Effects and underlying mechanisms of novel phenothiazine derivative DA414 in rats with intracerebral hemorrhage

To explore the efficacy and action mechanism of a novel phenothiazine derivative, DA414, in rats with intracerebral hemorrhage (ICH).

Male Sprague-Dawley (SD) rats (aged 8~10 weeks, weighing 250~300 g) were randomly divided into sham operation, model, and low and high dose DA414 groups [5 and 10 mg/(kg·d)], with 20 animals per group. The size and extent of injury in the ICH area were measured by magnetic resonance imaging (MRI) and histopathological slices. Neurological function was evaluated with a behavioral grading system. Western blotting was used to detect the expression of ferroptosis-related factor, glutathione peroxidase 4 (GPX4). Multiplex immunohistochemistry was employed to quantitatively evaluate microglial activation in perihematomal tissue. RT-qPCR was applied to measure the mRNA expression levels of NLRP3 inflammasome components (NLRP3, Caspase-1, IL-1β), pro-inflammatory (IL-18, TLR4, IL-6, TNF-α) and anti-inflammatory cytokines (IL-4, IL-10). The integrity of the blood-brain barrier (BBB) was assessed by Evans blue staining, and the biosafety of DA414 for the liver, kidneys and heart was assessed by HE staining and clinical biochemical tests.

DA414 significantly promoted the absorption of hematoma, reduced neuronal injury, and improved neurological function scores. DA414 significantly up-regulated the ferroptosis regulatory factor GPX4 (P<0.01), and also significantly inhibited the activation of microglia after ICH (P<0.001). RT-qPCR indicated that DA414 treatment resulted in downregulation of mRNA expression in the inflammasome pathway (NLRP3, Caspase-1, IL-1β, all P<0.01) and pro-inflammatory cytokines (TLR4, IL-6, both P<0.05; IL-18, TNF-α, both P<0.01) and up-regulation of anti-inflammatory cytokines (IL-4, IL-10, both P<0.05), suggesting that DA414 exerts its neuroprotective effect probably by regulating ferroptosis and inflammation. Safety assessment revealed that DA414 had no significant effect on BBB integrity or damage to the liver, kidneys, and heart in rats.

DA414 exerts significant neuroprotective effects in ICH model by targeted inhibition for ferroptosis and modulating inflammatory response. Our study provides an experimental foundation for ICH treatment.