Sonic Hedgehog Protects Against Ischemic Brain Injury Via Modulating DRP1-Dependent Microglial Pyroptosis

Abstract

Background

Microglial pyroptosis plays a critical role in the neuroinflammation following ischemic stroke. Although Sonic Hedgehog (SHH) signaling has been shown to exert neuroprotective effects in cerebral ischemia, whether SHH regulates microglial pyroptosis and the underlying mechanisms remain largely unclear.

Methods

Serum samples from patients with acute ischemic stroke (AIS) and healthy controls were analyzed using ELISA. Middle cerebral artery occlusion/reperfusion (MCAO/R) mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated BV2 microglia were established as in vivo and in vitro ischemic models, respectively. SHH and DRP1 expression were manipulated using adeno-associated virus (AAV)-mediated knockdown or siRNA transfection. Pyroptosis-related molecules were evaluated by qRT-PCR, western blotting, immunofluorescence staining, and scanning electron microscopy. Infarct volume and neurological function were assessed by TTC staining, modified neurological severity score (mNSS), and corner test.

Results

Serum SHH levels were significantly reduced in AIS patients and negatively correlated with circulating inflammatory cytokines. Separately, GEPIA2 analysis of human brain tissue datasets revealed that SHH expression was negatively correlated with the pyroptosis-related genes IL1B, GSDMD, and CASP1. Both in vivo and in vitro ischemic interventions markedly upregulated the expression of SHH, DRP1, and pyroptosis-associated proteins in microglia. SHH knockdown elevated DRP1 expression, aggravated microglial pyroptosis, enlarged cerebral infarct volume, and worsened neurological dysfunction. Rescue experiments confirmed that DRP1 knockdown effectively reversed the pro-pyroptotic and injury-aggravating effects of SHH inhibition in microglia after ischemic stroke.

Conclusions

SHH protects against cerebral ischemic injury by suppressing DRP1-mediated microglial pyroptosis. The SHH–DRP1 signaling axis represents a previously unrecognized regulatory pathway involved in ischemic stroke and may serve as a potential therapeutic target for limiting neuroinflammation and improving neurological outcomes.