Neuroprotective mechanism of acupuncture against brain injury during delayed thrombolysis for acute ischemic stroke.

Abstract

Objective: This study aimed to investigate the ferroptosis-related mechanism of brain injury in delayed thrombolysis for acute ischemic stroke (AIS) and clarify the neuroprotective role of acupuncture.

Methods: This study integrated transcriptomic and proteomic analyses, clinical retrospective analysis and animal experiments. Multi-omics analyses, including high-throughput transcriptome sequencing and liquid chromatography-tandem mass spectrometry-based proteomics, were performed on brain tissues from rats with thromboembolic stroke to identify differentially expressed genes and proteins, overlapping molecules, and ferroptosis-related enriched pathways. The clinical retrospective study included 110 participants (30 healthy volunteers and 80 AIS patients, stratified by thrombolysis time windows of <3 h and 4.5-6 h) to assess neurological outcomes and iron metabolism markers. A rat model of thromboembolic stroke was used for in vivo experiments. Sprague-Dawley rats were subjected to delayed intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA) at 6 h post-modeling, followed by ferroptosis modulators (liproxstatin-1 and iron dextran) and acupuncture. Ferroptosis-related markers, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), iron deposition, malondialdehyde (MDA) and glutathione (GSH), were detected. Additionally, functional and structural assessments covering mitochondrial morphology and neurological function were conducted.

Results: Multi-omics analysis revealed significant enrichment of ferroptosis-related pathways, including glutamate metabolism and lipid peroxidation, along with abnormal expression of molecules associated with oxidative stress and blood-brain barrier (BBB) disruption. In this retrospective study, reduced serum iron and transferrin levels, as well as total iron-binding capacity, were observed in AIS patients. Serum iron level was negatively correlated with the National Institutes of Health Stroke Scale score (r = -0.709, P < 0.01) and had the highest predictive efficacy for symptomatic intracerebral hemorrhage among the three iron metabolism indicators, with the area under the curve value of 0.803, indicating that delayed thrombolysis triggers ferroptosis. In vivo experiments demonstrated that acupuncture upregulated the expression of anti-ferroptosis proteins SLC7A11 and GPX4 and downregulated the pro-ferroptosis protein ACSL4. It alleviated ferroptosis-related pathological changes by reducing iron deposition and MDA content and increasing GSH level. Additionally, it improved mitochondrial structural integrity and neurological function and protected the BBB from disruption. Moreover, it exerted a synergistic effect with liproxstatin-1 against ferroptosis and brain injury.

Conclusion: Delayed thrombolysis for AIS triggered ferroptosis, accompanied by lipid peroxidation and BBB disruption. The acupuncture exerted neuroprotective effects by regulating ferroptosis, improving iron metabolism and protecting BBB integrity, which is a promising non-pharmacological strategy to enhance the safety of delayed rt-PA thrombolysis for AIS. Please cite this article as: Huang Z, Liu XY, Gao JF, Zhang HT, Huang Y, Wu R, Cheng YY, Zhang ZH, Xu XP, Zhang XC, Ni GX. Neuroprotective mechanism of acupuncture against brain injury during delayed thrombolysis for acute ischemic stroke. J Integr Med. 2026; Epub ahead of print.