An-Gong-Niu-Huang-Wan ameliorates neurovascular uncoupling and facilitates recovery after cerebral ischemic stroke by mediating P2X7 receptor inhibition.

BACKGROUND: Neurovascular uncoupling is a critical yet underrecognized pathological mechanism that exacerbates neurological injury after cerebral ischemic stroke. An-Gong-Niu-Huang-Wan (AGNHW), a well-established traditional Chinese formula, has been clinically utilized for stroke management for over two centuries owing to its multi-target neuroprotective properties. However, its potential to ameliorate impaired neurovascular coupling following ischemic insult remains largely unexplored.

PURPOSE: This study aimed to elucidate the therapeutic mechanism of AGNHW in restoring neurovascular coupling, with a focus on the purinergic P2X7 receptor (P2X7R) signaling pathway.

METHODS: A distal middle cerebral artery occlusion model was induced in male C57BL/6 mice. AGNHW was administered intragastrically 1 hour post-occlusion. Cortical neurovascular coupling was evaluated using laser speckle contrast imaging during whisker stimulation. Functional recovery was assessed via gait analysis and grip strength, while tissue injury was examined through hematoxylin and eosin staining. Neuronal activity, monitored in rAAV2/9-CaMKIIα-GCaMP6s mice, and arterial hemodynamics were visualized using two-photon microscopy during functional hyperemia. RNA sequencing was employed to identify potential mechanistic targets of AGNHW, followed by molecular biology techniques to investigate the role of purinergic signaling, particularly P2X7R-mediated regulation of neurovascular coupling.

RESULTS: Compared with the control group, AGNHW treatment significantly enhanced the cerebral blood flow response and increased the area under the curve during whisker stimulation, accompanied by alleviated neuronal damage and improved motor function-reflected by prolonged stance time and elevated peak pressure in the affected forelimb and enhanced grip strength. AGNHW also markedly increased the signal intensity of calcium activity (ΔF/F₀) following ischemia. Furthermore, AGNHW improved arterial hemodynamics by promoting arterial dilation, increasing red blood cell velocity, and elevating red blood cell flux during functional hyperemia. It also ameliorated blood-brain barrier integrity and upregulated the expression of tight junction proteins (ZO-1, occludin, claudin5). Mechanistically, AGNHW downregulated the genes expression of P2rx7, P2ry12, and Adora2a, and inhibited P2X7R activation by reducing ATP release, thereby inhibited inflammatory cell infiltration and alleviating the secretion of inflammatory mediators such as IL-1β, IL-18, and TNF-α. Notably, activation of P2X7R signaling abolished the protective effects of AGNHW on cerebral blood flow response and blood-brain barrier integrity.

CONCLUSIONS: AGNHW attenuated neurovascular uncoupling and improved cerebral blood flow response and neuronal activity during whisker stimulation after cerebral ischemia, mediated by inhibition of the P2X7R-driven purinergic-inflammatory cascade. These findings elucidated a mechanism through which AGNHW preserves neurovascular function.