Therapeutic effects of Naoshuan Kangfu Capsule on post-stroke spasticity in normothermic and cold-exposed rat models
Zhiyuan Du, Kexin Wang, Zijia Liu, Yifei Wang, Bosi Sun, Yu Wang, Chenchen Yin, Tianjiao Zhao, Dan Yin, Ajiao Yu, Weijun Li, Yang Qu, Yu Liu, Lei Wang, Lei Jiao, Baofeng YangAbstract
Objective
Post-stroke spasticity (PSS), a common and debilitating complication after stroke, severely impacts patients' motor function and quality of life. Naoshuan Kangfu Capsule (NSKF) is traditionally used for the treatment of stroke, hemiplegia, aphasia, and facial paralysis caused by blood stasis obstructing collaterals; however, its therapeutic effects and underlying mechanisms in PSS remain unclear. This study investigated the protective and reparative effects of NSKF on PSS following cerebral ischemia under both normal and cold exposure conditions.
Methods
A global cerebral ischemia model was established in rats using bilateral two-vessel occlusion (2VO). PSS was evaluated using behavioral assessments, including the modified Neurological Severity Score (mNSS), balance beam test, Ashworth Scale, and sticky dot test, in 2VO rats maintained under normal and cold temperature conditions. Hematoxylin-eosin (HE) staining was used to assess cerebral histomorphological changes. RNA sequencing was conducted to identify differentially expressed genes in brain tissues. Cerebral cortex tissue neuron-specific enolase (NSE), brain vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), glutamate (Glu), and γ-aminobutyric acid (GABA) levels were measured using ELISA. qRT-PCR was used to determine the mRNA expression levels of Grm1a, Gabbr1, Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α). Immunofluorescence staining was performed to assess ionized calcium-binding adaptor molecule 1(IBA-1) and cluster of differentiation 31 (CD31) expression. Apoptosis was evaluated using TUNEL staining and Caspase-3 assay.
Results
NSKF significantly alleviated PSS in 2VO rats at 5 and 10 weeks after cerebral ischemia, as evidenced by multiple behavioral assessments, with effects comparable to those of the positive control drug Baclofen. NSKF suppressed NSE and IBA-1 activation, indicating attenuation of neuronal injury and neuroinflammation. RNA sequencing revealed that NSKF regulated pathways associated with apoptosis, angiogenesis, and inflammatory responses. Subsequent validation confirmed that NSKF inhibited neuronal apoptosis, as evidenced by reduced cell death and caspase-3 activity, while promoting angiogenesis through increased CD31 and VEGF expression. In addition, NSKF modulated neurotransmitter homeostasis by decreasing cortical Glu/GABA levels, and upregulating the expression of metabotropic glutamate receptor 1a (Grm1a) and GABA B receptor 1 (Gabbr1). However, NSKF did not significantly affect neuronal proliferation. Furthermore, NSKF suppressed neuroinflammation by reducing IBA-1 activation and pro-inflammatory cytokine expression (IL-1β, IL-6, IL-18, and TNF-α), potentially through inhibition of the NF-κB signaling pathway. Notably, NSKF maintained its therapeutic efficacy against PSS in 2VO rats exposed to chronic cold conditions. Long-term administration of NSKF (50, 400, 800, and 1600 mg/kg for 10 weeks) produced no observable toxic effects.
Conclusions
NSKF improves neurological deficits and alleviates post-stroke spasticity after cerebral ischemic injury by reducing neuronal apoptosis, enhancing angiogenesis, suppressing neuroinflammation, and restoring Glu/GABA neurotransmitter balance. Its sustained efficacy under cold exposure conditions suggests potential advantages for patients living in cold environments. These findings provide experimental evidence for the therapeutic potential of NSKF as a candidate intervention for post-stroke spasticity.