Synergistic Neuroprotection of Combined Shenmai and Hypothermia in Cerebral Ischemia-Reperfusion Injury via ERK-Dependent Anti-inflammatory Effects.

Cerebral ischemia-reperfusion (I/R) injury remains a major challenge in patients with ischemic stroke undergoing endovascular thrombectomy (EVT). Although selective intraarterial hypothermia has shown neuroprotective potential, its therapeutic efficacy is limited, highlighting the need for effective pharmacological adjuncts. This study investigated whether combining intracarotid hypothermia with Shenmai could synergistically enhance neuroprotection against cerebral I/R injury. Cold Shenmai (4 °C) or saline was infused into rat brain. Systemic toxicity was assessed by body weight, serum biochemistry, organ morphology, and indices. Brain toxicity was evaluated with 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin (H&E), and Fluoro-Jade B (FJB) staining. Cerebral I/R injury was induced by middle cerebral artery occlusion (MCAO). Neuroprotection was assessed by TTC staining, neurological deficit score, rotarod, adhesive removal, and by H&E, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Nissl staining. RNA sequencing explored mechanisms. Inflammatory cytokines were quantified by quantitative real-time polymerase chain reaction (qRT-PCR), while extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-κB) signaling were examined by Western blot and immunofluorescence. The ERK inhibitor PD98059 verified mechanistic contributions. Cold Shenmai infusion showed no evidence of systemic or cerebral toxicity. Compared with cold saline, it significantly reduced infarct volume, improved neurological function and behavioral outcomes, and attenuated neuronal damage. Transcriptomic analysis revealed downregulation of pro-inflammatory pathways and reduced expression of microglial activation-related signaling. Mechanistically, cold Shenmai enhanced ERK1/2 phosphorylation, which was associated with reduced microglial marker expression and suppressed NF-κB P65 nuclear translocation. Importantly, these protective effects were markedly attenuated by the ERK1/2 inhibitor PD98059, indicating that ERK signaling plays a critical role in mediating the anti-inflammatory and neuroprotective effects of cold Shenmai during cerebral I/R injury. Intracarotid Shenmai with hypothermia synergistically attenuates cerebral I/R injury through ERK-mediated anti-inflammatory effects, including reduced microglial activation markers. These findings provide mechanistic preclinical evidence supporting Shenmai as an effective pharmacological adjunct to intraarterial hypothermia and suggest a promising therapeutic approach for mitigating reperfusion injury following EVT in acute ischemic stroke.