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Comprehensive analysis of autophagy status and its relationship with immunity and inflammation in ischemic stroke through integrated transcriptomic and single-cell sequencing

Genes & Immunity volume 26, pages 111–123 (2025)Cite this article

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Abstract

Ischemic stroke (IS) significantly impacts patients’ health and quality of life, with the roles of autophagy and autophagy-related genes in IS still not fully understood. In this study, IS datasets were retrieved from the GEO database. Autophagy-related genes(ARGs) were identified and screened for differential expression. A prediction model was constructed using machine learning algorithm. WGCNA was employed to analyze differential regulation modules among different clusters of stroke patients. The analysis results were validated using single-cell sequencing data. Finally, autophagy hub genes were validated in an external cohort and an IS mouse model. We observed suppressed autophagy states in IS patients. A diagnostic model with good clinical efficacy for stroke diagnosis was constructed based on the selected key genes (AUC = 0.87). Consensus clustering identified two IS subtypes with distinct gene expression patterns and immune cell infiltration. scRNA-seq data analysis confirmed downregulation of pexophagy in IS. CellChat analysis identified key signaling pathways and intercellular interactions related to pexophagy. Validation in an external cohort and IS mouse model confirmed differential gene expression, supporting the involvement of pexophagy in IS pathogenesis. The identified key genes, molecular subtypes, and cellular interactions provide a foundation for further research into targeted therapies and precision medicine approaches for IS patients.

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Fig. 1
Fig. 2: Integration and analysis of different RNA-seq data.
Fig. 3: Differential analysis of gene expression.
Fig. 4: Identification of key genes and model construction.
Fig. 5: Correlations between hub genes and immune cells and inflammatory factors.
Fig. 6: Clustering of samples and clinical significance analysis.
Fig. 7: WGCNA weighted gene co-expression network analysis.
Fig. 8: Distribution of pexophagy-related genes in IS based on single-cell analysis.
Fig. 9: Cell trajectory analysis and cell communication analysis.
Fig. 10: Validation of expression of the hub pexophagy-related genes.

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Data availability

The data underlying this article are available in the GEO (https://www.ncbi.nlm.nih.gov/geo/) database.

Code availability

The code used to generate the results central to this study is available upon request.

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Funding

The study was supported by the National Natural Science Foundation of China (82072159), Specialized Capacity Building Project ((2021)79), and Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2022058).

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Author notes

  1. These authors contributed equally: Xiaole Zhu, Zhongman Zhang, Yi Zhu.

Authors and Affiliations

  1. Department of Emergency, Jiangsu Province Hospital and The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China

    Xiaole Zhu, Zhongman Zhang, Yi Zhu, Yanlong Chen, Wei Li & Xufeng Chen

  2. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Xiaole Zhu

  3. Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing, China

    Huae Xu

Authors
  1. Xiaole Zhu
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  2. Zhongman Zhang
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Contributions

Xufeng Chen and Huae Xu designed and managed the study. Yi Zhu and Wei Li responsible for designing and developing the research methods and experiments in the study. Xiaole Zhu and Zhongman Zhang conducted the statistical analysis, data interpretation, Yanlong Chen drew conclusions based on the collected data.

Corresponding author

Correspondence to Xufeng Chen.

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The authors declare no competing interests.

Ethics approval

We confirm that all methods in this study were performed in accordance with the relevant guidelines and regulations. Animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval No. IACUC-2408015), and all procedures strictly followed institutional and international ethical guidelines for animal research.

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Zhu, X., Zhang, Z., Zhu, Y. et al. Comprehensive analysis of autophagy status and its relationship with immunity and inflammation in ischemic stroke through integrated transcriptomic and single-cell sequencing. Genes Immun 26, 111–123 (2025). https://doi.org/10.1038/s41435-025-00320-y

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