Journal of orthopaedic translation · 2026
The PTBP1-EZH2-DOCK2 axis promotes M1 microglial polarization and exacerbates neuronal apoptosis following spinal cord injury.
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Sha Chunhe, Pan Feng, Liu Xiaodong, Wang Zhiqing, Dai Hongbin, Huang Kai
Аннотация
OBJECTIVE: Microglial M1 polarization plays a critical role in secondary injury following spinal cord injury (SCI) by promoting the release of inflammatory factors and inducing neuronal apoptosis. This study aims to investigate the regulatory mechanism of the polypyrimidine tract-binding protein 1 (PTBP1)-enhancer of zeste homolog 2 (EZH2)-dedicator of cytokinesis 2 (DOCK2) axis in microglial polarization and to analyze its impact on neuronal apoptosis, thereby uncovering potential mechanisms underlying SCI pathophysiology.
METHODS: A SCI rat model was established, with expression regulated via lentiviral infection. Motor function was evaluated using behavioral assessments, while hematoxylin and eosin (HE) staining, immunofluorescence (IF), co-staining of TUNEL/NeuN, Nissl staining, and Western blotting (WB) were performed to evaluate spinal cord pathology, microglial polarization, inflammation, and neuronal apoptosis, respectively. In HMC3 cells, flow cytometry, IF, WB, actinomycin D treatment, RIP-qPCR, ChIP-qPCR, and dual-luciferase reporter assays were utilized to investigate the regulatory interactions among PTBP1, EZH2, and DOCK2 and their impacts on microglial polarization. Co-culture of HMC3 with SH-SY5Y cells were conducted to evaluate the effect of microglial polarization on neuronal apoptosis.
RESULTS: In both SCI rats and lipopolysaccharide (LPS)-treated HMC3 cells, expression of PTBP1, EZH2, and DOCK2 was significantly upregulated. Mechanistic analysis revealed that PTBP1 enhanced EZH2 mRNA stability, increasing EZH2 expression. Moreover, EZH2 promoted DOCK2 transcription in manner independent of the polycomb repressive complex 2 (PRC2), histone methylation, and DNA methylation. Inhibition of the PTBP1-EZH2-DOCK2 axis promoted the shift of HMC3 cells toward M2 polarization. Restoration of the M1/M2 microglial balance attenuated neuronal apoptosis and inflammatory responses. In vivo studies further confirmed that inhibiting this pathway ameliorated motor function and facilitated SCI recovery.
CONCLUSION: PTBP1 enhances EZH2 mRNA stability to upregulate DOCK2 transcription, driving microglial polarization toward the M1 phenotype. This mechanism aggravates neuronal apoptosis and inflammation, ultimately contributing to the progression of SCI pathology.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study reveals the PTBP1-EZH2-DOCK2 axis as a potential molecular basis for intervening in aberrant microglial polarization and developing novel therapeutic targets for SCI. This research possesses significant potential for clinical translation.
METHODS: A SCI rat model was established, with expression regulated via lentiviral infection. Motor function was evaluated using behavioral assessments, while hematoxylin and eosin (HE) staining, immunofluorescence (IF), co-staining of TUNEL/NeuN, Nissl staining, and Western blotting (WB) were performed to evaluate spinal cord pathology, microglial polarization, inflammation, and neuronal apoptosis, respectively. In HMC3 cells, flow cytometry, IF, WB, actinomycin D treatment, RIP-qPCR, ChIP-qPCR, and dual-luciferase reporter assays were utilized to investigate the regulatory interactions among PTBP1, EZH2, and DOCK2 and their impacts on microglial polarization. Co-culture of HMC3 with SH-SY5Y cells were conducted to evaluate the effect of microglial polarization on neuronal apoptosis.
RESULTS: In both SCI rats and lipopolysaccharide (LPS)-treated HMC3 cells, expression of PTBP1, EZH2, and DOCK2 was significantly upregulated. Mechanistic analysis revealed that PTBP1 enhanced EZH2 mRNA stability, increasing EZH2 expression. Moreover, EZH2 promoted DOCK2 transcription in manner independent of the polycomb repressive complex 2 (PRC2), histone methylation, and DNA methylation. Inhibition of the PTBP1-EZH2-DOCK2 axis promoted the shift of HMC3 cells toward M2 polarization. Restoration of the M1/M2 microglial balance attenuated neuronal apoptosis and inflammatory responses. In vivo studies further confirmed that inhibiting this pathway ameliorated motor function and facilitated SCI recovery.
CONCLUSION: PTBP1 enhances EZH2 mRNA stability to upregulate DOCK2 transcription, driving microglial polarization toward the M1 phenotype. This mechanism aggravates neuronal apoptosis and inflammation, ultimately contributing to the progression of SCI pathology.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study reveals the PTBP1-EZH2-DOCK2 axis as a potential molecular basis for intervening in aberrant microglial polarization and developing novel therapeutic targets for SCI. This research possesses significant potential for clinical translation.