Electroacupuncture modulates P300-mediated STAT6/PPARγ binding to promote M2 polarization of microglia following cerebral ischemic injury.

Abstract

Microglia-mediated neuroinflammation is a key determinant of ischemic brain injury. Electroacupuncture (EA) is known to promote M2 polarization of microglia by upregulating Signal Transducer and Activator of Transcription 6 (STAT6) / Peroxisome Proliferator-Activated Receptor γ (PPARγ), however, the epigenetic mechanism governing the transcriptional stability and recruitment efficiency of this pathway remain poorly understood. We hypothesized that the transcriptional coactivator E1A-binding protein P300 (P300) may serve as a functional partner involved in facilitating this process. To test this, we established a middle cerebral artery occlusion (MCAO) model in mice and evaluated the neuroprotective effects of EA through laser speckle blood flow imaging, gait analysis, and triphenyl tetrazolium chloride (TTC) and Hematoxylin and Eosin (HE) staining. The effects of EA on microglial neuroinflammation were verified via transmission electron microscopy (TEM), immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA). Furthermore, we utilized Western Blot (WB), co-Immunoprecipitation (Co-IP), chromatin immunoprecipitation followed by quantitative polymerase chain reaction (CHIP-qPCR), and CD68 promoter-driven adeno-associated virus (AAV) with P300 shRNA knockdown technology to deeply explore the core mechanism by which P300 regulates the assembly of the STAT6/PPARγ transcriptional complex during EA treatment. Results demonstrated that EA significantly restored cerebral blood flow, reduced infarct volume, and restore neurological function. Moreover, local knockdown of P300 in the ischemic penumbra via CD68 promoter driven AAV significantly attenuated the EA-induced M2 transition. Mechanistically, EA promoted the functional recruitment of P300 to STAT6, which was associated with the enhanced assembly of the STAT6/PPARγ complex on target promoters. In summary, EA promotes M2 polarization by enhancing the stability of the P300 associated STAT6/PPARγ transcriptional complex, providing a novel epigenetic intervention target for ischemic stroke treatment.