1. Academic Validation
  2. Single-cell sequencing and transcriptomic data reveal that P65 activation significantly promotes microglia-mediated neuroinflammation after ischemic stroke

Single-cell sequencing and transcriptomic data reveal that P65 activation significantly promotes microglia-mediated neuroinflammation after ischemic stroke

  • Sci Rep. 2025 May 22;15(1):17760. doi: 10.1038/s41598-025-00524-2.
Ruiyu Wang # 1 Yilun Qian # 1 Xinchen Zhou # 2 Hang Xu 3 Yu Wang 1 Yao Geng 4 Tianshu Wang 5 Binxiu Sha 6
Affiliations

Affiliations

  • 1 The First Affiliated Hospital With Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of China.
  • 2 School of Nursing and Rehabilitation, Nantong University, Nantong, Jiangsu Province, People's Republic of China.
  • 3 Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of China.
  • 4 The First Affiliated Hospital With Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of China. gengyao19970821@163.com.
  • 5 The First Affiliated Hospital With Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of China. wts15951928785@gmail.com.
  • 6 The First Affiliated Hospital With Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of China. njsbx2011@163.com.
  • # Contributed equally.
Abstract

The pathophysiological mechanisms underlying cerebral ischemia-reperfusion (I/R) injury are highly complex. Previous studies have indicated phenotypic changes in various cell types following stroke but have failed to identify the key regulatory genes and cell subtypes associated with the disease. The study utilized five datasets: GSE227651, GSE104036, GSE116878, GSE249957, and GSE22255. The Seurat pipeline was employed for standard quality control and single-cell data analysis. Monocle2 and CytoTRACE were used for trajectory analysis, while Mfuzz was applied to identify time-series gene expression patterns. Middle cerebral artery occlusion (MCAO) mice served as the animal model for cerebral I/R injury, and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated BV2 cells were used to simulate microglial phenotypic changes following ischemia-reperfusion. qPCR, Western blotting, and immunofluorescence staining were used to detect key gene and protein alterations. P65 was identified as a key transcription factor driving inflammatory responses and transcriptional changes following ischemic stroke. Two microglial subtypes, CX3CR1 + and Cdk1+, were identified, with their proportions significantly increasing on days 1 and 3 after MCAO. Increased levels of inflammation, neuronal Apoptosis, and P65 phosphorylation in microglia were observed in both the MCAO animal model and OGD/R cell model. Notably, inhibition of P65 phosphorylation effectively suppressed the progression of inflammation during cerebral I/R injury. We identified microglial subtypes associated with inflammatory responses following cerebral ischemia-reperfusion injury, with their proportions increasing post-injury. P65 was confirmed as a critical regulator of the inflammatory response, contributing to neuronal protection and the restoration of neurological function.

Keywords

Middle cerebral artery occlusion; Single cell; Stroke; Transcription factor.

Figures
Products