Scalp acupuncture modulates RIP1/RIP3/MLKL-mediated necroptosis and neuroinflammation after intracerebral hemorrhage in male rats.

BACKGROUND

Secondary brain injury following intracerebral hemorrhage (ICH) is largely driven by regulated cell death and neuroinflammation. Necroptosis, a form of programmed necrotic cell death mediated by the RIP1/RIP3/MLKL pathway, has emerged as a key contributor to tissue injury after ICH; however, effective strategies for its modulation remain limited.

OBJECTIVE

This study aimed to determine whether RIP1/RIP3/MLKL-dependent necroptosis is functionally activated after ICH and to investigate whether scalp acupuncture (SA) modulates this pathway, using the necroptosis inhibitor necrostatin-1 (Nec-1) as a mechanistic comparator.

METHODS

A total of 168 adult male Sprague-Dawley rats were randomly assigned to sham, ICH, SA + ICH, and Nec-1 + ICH groups. Neurological function, necroptotic cell death, ultrastructural alterations, necroptosis-related signaling molecules, inflammatory mediators, and brain edema were systematically evaluated using behavioral assessments, propidium iodide staining, immunofluorescence, transmission electron microscopy, Western blotting, RT-qPCR, ELISA, and wet/dry weight analysis.

RESULTS

Following ICH, multiple independent assays consistently demonstrated marked activation of necroptosis in perihematomal brain tissue, including increased PI-positive cells, RIP1/RIP3 colocalization, mitochondrial structural disruption, and upregulation of RIP1, RIP3, MLKL, PGAM5, DRP1, and HMGB1. Both SA and Nec-1 significantly attenuated necroptosis-related signaling and were associated with improved neurological outcomes. Notably, compared with Nec-1, SA exerted stronger inhibitory effects on proinflammatory cytokine release and brain edema.

CONCLUSION

These findings support a prominent role for RIP1/RIP3/MLKL-mediated necroptosis in secondary injury after ICH in male rats. SA effectively attenuates necroptosis-related signaling and associated neuroinflammation, highlighting its potential as a multimodal biological intervention. Given that only male animals were included, further studies incorporating female subjects are warranted to determine whether sex-specific differences influence necroptotic signaling or therapeutic responsiveness.