Analyzing the Spatiotemporal Regulatory Mechanism of Ferroptosis-Related GPX4 in TBI Based on Single-Cell and Spatial Transcriptome Sequencing

Introduction: TBI is a major global public health issue, and its secondary injury involves multiple pathological processes. In recent years, ferroptosis has attracted increasing attention for its role in TBI. This study aims to systematically analyze the spatiotemporal regulatory mechanism of GPX4 in TBI using single-cell sequencing and spatial transcriptome sequencing technologies.

Methods: Sequencing was performed using the 10×Genomics and Visium platforms. The data were integrated, and the expression of GPX4 was analyzed in combination with ferroptosis-related databases. At the cellular level: Primary neurons were cultured; GPX4 expression was regulated using siRNA and lentivirus; ferroptosis was induced by Erastin or RSL3; and cell viability and ferroptosis markers were detected. At the animal level: AAV was injected via stereotaxis to regulate GPX4 in the injured area, followed by behavioral evaluations and histological analyses. Co-IP was used to explore the molecular interaction mechanism of GPX4.

Results: Single-cell sequencing identified dynamic changes in cell subsets after TBI, and differential expression of ferroptosis-related genes was observed in all subsets. Spatial transcriptome sequencing revealed the specific distribution of these genes in the injured area. Integrated analysis showed that GPX4 expression changed significantly in the injured area, with obvious spatiotemporal specificity in neurons. Cellular experiments confirmed that GPX4 silencing exacerbated damage caused by ferroptosis inducers and increased lipid peroxidation levels, while GPX4 overexpression alleviated ferroptosis. Animal experiments indicated that regulating GPX4 significantly affected the recovery of neurological function and cognitive ability in TBI mice.

Conclusion: This study is the first to integrate the two sequencing technologies, revealing the spatiotemporal regulatory pattern of GPX4 in TBI and its cell-specific role. It confirms that GPX4 plays an important role in TBI-induced secondary injury, affects ferroptosis by regulating iron metabolism, and provides a new approach for TBI treatment—with significant theoretical and clinical value.