After Traumatic Brain Injury, EPHA4 Enhances Endoplasmic Reticulum Stress to Promote M1 Microglial Polarization Through the MAPK Signaling Pathway

Background: Traumatic brain injury (TBI) is an important cause of disability and death worldwide. The development of neuroinflammation after TBI is related to the brain parenchyma. M1-type microglia play important roles in this process, but the specific mechanism through which regulating microglia M1 polarization is still not fully understood. This study aimed to investigate the role of Ephrin Receptor A4 (EphA4) in the M1 polarization of microglia after TBI.

Methods: A TBI rat model was established by the controlled cortical impact (CCI) method, and M1 polarization of GMI-R1 cells was induced by lipopolysaccharide (LPS) treatment. Target genes associated with the progression of TBI were screened by transcriptome sequencing; the expression of key genes and proteins was detected by real-time quantitative PCR (RT‒qPCR), Western blot, ELISA, and immunofluorescence, and the damage of the rat brain tissue and the blood-brain barrier (BBB) was evaluated by hematoxylin‒eosin (HE) staining and Evans blue staining.

Results: This study revealed that EphA4 expression is upregulated in the brain tissue of TBI rats and that treatment with its inhibitor, KYL peptide, can improve the progression of TBI. KYL peptide intervention downregulated the levels of the proinflammatory cytokines and upregulated the levels of the anti-inflammatory cytokines and inhibited the M1 polarization of microglia. The levels of the endoplasmic reticulum stress (ERS)-related proteins and CA²⁺ were upregulated in TBI rats and downregulated after KYL peptide treatment but subsequently increased after 123C4 treatment. Our results showed that EphA4 promoted the M1 polarization of microglia by enhancing ERS. Notably, mitogen-activated protein kinase (MAPK) signaling was significantly enriched in TBI. In vitro studies revealed that LPS treatment promoted the activation of MAPK signaling in GMI-R1 cells. A mechanistic study revealed that EphA4 may activate ERS by activating the MAPK signaling pathway and promote M1 polarization of microglia after TBI.

Conclusion: Our study revealed that the EphA4/MAPK axis may be a key regulatory factor in controlling microglial M1 polarization during brain injury. Blocking this signaling axis may represent a potential therapeutic approach for improving TBI.

EPHA4; M1 polarization of microglia; MAPK signaling pathways; MI-5595023; endoplasmic reticulum stress; traumatic brain injury.