Anlotinib ameliorates myositis-associated interstitial lung disease (MAILD) via suppression of the NETs-PI3K/Akt-driven epithelial-mesenchymal transition

Abstract

Background

Myositis-associated interstitial lung disease (MAILD) is one of the most severe complications of idiopathic inflammatory myopathy, characterised by rapidly progressive pulmonary fibrosis and high mortality. Treatment options are limited, and mechanisms driving epithelial-mesenchymal transition (EMT) in MAILD are incompletely understood. Anlotinib, a multitarget tyrosine kinase inhibitor, shows potential in fibrotic diseases; however, its role and mechanism in MAILD need clarification.

Methods

To assess anlotinib’s therapeutic effects, we established a MAILD mouse model and a neutrophil extracellular trap (NET)-induced human alveolar epithelial cell (A549) model. H&E and Masson staining analysed lung pathological changes and collagen deposition. Immunohistochemistry, immunofluorescence and Western blot detected expressions of NETs markers (myeloperoxidase, citrullinated histone H3), phosphatidylinositol 3-kinase/protein kinase B (PI3K)/Akt components and EMT markers (E-cadherin, α-smooth muscle actin). RNA sequencing and gene set enrichment analysis identified differentially expressed genes and signalling pathways. Cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) and wound healing assays assessed cellular repair and migration.

Results

MAILD mouse lungs showed structural damage, inflammatory infiltration, collagen deposition, increased NETs formation, PI3K/Akt activation and enhanced EMT. Anlotinib significantly ameliorated pulmonary fibrosis and reduced pro-inflammatory cytokines (tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β). In vivo and in vitro, anlotinib suppressed NETs formation, PI3K/Akt activation and EMT, while enhancing alveolar epithelial cell repair and migration.

Conclusion

Anlotinib alleviates MAILD progression by inhibiting the NETs–PI3K/Akt axis and subsequent EMT, providing a theoretical basis for drug repurposing and supporting its clinical translation potential in MAILD.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Myositis-associated interstitial lung disease (MAILD) is a severe, treatment-refractory condition. While neutrophil extracellular traps (NETs) and PI3K/Akt signalling are individually linked to fibrosis, their integrated role in driving epithelial-mesenchymal transition (EMT) within MAILD was not defined.

WHAT THIS STUDY ADDS

• This study establishes a novel ‘NETs–PI3K/Akt–EMT’ axis as a core pathogenic mechanism in MAILD. We show that anlotinib exerts a coordinated anti-fibrotic effect by concurrently inhibiting NETosis, PI3K/Akt activation and EMT progression.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• These findings provide a robust mechanistic basis for repurposing anlotinib in MAILD. The defined axis offers a new, stratified target for clinical trials, potentially guiding therapy for patients with this high-mortality disease.

Introduction

Idiopathic inflammatory myopathies (IIMs) represent a heterogeneous group of disorders characterised by chronic muscle inflammation and multisystem involvement. Myositis-associated interstitial lung disease (MAILD) ranks among the most common and devastating complications of IIMs. Notably, in patients with anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody-positive dermatomyositis, the incidence of MAILD is approximately 70–90%, often manifesting as rapidly progressive interstitial lung disease with a mortality rate exceeding 40% within 6 months of diagnosis.1,3 Current clinical management primarily depends on glucocorticoids combined with immunosuppressants (eg, tacrolimus, cyclophosphamide) or biologic agents (eg, rituximab). However, a significant proportion of patients show inadequate responses or are intolerant to drug-related adverse effects, leading to disease progression.^{4 5} Therefore, elucidating the cellular and molecular mechanisms underlying MAILD and identifying novel therapeutic targets are urgently needed.

Epithelial-mesenchymal transition (EMT) is widely acknowledged as a pivotal biological process in pulmonary fibrosis. Under conditions of persistent injury and an inflammatory microenvironment, alveolar epithelial cells lose epithelial polarity and downregulate epithelial markers (eg, E-cadherin (E-Ca)) while acquiring mesenchymal characteristics (eg, increased expression of α-smooth muscle actin (α-SMA) and vimentin). This transition enhances cell migration, invasion and extracellular matrix production, ultimately leading to pulmonary structural remodelling and fibrosis.6,8 Recent studies have demonstrated that neutrophil extracellular traps (NETs) are significantly elevated in the serum and bronchoalveolar lavage fluid of MAILD patients, with levels positively correlating with disease severity and prognosis.^{9 10} Components of NETs, such as histones and myeloperoxidase (MPO), can function as damage-associated molecular patterns (DAMPs) to activate signalling pathways (eg, via Toll-like receptor 9 (TLR9)), thereby inducing epithelial inflammation, apoptosis and EMT.^{11 12}

The phosphatidylinositol 3-kinase/protein kinase B (PI3K)/Akt signalling pathway is a critical intracellular signalling axis that regulates cell survival, proliferation, metabolism and EMT. Studies have demonstrated aberrant activation of the PI3K/Akt pathway in idiopathic pulmonary fibrosis (IPF) and various connective tissue disease-associated ILDs. This pathway serves as a core downstream mediator of profibrotic factors (eg, transforming growth factor-β (TGF-β)), directly participating in the transcriptional regulation of EMT.^{13 14} Importantly, emerging evidence indicates that NETs can activate PI3K/Akt signalling, thereby promoting fibrotic processes.¹⁵ Based on these observations, we hypothesise that in MAILD, NETs induce EMT in alveolar epithelial cells via activation of the PI3K/Akt pathway, thereby promoting pulmonary fibrosis.

Anlotinib is a novel multitargeted tyrosine kinase inhibitor (TKI) that potently inhibits receptors including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and c-Kit.¹⁶ Preclinical studies have shown that anlotinib mitigates bleomycin-induced pulmonary fibrosis in mice by suppressing fibroblast activation and glycolytic reprogramming.¹⁷ However, whether anlotinib holds therapeutic potential for MAILD and whether it exerts its effects by modulating the ‘NETs–PI3K/Akt–EMT’ axis remains unknown. This study aims to systematically assess the antifibrotic effects of anlotinib using in vivo and in vitro MAILD models, with a focus on elucidating its mechanism of action by investigating NETs formation, PI3K/Akt pathway activation and EMT regulation. Our findings are anticipated to provide a robust theoretical and experimental basis for the clinical translation of anlotinib in MAILD therapy.

Materials and methods

Animal model establishment and experimental grouping

Female BALB/c wild-type mice (6–8 weeks old) were purchased from the Lanzhou Veterinary Research Institute. All animals were housed under specific pathogen-free conditions at the Animal Experiment Center of the Second Hospital of Lanzhou University, with free access to food and water. The housing environment was maintained on a 12/12-hour light/dark cycle at a temperature of 22±2°C. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Second Hospital of Lanzhou University (approval No.: D2025-139).

The MAILD mouse model was established as previously described by our team.¹⁸ Briefly, mice were immunised by subcutaneous injection of allogeneic rat skeletal muscle homogenate (200 µg per mouse) emulsified in complete Freund’s adjuvant (Sigma-Aldrich, Missouri, USA), followed by intraperitoneal injections of the TLR9 agonist CpG-ODN (InvivoGen, California, USA; 50 µg per mouse) on days 1, 7, 14 and 21 to simulate myositis and subsequent interstitial lung disease. Mice were randomly divided into the following four groups (n=5 per group, total 20): (1) control group: injected with an equal volume of physiological saline; (2) MAILD model group: subjected to the aforementioned modelling protocol; (3) anlotinib treatment group: received daily oral gavage of anlotinib (dissolved in corn oil containing 5% DMSO) at 1 mg/kg/day concurrently with model induction; (4) Anlotinib drug control group: received daily oral gavage of anlotinib (1 mg/kg/day) alone as a drug control.

All interventions continued until the end of the experiment on day 28. Body weight and general condition were monitored daily. On day 28, all mice were euthanised via anaesthesia with an overdose of sodium pentobarbital administered intraperitoneally. Blood and lung tissues were collected. Parts of the lung tissue were fixed in 4% paraformaldehyde for histological analysis, while other parts were rapidly frozen and stored at −80°C for subsequent molecular biology assays.

Immunohistochemistry

Paraffin-embedded sections were deparaffinised, rehydrated and subjected to antigen retrieval. Staining was performed using the following primary antibodies: anti-E-Ca (Bioss, Cat# bs-10009R, 1:500), anti-α-SMA (Abcam, Cat# ab5694, 1:500), anti-Akt (Bioss, Cat# bsm-33337M, 1:500) and anti-p-Akt (Affinity Biosciences, Cat# AF1015, 1:500). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, sections were developed using 3,3′-diaminobenzidine substrate, mounted and observed under a microscope for positive signal assessment.

Cell culture and treatment

Primary A549 cells were purchased from Shanghai Fuheng Biotechnology. Cells from the second or third passage were used for experiments. A549 cells were maintained in RPMI-1640 medium (Gibco, Grand Island, New York, USA). The experimental groups were as follows: (1) NET group: cells stimulated with 100 ng/mL NETs for 24 hours; (2) Anlotinib intervention group: cells co-treated with 100 ng/mL NETs and 200 ng/mL anlotinib for 24 hours; (3) Omipalisib (U0216) group: cells pretreated with the PI3K inhibitor omipalisib for 30 min before co-culture with 100 ng/mL NETs for 24 hours.

RNA-seq sample preparation

A549 cells in the control group were cultured in RPMI-1640 medium (Gibco) for 24 hours. In the NETs group, A549 cells were stimulated with 100 ng/mL NETs for 24 hours. Total RNA was extracted from cells using Trizol reagent (Invitrogen, California, USA) and stored at −80°C. Samples from three independent experiments were sent to Shanghai OE Biotech. for transcriptome sequencing and analysis.

Immunofluorescence

Cells or tissue sections were fixed, permeabilised and blocked. They were then incubated overnight at 4°C with the following primary antibodies: anti-E-Ca (E-Ca, Cat# bs-10009R, 1:500), anti-α-SMA (Cat# ab5694, 1:500), anti-Akt (Cat# 10 176–2-AP, 1:500) and anti-p-Akt (Cat# 66444-1-1g, 1:500). Primary antibodies were detected using appropriate fluorescently labelled secondary antibodies incubated at 37°C for 1 hour. Confocal fluorescence images were acquired using a Zeiss LSM 880 laser scanning microscope with a 63× oil immersion objective.

Western blot analysis

Proteins were extracted from cells using RIPA lysis buffer (Cat# R0030) supplemented with protease and phosphatase inhibitors (Cat# P1260). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were washed with Tris-buffered saline (TBS), blocked with 5% skim milk for 1 hour and then incubated overnight at 4°C with the following primary antibodies: anti-E-Ca (E-Ca, Cat# bs-10009, 1:1000), anti-α-SMA (Cat# ab5694, 1:1000), anti-Akt (Cat# 10 176–2-AP, 1:500) and anti-p-Akt (Cat# 66444-1-1g, 1:500). After washing with TBS, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Protein bands were visualised using a chemiluminescence imaging system and an ECL detection kit.

Statistical analysis

GraphPad Prism V.8 software was used for intergroup comparisons and graph generation. Data are presented as mean±SD Statistical significance was determined by one-way analysis of variance followed by Dunnett’s multiple comparisons test.

Results

Anlotinib ameliorates pulmonary pathological injury and systemic inflammation in MAILD mice

To evaluate the therapeutic effect of anlotinib on MAILD, we first established a mouse model and conducted in vivo pharmacodynamic studies. As shown in figure 1A, H and E staining revealed that compared with the control group, lung tissues from the MAILD model group exhibited typical pathological features of interstitial pneumonia, including severe destruction of alveolar architecture and extensive diffuse inflammatory cell infiltration. In stark contrast, anlotinib treatment markedly ameliorated these pathological injuries and significantly reduced the degree of inflammatory cell infiltration. Quantitative analysis (figure 1C) confirmed that anlotinib treatment significantly lowered the lung inflammation score (p<0.001).

Figure 1. Anlotinib ameliorates pulmonary pathology and systemic inflammation in MAILD mice. (A) Representative H&E-stained lung tissue sections from each group. (B) Dynamic changes in body weight of mice in each group during the experimental period (n=5). (C) Quantitative analysis of inflammatory cell infiltration scores in lung tissues (n=5). (D) Representative Masson’s trichrome-stained lung tissue sections showing collagen deposition. (E) Quantitative analysis of lung fibrosis scores (n=3). (F–H) Serum levels of CK, AST and ALT detected by biochemical assays (n=3). (I–K) Serum concentrations of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β measured by ELISA (n=3). Data are presented as mean±SD; *p<0.05, **p<0.01, ***p<.001; statistical significance was analysed by one-way ANOVA followed by Tukey’s post-hoc test. ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; CK, creatine kinase; IL, interleukin; MAILD, myositis-associated interstitial lung disease; NETs, neutrophil extracellular traps; TNF-α, tumour necrosis factor-α.

Body weight was monitored throughout the experimental period (figure 1B). Mice in the MAILD model group showed significant weight loss following multiple immunisations, consistent with a systemic wasting state associated with the disease. Anlotinib intervention effectively mitigated this weight loss, with the body weight change trajectory resembling that of the control group, suggesting an improvement in the overall health status of the treated mice.

Pulmonary fibrosis is a hallmark pathological feature of MAILD. Masson’s trichrome staining revealed substantial blue collagen fibre deposition in the lung interstitium of the MAILD model group (figure 1D,E). Anlotinib treatment significantly reduced the area of collagen deposition and the fibrosis score (p<0.01), indicating its efficacy in inhibiting collagen production and fibrotic progression in MAILD.

Given the frequent multi-system involvement in MAILD, we measured serum indicators reflecting muscle injury and organ function. Serum levels of creatine kinase, aspartate aminotransferase and alanine aminotransferase were significantly lower in the anlotinib-treated group compared with the model group (figure 1F–H), suggesting a potential protective effect of anlotinib on muscle and liver. Furthermore, ELISA results demonstrated that anlotinib treatment significantly reduced the serum levels of key pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in MAILD mice (figure 1I–K), confirming its systemic anti-inflammatory effect.

Anlotinib effectively suppresses NET formation in vivo and in vitro

To investigate whether anlotinib modulates the formation of NETs, we first performed relevant detections in lung tissues of MAILD model mice. Immunohistochemistry (IHC) and immunofluorescence (IF) analyses revealed that the expression levels of specific NETs markers—MPO and citrullinated histone H3 (Cit-H3)—were significantly elevated in the lung tissues of MAILD model mice compared with the control group (figure 2A–G), indicating robust NETs formation in the disease model. Notably, compared with the MAILD model group, the fluorescence signal intensity and positive area for both MPO and Cit-H3 in lung tissues showed a marked decrease (figure 2A–G), preliminarily suggesting that anlotinib can effectively inhibit aberrant NETs formation in vivo.

Figure 2. Anlotinib inhibits NET formation in vivo and mitigates NET-induced alveolar epithelial cell dysfunction in vitro. (A) IHC staining of MPO in mouse lung tissues. (B) Quantitative analysis of MPO-positive area in IHC staining (n=3). (C) IHC staining of Cit-H3 in mouse lung tissues. (D) Quantitative analysis of Cit-H3-positive area in IHC staining (n=3). (E) IF staining of MPO (green) and Cit-H3 (red) in mouse lung tissues; nuclei were counterstained with DAPI (blue). (F) Quantitative analysis of MPO fluorescence intensity in IF staining (n=3). (G) Quantitative analysis of Cit-H3 fluorescence intensity in IF staining (n=3). (H) Quantification of NETs fluorescence intensity (MPO⁺Cit-H3⁺ double-positive signals) in vitro (n=3). (I) High-content imaging analysis of NETs formation in primary mouse bone marrow-derived neutrophils stimulated with PMA with or without anlotinib treatment. Data are presented as mean±SD; *p<0.05, **p<0.01, ***p<0.001. AOD, average optical density; Cit-H3, citrullinated histone H3; DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; IHC, immunohistochemistry; MAILD, myositis-associated interstitial lung disease; MFI, mean fluorescence intensity; MPO, myeloperoxidase; NC, negative control; NETs, neutrophil extracellular traps; PMA, phorbol 12-myristate 13-acetate.

To further confirm that the reduced expression of MPO and Cit-H3 in anlotinib-treated mice truly reflects suppressed NET formation (rather than altered neutrophil infiltration or intracellular protein expression), we validated the morphological characteristics of NETs. Notably, a typical morphological feature of NETs is that activated neutrophils release intracellular chromatin and antimicrobial proteins (eg, MPO, Cit-H3) into the extracellular environment via NETosis, forming reticular fibrous structures—thus, immunostaining signals for NETs components should localise in the extracellular region. This is further validated by the IF results in figure 3E, which show extracellular MPO⁺/Cit-H3⁺ reticular structures, confirming the extensive accumulation of NETs in lung tissue interstitia.

Figure 3. Anlotinib restores the repair function of alveolar epithelial cells impaired by NETs in vitro. (A) EdU staining showing cell proliferation of alveolar epithelial cells (nuclei stained with DAPI, blue) after NETs stimulation with or without anlotinib treatment. (B) Scratch assay images showing wound healing of alveolar epithelial cells at 0 hours and 24 hours after NETs challenge with or without anlotinib intervention. (C) Quantitative analysis of EdU-positive cells (proliferation rate) (n=3). (D) Quantitative analysis of wound closure rate at 24 hours (n=9). (E) CCK-8 assay showing the viability of alveolar epithelial cells after NETs stimulation and anlotinib treatment (n=3). Data are presented as mean±SD; *p<0.05, **p<0.01, ***p<0.001. CCK, cell counting kit; DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine; NETs, neutrophil extracellular traps; ns, no significance; OD, optical density.

To validate this in vivo finding and exclude potential interference from the complex in vivo microenvironment (eg, interactions with other cell types or cytokines), we further conducted in vitro experiments using primary neutrophils. Neutrophils isolated from mouse bone marrow were stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NET formation. As shown in figure 2H–I, PMA stimulation successfully triggered the release of extensive NETs, characterised by fibrous web-like structures composed of Cit-H3 and DNA. However, when anlotinib was added during co-incubation, PMA-induced NETs formation was significantly suppressed compared with the PMA-only group. Neutrophils maintained a more intact cellular morphology, and the production of Cit-H3/DNA-positive reticular structures (a hallmark of NETs) was substantially reduced (figure 2H–I).

In summary, our in vivo and in vitro data are mutually corroborative, collectively demonstrating that anlotinib possesses the capacity to inhibit NET formation. This finding provides a novel mechanistic perspective—that is, inhibition of NET formation—for elucidating the potential protective role of anlotinib in MAILD.

Effects of anlotinib on NET-induced dysfunction in alveolar epithelial cells

To evaluate the impact of anlotinib on NET-induced dysfunction in alveolar epithelial cells, a series of functional assays were conducted. NET exposure exerted severe cytotoxic effects and functional suppression on the cells. EdU and CCK-8 assays demonstrated that NETs significantly inhibited cell proliferation and viability (figure 3A,C). These findings suggest that under this specific microenvironment, the primary effect of anlotinib shifts from inhibiting proliferation to rescuing cells from the NETs-induced suppressed state. This rescue effect is potentially mediated through its anti-inflammatory, antioxidant or antiapoptotic properties, thereby restoring normal physiological function. This observation underscores a fundamental principle of drug action: the net pharmacological effect is contingent on the cellular microenvironment.

Furthermore, we performed a scratch wound healing assay to assess the influence of NETs on the migratory capacity of alveolar epithelial cells. Contrary to expectations, NETs stimulation impeded the wound closure rate compared with the control group, as indicated by a larger remaining scratch area (figure 3B,D). We hypothesise that this apparent inhibition of migration may be attributed to the cytotoxic effects of NET components, such as histones and proteases, which reduce cell numbers or overall viability, thereby masking any potential promigratory effect. Consistent with this interpretation, the CCK-8 assay confirmed that NETs treatment significantly reduced cell viability (figure 3E). Co-treatment with anlotinib mitigated this NETs-induced cytotoxicity, restored cell viability and consequently significantly enhanced the wound healing process.

Transcriptomic sequencing reveals that NETs drive fibrotic phenotypes through activation of the PI3K/Akt signalling pathway

To elucidate the molecular mechanisms underlying NETs-induced epithelial cell dysfunction, we performed RNA-seq transcriptomic analysis on A549 cells following NETs stimulation. Differential expression analysis (Volcano plot, figure 4A) identified 2007 significantly differentially expressed genes (DEGs), comprising 1066 upregulated and 941 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these DEGs were significantly enriched in biological processes such as inflammatory response, immune regulation, extracellular matrix organisation and cellular stress response (figure 4C). Notably, several key genes of the PI3K/Akt signalling pathway were significantly upregulated in the NETs-stimulated group, including IL3RA, CSF3 and PIK3CG (figure 4B). These genes are directly involved in the regulation of the PI3K/Akt pathway activation, providing transcriptomic evidence for the involvement of this pathway in NET-induced cellular responses. The KEGG pathway enrichment bubble plot further demonstrated that, compared with the control group, DEGs after NETs stimulation were predominantly enriched in fibrosis-related/inflammation-related pathways, including the PI3K-Akt signalling pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway, TNF signalling pathway, Janus kinase–signal transducer and activator of transcription signalling pathway and extracellular matrix (ECM)-receptor interaction (figure 4C). It is worth noting that the NF-κB pathway showed higher enrichment significance in the KEGG analysis; however, this is consistent with the known hierarchical regulatory relationship between signalling pathways—PI3K-Akt acts as an upstream key node that regulates the activation of downstream pathways such as NF-κB, and the signal amplification effect may lead to higher enrichment significance of downstream pathways in transcriptomic analysis.

Figure 4. Transcriptomic analysis reveals that NETs activate the PI3K/Akt signalling pathway in alveolar epithelial cells. (A) Volcano plot showing differentially expressed genes (DEGs) in alveolar epithelial cells with versus without NET stimulation. (B) Heatmap of upregulated DEGs involved in the regulation of the PI3K/Akt signalling pathway. (C) Bubble plot of KEGG pathway enrichment analysis of DEGs; the size of bubbles represents the number of DEGs and the colour represents the p value. (D) Heatmap of DEGs associated with the PI3K/Akt signalling pathway. (E) GO gene set enrichment analysis. (F) Sankey diagram depicting the regulatory relationships between transcription factors (TFs) and their target DEGs. (G) Distribution of differentially expressed TF families. (H) Protein-protein interaction (PPI) network of key DEGs (nodes represent proteins, edges represent interaction relationships). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; NETs, neutrophil extracellular traps; PI3K/Akt, phosphatidylinositol 3-kinase/protein Kinase B.

Heatmap visualisation of the DEGs indicated distinct expression patterns of 20 genes in the NETs group compared with the negative control group (figure 4D). A chord diagram of KEGG pathway enrichment illustrated a strong bias towards functions promoting inflammation, immune cell recruitment, extracellular matrix remodelling and cell migration among the upregulated genes in the NETs-stimulated group, indicating the establishment of a highly inflammatory microenvironment (figure 4E). A Sankey diagram (figure 4F) depicted the regulatory relationships between transcription factors (TFs) and their predicted target genes, implicating these TFs in processes such as cell proliferation, immune evasion/inflammatory response and aberrant cell differentiation. The family distribution of differentially expressed TFs (figure 4G) confirmed systematically that NET formation is not driven by individual TFs but results from the coordinated regulation of an entire transcriptional network. Specific TF families (eg, ETS, AP-1) were extensively activated, working in concert to coregulate downstream target genes related to inflammation, invasion and metabolism—as suggested by the Sankey diagram and DEG heatmap—thereby collectively shaping the malignant phenotype associated with NETs. The protein-protein interaction network (figure 4H) showed that this network was primarily dominated by genes from two major modules: inflammation (eg, TNF, TLRs) and invasion (eg, matrix metalloproteinase). This finding is highly consistent with and mutually validates the results from the prior KEGG pathway analysis (eg, NF-κB pathway, TNF pathway, ECM-receptor interaction).

Anlotinib suppresses PI3K/Akt pathway activation and ameliorates EMT progression in vivo

Based on the findings from in vitro transcriptomics, we sought to validate the regulatory effects of anlotinib on the PI3K/Akt pathway and EMT in an animal model. IHC and IF staining revealed that lung tissues from MAILD model mice exhibited significantly downregulated expression of the epithelial marker E-Ca and markedly upregulated expression of the mesenchymal marker α-SMA (figure 5A–F), confirming the occurrence of EMT in vivo. More importantly, compared with the model group, anlotinib treatment partially restored E-Ca expression and significantly suppressed α-SMA expression in the lung tissues of MAILD mice (figure 5A–F).

Figure 5. Anlotinib inhibits PI3K/Akt pathway activation and reverses EMT in the lungs of MAILD mice. (A) IHC staining of E-Ca and α-SMA in mouse lung tissues. (B) IF staining of E-Ca (green) and α-SMA (red) in mouse lung tissues; nuclei were counterstained with DAPI (blue). (C–D) Quantitative analysis of E-Ca (C) and α-SMA (D) positive areas in IHC staining (n=3). (E–F) Quantitative analysis of E-Ca (E) and α-SMA (F) fluorescence intensities in IF staining (n=3). (G) IHC staining of p-Akt and t-Akt in mouse lung tissues. (H) IF staining of p-Akt (red) and t-Akt (green) in mouse lung tissues; nuclei were counterstained with DAPI (blue). (I–J) Quantitative analysis of p-Akt (I) and t-Akt (J) positive areas in IHC staining (n=3). (K–L) Quantitative analysis of p-Akt (K) and t-Akt (L) fluorescence intensities in IF staining (n=3). Data are presented as mean±SD; *p<0.05, **p<0.01, ****p<.0001. AOD, average optical density; E-Ca, E-cadherin; IF, immunofluorescence; IHC, immunohistochemistry; MAILD, myositis-associated interstitial lung disease; MFI, mean fluorescence intensity; α-SMA, α-smooth muscle actin.

Concurrently, assessment of key signalling pathway proteins showed that p-Akt levels were significantly elevated in the lung tissues of the MAILD model group, while total Akt protein levels remained largely unchanged (figure 5G–L). Anlotinib treatment effectively inhibited Akt phosphorylation. These in-vivo data strongly demonstrate that anlotinib alleviates pulmonary fibrosis primarily by suppressing the aberrant activation of the PI3K/Akt signalling pathway and consequently reversing the EMT process.

Anlotinib alleviates NETs-induced EMT in vitro: mechanistic validation

Finally, we conducted in vitro mechanistic validation in the A549 cell line. Anlotinib intervention effectively inhibited NETs-induced Akt phosphorylation (p<0.01), partially restored E-Ca expression and reduced α-SMA expression (p<0.05) (figure 6A–F). IF staining visually confirmed these findings: after NETs stimulation, the fluorescence signal of E-Ca was weakened, while signals for α-SMA and p-Akt were enhanced. In contrast, anlotinib treatment restored the membrane localisation of E-Ca and attenuated the fluorescence signals of α-SMA and p-Akt.

Figure 6. Anlotinib inhibits NETs-induced EMT by suppressing the PI3K/Akt signalling pathway in A549 cells in vitro. (A) IF staining of E-Ca (green) and α-SMA (red) in A549 cells after NETs stimulation with or without anlotinib treatment; nuclei were counterstained with DAPI (blue). (B) IF staining of t-Akt (green) and p-Akt (red) in A549 cells; nuclei were counterstained with DAPI (blue). (C–D) Quantitative analysis of E-Ca (C) and α-SMA (D) fluorescence intensities in IF staining (n=3). (E–F) Quantitative analysis of t-Akt (E) and p-Akt (F) fluorescence intensities in IF staining (n=3). (G) Western blot analysis of protein levels of PI3K and p-PI3K; GAPDH was used as an internal reference. (H) Western blot analysis of protein levels of t-Akt and p-Akt; GAPDH was used as an internal reference. Data are presented as mean±SD; *p<0.05, **p<0.01, ***p<.0001. α-SMA, α-smooth muscle actin; E-Ca, E-cadherin; IF, immunofluorescence; MFI, mean fluorescence intensity; NETs, neutrophil extracellular traps.

Furthermore, western blot analysis (figure 6G,H) demonstrated that NETs stimulation significantly upregulated the levels of p-PI3K and p-Akt, while inducing characteristic EMT-related protein expression changes—specifically, downregulation of E-Ca and upregulation of α-SMA. These results are consistent with the transcriptomic data, confirming that NETs can activate the PI3K/Akt signalling pathway and induce EMT in alveolar epithelial cells, while anlotinib can block this process by inhibiting PI3K/Akt phosphorylation.

Discussion

This study systematically delineates the mechanism by which the multitarget TKI anlotinib ameliorates fibrosis in MAILD via the NETs–PI3K/Akt–EMT axis—a previously unreported pathogenic pathway in this disease. By integrating in vivo murine model data, in vitro epithelial cell experiments and transcriptomic profiling, we demonstrate that anlotinib exerts pleiotropic anti-fibrotic effects: attenuating pulmonary histopathological damage, inflammatory cell infiltration and collagen deposition, suppressing NETs formation, blocking PI3K/Akt pathway activation and reversing EMT. These findings not only provide a mechanistic rationale for repurposing anlotinib in MAILD—an orphan disease with limited therapeutic options and poor prognosis^{19 20}—but also uncover a novel link between NETs and EMT mediated by PI3K/Akt signalling in fibrotic lung disorders.

NETs have emerged as central mediators of tissue fibrosis and autoimmunity, with clinical relevance in anti-MDA5+dermatomyositis-associated ILD—where elevated circulating NETs correlate with disease severity, rapid progression and poor prognosis.^{21 22} Notably, this study has confirmed the abnormal activation status of NETs in the MAILD mouse model through multidimensional detection, and its changes are highly synchronised with the core pathological phenotypes of MAILD, indirectly supporting the driving role of NETs in MAILD phenotypes. On one hand, the expression of NETs-specific markers (MPO, Cit-H3) in the lung tissues of MAILD model mice was significantly increased, and IF confirmed that these markers existed in the form of ‘extracellular reticular structures’ (a characteristic feature of functional NETs), ruling out protein upregulation caused by simple neutrophil infiltration (figure 2A–E). On the other hand, the regions with high NETs expression highly overlapped with the regions of pulmonary inflammatory infiltration, collagen deposition and EMT occurrence. Moreover, after anlotinib intervention, the reduction in NETs levels was synchronised with the alleviation of pulmonary fibrosis and the reversal of EMT (figure 2B,D; figure 5C,D). These results indicate that the abnormal activation of NETs is not a concomitant phenomenon of MAILD, but may be involved in driving the inflammatory and fibrotic processes of MAILD, providing preliminary evidence for further in-depth verification of the causal role of NETs. Beyond their canonical role as inflammatory end-products, NETs act as DAMPs via components such as Cit-Hs and MPO, shaping the profibrotic microenvironment through paracrine signalling.¹⁸ In multiple fibrotic contexts (including IPF and systemic sclerosis), NETs have been shown to directly induce EMT in epithelial cells, a process dependent on downstream kinase signalling cascades.^{20 23} In the present study, we extend this paradigm by showing that NETs exert dual pathogenic effects on alveolar epithelial cells: acute cytotoxicity (impaired proliferation and migration) and chronic pro-fibrotic reprogramming (induction of EMT). This aligns with the ‘double-edged sword’ role of NETs in pulmonary pathology²⁴ and highlights their potential as a therapeutic target in MAILD. Notably, anlotinib partially reversed NETs-induced epithelial dysfunction, suggesting its ability to mitigate oxidative stress and inflammatory damage—complementing its known antiangiogenic and antiproliferative activities.^{16 17 19}

A key mechanistic insight from this work is the identification of NETs as an upstream activator of the PI3K/Akt signalling pathway in MAILD. The PI3K/Akt axis is a well-recognised central regulator of EMT, transducing signals from cytokines (eg, TGF-β), growth factors and DAMPs to drive epithelial dedifferentiation via TFs including Snail, Zeb1 and Twist.^{8 13} Notably, PI3K/Akt signalling serves as a critical node in fibrosis, driving not only EMT but also fibroblast proliferation, survival and extracellular matrix synthesis—core processes in fibrotic remodelling.²⁵ Recent studies in silicosis and bleomycin-induced pulmonary fibrosis have confirmed that PI3K/Akt activation is indispensable for NETs-induced fibroblast transdifferentiation and EMT,^{4 26} consistent with our observations in MAILD. Our transcriptomic and protein validation data confirm that NETs stimulation upregulates PI3K/Akt phosphorylation, accompanied by EMT-characteristic changes (downregulated E-Ca, upregulated α-SMA)—a phenomenon reversed by anlotinib. Intriguingly, KEGG enrichment analysis identified the NF-κB pathway as more significantly enriched than PI3K/Akt, which is consistent with the hierarchical regulatory network of signalling pathways: PI3K/Akt serves as an upstream node that modulates downstream cascades including NF-κB and TNF via IκBα phosphorylation and nuclear translocation.^{15 27} The signal amplification effect likely contributes to the more prominent enrichment of downstream pathways in transcriptomics, while functional validation (Western blot, IF) unequivocally confirms that PI3K/Akt activation is functionally critical for NETs-induced EMT in MAILD. Additionally, bioinformatic analyses revealed broad activation of NETs-regulated TF families (eg, ETS, AP-1), which may sustain fibrotic phenotypes through epigenetic modifications and direct transcriptional regulation—providing a potential secondary mechanism for future exploration.

The NETs-PI3K/Akt-EMT axis identified here is supported by a comprehensive evidence chain: (1) initiating event: extensive NET formation and accumulation in MAILD lung lesions (in vivo validation); (2) phenotypic consequence: NETs-induced EMT in alveolar epithelial cells (in vivo and in vitro confirmation); (3) signalling bridge: NETs-driven PI3K/Akt pathway activation (transcriptomic and protein-level validation); (4) therapeutic intervention: anlotinib simultaneously inhibits NETs formation, blocks PI3K/Akt phosphorylation, reverses EMT and alleviates pulmonary fibrosis—forming a tripartite regulatory effect that confirms the functional relevance of this axis. While the precise molecular link between extracellular NETs and intracellular PI3K/Akt activation remains to be fully elucidated, current literature supports a model wherein NETs components (eg, histones, HMGB1) bind to pattern recognition receptors (TLR2/4/9) or RAGE on epithelial cells, initiating downstream PI3K/Akt signalling.^{11 12 28} In breast cancer and hepatocellular carcinoma, NETs have been shown to activate PI3K/Akt via TLR4-RAGE heterodimerisation,^{22 23} a mechanism that may be conserved in MAILD. We propose this mechanism based on existing evidence, with future studies focusing on validating specific receptor-ligand interactions.

Beyond confirming the NETs-PI3K/Akt-EMT axis, our findings also highlight the multifaceted anti-fibrotic potential of anlotinib in MAILD. Anlotinib’s anti-fibrotic activity in MAILD is multifaceted, extending beyond its established inhibition of VEGFR, PDGFR and FGFR.^{16 17 20} Previous work showed that anlotinib attenuates pulmonary fibrosis by inhibiting TGF-β1-mediated fibroblast activation;²⁹ this aligns with the central role of TGF-β as a fibrosis driver, as demonstrated by clinical progress of TGF-β receptor I (ALK5) inhibitors in fibrotic diseases.²⁷ We build on this by demonstrating that anlotinib targets the epithelial compartment via PI3K/Akt-EMT suppression, reduces NETs formation and pro-inflammatory cytokine production (TNF-α, IL-6), and likely restrains myofibroblast activation through PDGFR/FGFR inhibition. Notably, anlotinib has also been shown to inhibit PI3K/Akt/mTOR signalling in non-small cell lung cancer and renal fibrosis,^{20 26} supporting the generality of its pathway-targeting activity. This multi-target action is particularly relevant for MAILD—a complex disease involving epithelial dysfunction, immune dysregulation and stromal remodelling. Notably, NETs-induced EMT has been reported in other fibrotic and malignant contexts (eg, hepatocellular carcinoma via TLR4/NF-κB),²⁸ highlighting the generality of NETs as pathogenic triggers and supporting the broader applicability of our findings. Compared with selective PI3K inhibitors—whose efficacy may be limited by subtype-specific dependencies and off-target toxicity²⁷—anlotinib offers translational advantages including a well-established safety profile, oral bioavailability and dual targeting of immune and stromal components—making it suitable for chronic fibrosis management.¹⁷

Several limitations of this study warrant consideration. First, the specific molecular mechanism by which anlotinib inhibits NETosis (eg, direct effects on neutrophil activation, ROS production or PAD4 activity) remains unclear and requires further investigation.^{10 12} Second, while we propose a receptor-mediated model for NETs-PI3K/Akt crosstalk, the specific receptors involved (eg, TLR4, RAGE) have not been validated in MAILD. Third, cell-specific contributions to the NETs-PI3K/Akt-EMT axis (eg, alveolar epithelial cells vs fibroblasts) require clarification using conditional knockout models (eg, epithelial-specific Akt knockout mice). Fourth, our findings are based on preclinical models, and prospective clinical trials are needed to validate anlotinib’s efficacy in MAILD patients—particularly those with high NETs levels or PI3K/Akt activation.

Future directions should address these gaps: (1) investigate the direct effect of anlotinib on neutrophil function (eg, NETosis, ROS production, PAD4 expression); (2) validate NETs receptors (TLR2/4/9, RAGE) and their role in PI3K/Akt activation using receptor-specific inhibitors or knockout models; (3) employ single-cell RNA sequencing to map lung cell heterogeneity and NETs-induced transcriptional remodelling in MAILD; (4) explore combination regimens of anlotinib with immunosuppressants (eg, tacrolimus, rituximab) to synergistically target immune and fibrotic pathways; (5) conduct phase II clinical trials to evaluate anlotinib’s efficacy in MAILD, with NETs markers (MPO, Cit-H3) and PI3K/Akt phosphorylation as predictive biomarkers.

In conclusion, our study identifies the NETs-PI3K/Akt-EMT axis as a novel pathogenic mechanism in MAILD and highlights anlotinib’s potential as a targeted therapy for this devastating disease. By simultaneously modulating epithelial function, immune responses and stromal remodelling, anlotinib addresses the multifactorial nature of MAILD—offering a promising translational strategy for fibrotic lung disorders driven by NETs and PI3K/Akt activation.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.