Acupuncture improves neurological function in ICH rats via suppression of WNK-SPAK-NKCC1 signaling activation

The WNK3-SPAK-NKCC1 signaling pathway has been implicated in the pathogenesis of brain injury. The aim of this study is to examine the involvement of this pathway in intracerebral hemorrhage (ICH) and evaluate the therapeutic potential of acupuncture in modulating its activity. A total of 210 Sprague Dawley rats were randomly assigned to experimental groups. ICH was induced in all groups except the sham group via autologous blood injection. Point-through-point acupuncture was administered on the Baihui (GV20) and Qubin (GB7) acupoints. Protein expression levels within the WNK3-SPAK-NKCC1 pathway, as well as inflammatory and apoptotic markers in the perihematomal region, were assessed using western blotting, immunohistochemistry, immunofluorescence co-localization, and enzyme-linked immunosorbent assay. Brain water content, hematoxylin-eosin staining, and neurological function scoring were used to evaluate histopathological changes and functional outcomes. Acupuncture significantly improved neurological function, alleviated cerebral edema, and reduced perihematomal pathological injury in ICH rats at all observed time points. Notably, the therapeutic effect was most pronounced after seven consecutive acupuncture treatments. Time-course analysis of WNK3 and cleaved caspase-3 expression, together with correlation analysis, revealed that the neuroprotective effects of acupuncture were potentially associated with WNK3-mediated apoptotic mechanisms. Based on these findings, day 7 was identified as the optimal time point for further mechanistic investigation. At this time point, continuous acupuncture treatment significantly improved neurological function and decreased brain water content. In addition, the expression levels of WNK3, phosphorylated SPAK and NKCC1, TNF-α, and cleaved caspase-3 were significantly reduced, accompanied by decreased NeuN/TUNEL co-localization (p < 0.05), suggesting that acupuncture may exert neuroprotective effects partly through inhibition of the WNK3-SPAK-NKCC1 signaling pathway. Acupuncture may alleviate brain injury following ICH by suppressing activation of the WNK3-SPAK-NKCC1 signaling pathway, thereby decreasing cerebral edema, inflammatory responses, and neuronal apoptosis.