sal can alleviate LPS induced cytotoxicity and improve cell survival rate. Subsequent experiments will be conducted using Sal at a concentration of 40 µg/ml.

Fig. 8.

Changes in proliferation rate of RLE-6TN cells treated with Sal. Mean ± SD, n = 3. ^*P < 0.05, ^##P < 0.01, ^**P < 0.01

Cell apoptosis assay

As depicted in Fig. 9, apoptosis detection across different groups revealed that the apoptosis rate in the LPS group was significantly higher than that of the control group (P < 0.001); following treatment with Sal, there was a decrease in apoptosis rate compared to the LPS group (P < 0.05). This indicates that Sal may inhibit LPS-induced apoptosis and improve cell viability.

Fig. 9.

Changes in apoptosis rate of RLE-6TN cells treated with Sal. Mean ± SD, n = 3. ^*P < 0.05, ^###P < 0.001

ROS detection

ROS levels were measured using an ROS detection kit across various groups as shown in Fig. 10; it was found that ROS content in the LPS group was significantly higher than that of controls (P < 0.001). After adding Sal, ROS levels decreased compared to those observed with LPS alone (P < 0.01), suggesting that Sal can suppress oxidative stress induced by LPS on cells.

Fig. 10.

Changes in ROS content in RLE-6TN cells treated with Sal. Mean ± SD, n = 3. ^###P < 0.001, ^**P < 0.01

ELISA detection of Fe²⁺ and MDA

As presented in Fig. 11, both Fe²⁺ and MDA levels were significantly elevated in comparison with controls within the LPS group (P < 0.0001); conversely, Fe²⁺ and MDA concentrations were markedly reduced after treatment with Sal when compared to those seen with just LPS exposure (P < 0.001). In addition, compared with the control group, SLC7A11 was significantly downregulated in the LPS group (P < 0.0001) and significantly upregulated after administration of salidroside (P < 0.001).These findings indicate that Sal may inhibit iron death and lipid peroxidation triggered by LPS.

Fig. 11.

Changes in Fe²⁺ and MDA content in RLE-6TN cells treated with Sal. A: Changes in Fe²⁺ content, B: Changes in MDA content; Mean ± SD, n = 3. ^***P < 0.001, ^####P < 0.0001

Immunofluorescence detection of GPX4

To verify changes regarding GPX4 among different cellular groups, immunofluorescence detection for GPX4 was performed as illustrated in Fig. 12; it were significantly reduced in immunofluorescence intensity for GPX4 within samples fromthe LPS group relative to controls (P < 0.0001); however, following Sal treatment, GPX4 immunofluorescence intensity increased substantially when comparedto LPS alone (P < 0.001). In addition, WB results also showed that LPS significantly reduced the expression of GPX4 (P < 0.0001), and after administration of salidroside, GPX4 significantly increased (P < 0.0001).

Fig. 12.

Changes in immunofluorescence intensity of GPX4 in RLE-6TN cells treated with Sal. Scale: 60×, Mean ± SD, n = 3. ^****P < 0.0001, ^###P < 0.001

Western blot

To elucidate the associated pathways, we conducted Western blot analysis on GPX4, gp130, IL-6, MAPK, and p-MAPK. As illustrated in Fig. 13, compared to the control group, the LPS group exhibited significantly elevated levels of gp130, IL-6, and p-MAPK (P < 0.01). Following the administration of Sal, there was a notable decrease in the levels of gp130, IL-6, and p-MAPK when compared to the LPS group (P < 0.01). Conversely, while GPX4 levels were significantly reduced in the LPS group relative to the control group (P < 0.01), a marked increase in GPX4 was observed in the Sal group compared to the LPS group (P < 0.01). These findings suggest that Sal may mitigate lung injury by modulating the MAPK/GPX4 pathway.

Fig. 13.

Western blot. A: Western blot was used to detect the protein expression of GPX4、gp130、IL-6、MAPK and p-MAPK in cells, B: Relative expression of GPX4、gp130、IL-6、MAPK and p-MAPK. Mean ± SD, n = 3. ^****P < 0.0001, ^###P < 0.001

Discussion

Acute lung injury arises from widespread damage to the pulmonary microvascular endothelium and alveolar epithelium triggered by severe infection, shock, trauma or burns. Clinically it manifests as diffuse interstitial and alveolar oedema culminating in acute hypoxaemic respiratory insufficiency that can progress to life-threatening ARDS [40–42]. To date, no pharmacological agent has been approved specifically for ALI, and off-label therapies frequently provoke coagulopathy, gastric ulceration or osteoporosis [43]. By contrast, syndrome-based Chinese medicine employs either purified herbal monomers or multi-compound formulae to prevent or treat ALI/ARDS with encouraging results [44]. Salidroside, the principal bioactive constituent of Rhodiola rosea, has been reported to attenuate ALI through antioxidant and anti-inflammatory actions [23, 45, 46]; nevertheless, the precise molecular circuitry remains ill-defined. Here we integrated network pharmacology with molecular docking to map the therapeutic interactome of salidroside in ALI and subsequently validated the predictions experimentally in vitro. Our data demonstrate that (1) salidroside exerts protective effects against ALI via a multi-target, multi-pathway mechanism, and (2) salidroside suppresses LPS-evoked accumulation of ROS, Fe²⁺, MDA and IL-6, dampens p-MAPK signalling, while restoring GPX4 expression.

GO enrichment analysis revealed that ALI-relevant biological processes are enriched in signal transduction and protein phosphorylation. At the cellular-compartment level, salidroside preferentially modulates proteins localized to the cytoplasm and nucleus. Molecular-function profiling further indicated that ALI perturbs both protein-binding and ATP-binding capacities. KEGG pathway mapping identified MAPK, mTOR and VEGF cascades as the most significantly represented signalling routes—networks intimately linked to cell proliferation, differentiation, apoptosis and immune–inflammatory responses—thereby positioning salidroside as a promising botanical monomer for ALI prophylaxis and therapy. Independent studies corroborate these in silico predictions. Guo et al. [45] reported that salidroside suppresses hyperoxia-induced AECⅡ ferroptosis via blockade of the p38 MAPK axis, reversing hyperoxic lung injury in neonatal rats. Similarly, in LPS-challenged rodents, salidroside down-regulated p38 MAPK phosphorylation and ameliorated ALI histopathology [47], findings that align closely with our observations. The mTOR pathway has also been implicated in ALI pathobiology; salidroside attenuated lung damage by curtailing mTOR phosphorylation in rat models of endotoxin shock [48]. Regarding VEGF signalling, earlier work suggests a contextual, time-dependent role: early VEGF release increases pulmonary vascular permeability, whereas sustained depletion of VEGF/VEGFR-1 compromises alveolar epithelial viability [49]. Conversely, activation of macrophage-specific VEGF-C/VEGFR-3 signalling ameliorated LPS-induced ALI by enhancing resolution of alveolar inflammation [50]. Collectively, these data implicate salidroside-mediated fine-tuning of MAPK, mTOR and VEGF networks as a central mechanism underlying its protective effects against ALI.

Molecular docking results revealed that Sal exhibited favorable binding affinities to MAPK14 (p38MAPK) and GPX4, with binding energies of -6.7 and − 6.4 kcal/mol, respectively. This suggests that Sal may exert therapeutic effects on ALI by modulating these two target genes. Yang et al. [51] demonstrated that in an LPS-induced ALI model, phosphorylation levels of p38MAPK were significantly elevated, and inhibition of p38MAPK phosphorylation markedly alleviated ALI symptoms. Similarly, Zhang et al. [52] reported that in pediatric sepsis-induced ALI, overexpression of MAPK14 enhanced oxidative stress, lipid peroxidation, iron accumulation, promoted neutrophil infiltration, and upregulated PD-L1 expression, leading to immunosuppression. Knockdown of MAPK14 reduced ferroptosis and attenuated ALI, which is consistent with our findings. Furthermore, Wu et al. [53] identified MAPK14 as a hub gene potentially playing a critical role in the inflammatory regulation of ALI through bioinformatic analysis. Numerous studies have also indicated that phosphorylated p38MAPK levels are significantly increased in ALI, and inhibition of its phosphorylation via traditional Chinese medicine substantially alleviated ALI symptoms [54, 55]. As a key marker of ferroptosis [56], GPX4 expression was found to be significantly reduced in ALI, promoting ferroptotic cell death. Treatment with herbal medicine was shown to upregulate GPX4 levels, suppress ferroptosis, and consequently ameliorate ALI [57–59].

In acute lung injury, the MAPK/GPX4 axis serves as a central signaling hub regulating ferroptosis in alveolar epithelial cells, and its dysregulation represents a key mechanism driving the progression of lung injury [60, 61]. Aberrant activation of the MAPK signaling pathway (e.g., p38, ERK) induces ferroptosis through multiple mechanisms that impair GPX4 function [62]. On one hand, activated MAPK signaling exacerbates oxidative stress, leading to the direct depletion of glutathione (GSH), an essential substrate for GPX4 activity. On the other hand, it modulates downstream transcription factors to suppress the expression of critical anti-ferroptotic proteins, including GPX4 and the cystine transporter SLC7A11 [63]. In specific ALI models, sepsis-related stimuli have been shown to activate the MAPK/ERK pathway, while hyperoxic lung injury demonstrates that IL-17 A promotes ferroptosis via the Act1-TRAF6-p38 MAPK signaling cascade [45]. This process is accompanied by the accumulation of lipid peroxides (e.g., malondialdehyde, MDA) and disruption of iron homeostasis, ultimately resulting in rupture of the alveolar epithelial cell membrane, cell death, and compromised pulmonary barrier function [64]. Therefore, therapeutic strategies targeting inhibition of the MAPK pathway or enhancement of GPX4 activity can effectively mitigate ferroptosis in ALI, providing a well-defined molecular target for the development of novel lung-protective agents, such as salidroside.

The present study demonstrated that Sal effectively reduced the expression levels of IL-6 and ROS in ALI. Wu et al. [65] reported that during sepsis, pulmonary capillary endothelial cells stimulated by cytokines such as IL-1, IL-6, and TNF-α exhibit increased vascular permeability, leading to the leakage of plasma components (including water and proteins) into the alveolar space and resulting in pulmonary edema. Inhibition of IL-6 expression has been shown to alleviate ALI [66–68]. Additionally, in ALI, excessive ROS production damages pulmonary vascular endothelial cells, promotes fluid leakage, and exacerbates lung injury [69]. Suppression of ROS generation has been found to reverse ALI symptoms [70–72]. These findings are consistent with our results, indicating that reduction in IL-6 and ROS levels can lead to the amelioration of ALI. Nevertheless, this study has certain limitations. Future investigations will focus on the following aspects: (1) Examining downstream proteins of MAPK signaling and other proteins associated with ferroptosis; (2) Establishing in vivo models to validate whether Sal mitigates acute lung injury through this pathway.

Previous studies have demonstrated that salidroside can alleviate acute lung injury (ALI) by suppressing inflammation [73] and modulating well-known signaling pathways such as p38 MAPK [45] and JAK2/STAT3 [74]. However, most of these investigations have primarily focused on observational phenomena or the validation of established pathways, leaving a clear mechanistic understanding of salidroside’s regulation of GPX4—the core executor molecule in ferroptosis—largely unexplored. In this study, we confirmed that salidroside directly targets MAPK14, inhibiting its phosphorylation and activation, thereby promoting GPX4 expression and ultimately suppressing ferroptosis in alveolar epithelial cells. This finding not only elucidates the pharmacological basis of salidroside but, more importantly, identifies MAPK14 as a critical node linking inflammatory stress to ferroptosis. These results provide a promising novel therapeutic target for ALI and offer clear direction for future research. For instance, future in vivo studies should employ MAPK14-specific agonists or genetically modified animal models to validate the central role of this axis in ALI pathogenesis and to investigate its crosstalk with other pathological processes such as inflammation. Additionally, structure-based drug design could be applied to develop salidroside derivatives or analogs targeting MAPK14, aiming to enhance potency and selectivity. Furthermore, combination therapies involving salidroside and conventional anti-inflammatory agents (e.g., corticosteroids) may be explored, potentially achieving synergistic effects through concurrent inhibition of inflammation and ferroptosis, thus offering new strategies for managing refractory ALI.

Although salidroside has been widely investigated in both in vitro and in vivo models of acute lung injury, its clinical translation faces several challenges. First, pharmacokinetic limitations remain a major obstacle: previous studies in rats indicate an oral bioavailability of only 28.74%, with P-glycoprotein (P-gp)-mediated efflux significantly impairing intestinal absorption [75], resulting in a short plasma half-life and rapid systemic clearance [76]. Second, there is insufficient evidence regarding its efficacy and safety in humans. Current clinical studies are limited by small sample sizes and variable methodological quality, lacking large-scale, high-quality randomized controlled trials. Moreover, potential drug interactions with antidepressants, antihyperglycemic agents, anticoagulants, and immunosuppressants raise additional safety concerns [77]. Therefore, further in-depth investigation of salidroside is warranted to bridge the gap between preclinical findings and clinical application.

Conclusion

In summary, Sal was identified through network pharmacology and molecular docking as a promising active compound for the treatment of ALI. Preliminary in vitro experiments demonstrated that Sal pretreatment attenuates inflammatory response and oxidative stress, thereby conferring protection to lung tissue, likely via modulation of the MAPK/GPX4 signaling pathway (Fig. 14). These findings suggest that salidroside may serve as a potential therapeutic agent against LPS-induced ALI.

Fig. 14.

Schematic of the protective mechanism of Sal against LPS-induced ALI. Sal inhibits the IL-6-gp130 interaction, thereby suppressing the MAPK/GPX4 pathway. This leads to the downregulation of ROS, MDA, and Fe²⁺, ultimately inhibiting ferroptosis in alveolar epithelial cells

Abbreviations

Sal

Salidroside

ALI

Acute lung injury

GO

gene ontology

IL-6

Interleukin-6

KEGG

Kyoto Encyclopedia of Genes and Genomes

LPS

Lipopolysaccharide

ARDS

Acute Respiratory Distress Syndrome

LPO

Lipid peroxides

MDA

Malondialdehyde

IL-1

Interleukin-1

GPX4

Glutathione Peroxidase 4

CCK8

Cell Counting Kit-8

mTOR

Mammalian target of rapamycin

TNF-α

Tumor Necrosis Factor-α

VEGF

Vascular endothelial growth factor

ROS

Reactive oxygen species

Authors’ contributions

M Z: Software, Validation, Formal analysis, Writing—review & editing. M Z and M Q W: Conceptualization, Writing—original draft. W F and M C: Conceptualization, Investigation, Supervision and Editing. W L: Supervision, Writing—review & editing.

Funding

This work was funded by the National Natural Science Foundation of China (82471061), Shandong Natural Science Foundation (ZR2023MH035), Youth Science Foundation Cultivation Support Program (Natural Science) of Shandong First Medical University &Shandong Academy of Medical Sciences (202201-009). The scientific research project of Yunnan Provincial Department of Education (2026J2412), the school level scientific research project of Lijiang University of culture and tourism (2024xyp06) and the third batch of young and middle-aged academic and technical reserve talents of Lijiang University of culture and tourism (2024xshb03)

Data availability

The Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.