Regulatory effects of head acupuncture on the GABAergic system and Wnt7a/β-catenin pathway in post-stroke spasticity rats

Abstract

To investigate the regulation of the GABAergic system and Wnt/β-catenin signaling pathway in rats with limb spasticity after stroke by Head Acupuncture therapy and its potential mechanism. A middle cerebral artery occlusion (MCAO) model was established by the thread bolus method, and randomly grouped into a blank group, a model group, and a needling group, the latter of which received the intervention of needling at the points of "Hundred Houses" and "Rate Valley" for 30 min a day for 7 consecutive days. The neurological deficits and muscle tone of rats were assessed by Zeo Longa score and modified Ashworth score, and the expression levels of key proteins of the GABAergic system (GAD65, GAD67, GABA-T, GAT-3, NKCC1) and Wnt/β-catenin signaling pathway were detected by immunohistochemistry, real-time fluorescence quantitative PCR and Western blot and Wnt/β-catenin signaling pathway key protein expression levels. Head Acupuncture significantly improved the neurological deficit score and muscle tone of rats in the model group, decreased the mRNA and protein expression levels of GABA-T and NKCC1, and significantly up-regulated the expression of GAD65, GAD67, and GAT-3. In addition, acupuncture promoted the expression of Wnt7a and β-catenin, and the effect of 7 days of treatment was better than that of 1 day. Head Acupuncture therapy may exert a therapeutic effect on limb spasticity after stroke by regulating the balance of the GABAergic system and activating the Wnt/β-catenin signaling pathway. This provides a new experimental basis for the application of acupuncture in post-stroke sequelae.Clinical trial number: 2024062814.

Keywords: GABAergic system; Head acupuncture; Limb spasticity; Spasticity rats; Wnt/β-catenin.

Fig. 1

Schematic diagram of acupuncture in rats. Note: GV20: in the middle of the parietal bone; GB8: above the tip of the pinna, on the line connecting the middle point of the parietal node

Fig. 2

Comparison of groups of rats Zeo Longa scores ( ± S, 10 rats/group). Note: ***P < 0.001 versus Con group, ***P < 0.001 versus Model group(1d + 7d), ***P < 0.001 versus Acu 1d group

Fig. 3

Comparison of modified Ashworth scores of rats in each group ( ± S, 10 rats/group). Note: ***P < 0.001 versus Con group, ***P < 0.001 versus model group (1d + 7d), ***P < 0.001 versus Acu 1d group

Fig. 4

Comparison of histopathological morphology of the cerebral cortex in the infarcted area of rats in each group (HE staining, original magnification × 400). Note: Morphological changes in ischemic penumbra detected by HE staining in the ipsilateral cortexscale, bar = 50 μm, the upper left panel shows the location of staining in the brain (small black box)

Fig. 5

Comparison of positive expression of GAD65, GAD67, GABA-T, GAT-3, and NKCC1 in brain tissues on the ischemic side of rats in each group ( ± S, 6 rats/group, original magnification × 400). Note: A Representative images of GAD65, GAD67, GABA-T, GAT-3, and NKCC1 expression assessed by immumohistochemical staining in ipsilateral cortex, scale bar = 50 μm, the upper left panel shows the location of staining in the brain (small black box). B–F ns: P > 0.05. *P < 0. 05, **P < 0.01, ***P < 0.001

Fig. 6

Comparison of the expression levels of GAD65, GAD67, GABA-T, GAT-3, and NKCC1 mRNA in the brain tissues of the ischemic side of the rats in the various groups ( ± S, 6 rats/group). Note: A–E **P < 0.01, ***P < 0.001

Fig. 7

Comparison of protein expression levels of GAD65, GAD67, GABA-T, GAT-3, NKCC1, Wnt7a, and β-catenin in brain tissues of the ischemic side of rats in each group ( ± S, 3 rats/group). Note: A–H Western blotting images and quantitative data of relative expression level of GAD65, GAD67, GABA-T, GAT-3, NKCC1, Wnt7a, and β-catenin. **P < 0.01, ***P < 0.001