This study aims to elucidate the pathogenesis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD) and develop novel therapeutic strategies. RA-ILD is a leading cause of mortality in patients with RA, and currently lacks effective intervention measures. The study focuses on the pathological evolution of lung-resident mesenchymal stem cells (LR-MSCs) in RA-ILD and their role in regulating the microenvironment, as well as exploring the reparative potential of menstrual blood-derived endometrial stem cells (MenSCs) and their extracellular vesicles (EVs).  

Specific research objectives include:  

1. Constructing an atlas of pathological subpopulations of LR-MSCs: Using spatial transcriptomics technology to map the spatiotemporal evolution of LR-MSCs in RA-ILD and identify key pathological subpopulations driving disease progression.  

2. Elucidating the mechanism of LR-MSC-mediated microenvironmental imbalance: Analyzing the abnormal interaction network between pathological subpopulations of LR-MSCs and alveolar epithelial cells/immune cells, and revealing the molecular pathways by which they drive the "immunity-fibrosis" vicious cycle.  

3. Validating the therapeutic effects of MenSCs/EVs: Through animal models and organoid systems, to confirm whether MenSCs and their EVs can restore pulmonary microenvironment homeostasis by remodeling pathological subpopulations of LR-MSCs, providing a basis for clinical translation.  

Starting from the new perspective of "stem cell niche imbalance," this study aims to provide theoretical foundations and regenerative intervention strategies for targeted therapy of RA-ILD.

This project adopts a multidisciplinary integrated approach, combining clinical cohorts, animal models, molecular biology, and organoid technologies. The research protocol is divided into four core components.  

1. Clinical Samples and Animal Models

Clinical Cohort: Relying on the RA-ILD patient cohort in Henan Province (n=1889), lung tissue, bronchoalveolar lavage fluid (BALF), and peripheral blood samples were collected from patients with mild, moderate, and severe disease. Samples were obtained via bronchoscopic lung biopsy and used for spatial transcriptomics and multiplex immunofluorescence analysis.  

Animal Model: An RA-ILD model was induced in SKG mice via intraperitoneal injection of zymosan combined with LPS aerosol inhalation. Disease progression was dynamically observed at 4 weeks (mild), 10 weeks (moderate), and 16 weeks (severe).  

2. Identification of Pathological Subpopulations of LR-MSCs and Construction of Spatial Atlas

Spatial Transcriptomic Sequencing: Lung tissue samples were embedded in OCT and sectioned into frozen slices. mRNA was captured using a chip with spatial barcodes to construct a spatiotemporal gene expression atlas.  

Cell Sorting and Identification: LR-MSCs were sorted by flow cytometry (human markers: CD73+CD90+CD105+; mouse markers: Sca-1+CD90+) and subjected to transcriptome sequencing and functional validation (e.g., immunomodulation, secretome analysis).  

3. Study on Intervention Mechanisms of MenSCs/EVs

Preparation of MenSCs and EVs: MenSCs were isolated from menstrual blood, and EVs were collected via serum-free culture, purified using the NanoEX system, and characterized for particle size via nanoparticle flow cytometry.  

In Vivo and In Vitro Experiments:  

Animal Intervention: MenSCs or aerosolized MenSC-EVs were administered via tail vein injection to evaluate lung function, inflammatory factors, and fibrosis degree.  

Organoid Model: Lung organoids were constructed using patient BALF and co-cultured with MenSCs. Cell interaction networks were analyzed via single-cell sequencing.  

4. Data Analysis and Statistics

Statistical analysis was performed using GraphPad Prism 8, with inter-group comparisons using ANOVA or Tukey tests (significance level P<0.05). Multi-omics data were integrated using tools such as CellChat and NicheNet to construct regulatory networks.

1. Expected Research Results  

Atlas of Pathological Subpopulations of LR-MSCs: Clarify the spatial distribution characteristics of LR-MSCs in RA-ILD and their correlation with disease severity, and screen 3–5 key marker molecules (such as specific chemokines or transcription factors).  

Elucidation of the Mechanism of Microenvironmental Imbalance: Reveal that pathological subpopulations of LR-MSCs drive alveolar epithelial apoptosis and macrophage polarization through aberrant paracrine signaling (e.g., TGF-β, IL-6), activating the NF-κB and Smad signaling pathways.  

Therapeutic Efficacy of MenSCs/EVs: In animal models, MenSC transplantation is expected to reduce the lung inflammation score by over 30% and delay fibrosis progression; aerosolized EVs can restore alveolar barrier integrity.  

2. Support from Preliminary Research Foundation  

The project team has accumulated key preliminary results:  

The RA-ILD cohort shows that advanced age and high disease activity (DAS-28 > 3.7) are risk factors.  

MenSCs and their EVs significantly inhibit inflammation (e.g., via the miR-671-5p/AAK1 axis) and fibrosis in lung injury models.

This project proposes an innovative hypothesis: the progression of RA-ILD depends on alveolar niche imbalance dominated by pathological subpopulations of LR-MSCs, while exogenous MenSCs and their EVs can restore microenvironment homeostasis through targeted remodeling of these subpopulations. From the perspective of the stem cell niche, the study will reveal new mechanisms underlying RA-ILD and lay the foundation for developing MenSC-based pulmonary regenerative therapies.  

The expected outcomes will not only deepen the understanding of autoimmune lung diseases but also hold promise for clinical translation, providing precise intervention strategies for RA-ILD patients. Ultimately, this study will establish the therapeutic paradigm of "exogenous stem cells regulating endogenous stem cells" and promote the application of cell therapy in refractory lung diseases.