Background: Ischemic stroke (IS) is one of the leading causes of death and disability worldwide. The narrow therapeutic window (within 4.5 h) and severe hemorrhagic potential limits therapeutic efficacy of recombinant tissue type plasminogen activator (rt-PA) intravenous thrombolysis for patients. Xingnao Kaiqiao (XNKQ) acupuncture is an integral part of traditional Chinese medicine, specifically designed to address acute ischemic stroke by targeting key acupoints such as Shuigou (GV26) and Neiguan (PC6). In this study, we explored the therapeutic potential of XNKQ acupuncture in extending the time window for thrombolysis and interrogated the molecular mechanisms responsible for this effect. Methods: The effect of extending the thrombolysis window by acupuncture was evaluated via TTC staining, neuronal score evaluation, hemorrhagic transformation assay, and H&E staining. RNA sequencing (RNA-seq) technology was performed to identify the therapeutic targets and intervention mechanisms of acupuncture. Evans blue staining and transmission electron microscopy were used to assess blood–brain barrier (BBB) integrity. Immunofluorescence staining and co-immunoprecipitation were performed to evaluate the level of autophagy and apoptosis and validate their interactions with BBB endothelial cells. Results: Acupuncture alleviated infarction and neurological deficits and extended the thrombolysis window to 6 h. The RNA-seq revealed 16 potential therapeutic predictors for acupuncture intervention, which related to suppressing inflammation and restoring the function of BBB and blood vessels. Furthermore, acupuncture suppressed BBB leakage and preserved tight junction protein expression. The protective effect was associated with regulation of the autophagy–apoptosis balance in BBB endothelial cells. Acupuncture intervention dissociated the Beclin1/Bcl-2 complex, thereby promoting autophagy and reducing apoptosis. Conclusion: XNKQ acupuncture could serve as an adjunctive therapy for rt-PA thrombolysis, aiming to extend the therapeutic time window and mitigate ischemia–reperfusion injury. Acupuncture suppressed BBB disruption by regulating the autophagy–apoptosis balance, which in turn extended the therapeutic window of rt-PA in IS. These findings provide a rationale for further exploration of acupuncture as a complementary candidate co-administered with rt-PA.

Thrombolytic therapy remains the standard treatment for acute ischemic stroke. The narrow window for thrombolysis means that delay in treatment worsens brain injury. Astrocytes regulate blood-brain barrier (BBB) integrity and neuroinflammation, yet their neurotoxic polarization exacerbates injury in neuropathological conditions, including ischemic stroke. Delayed recombinant tissue plasminogen activator (rt-PA) thrombolysis disrupts astrocyte homeostasis, further aggravating brain injury. This study investigates whether acupuncture, a key therapy in traditional Chinese medicine, alleviates delayed thrombolysis-induced injury by modulating astrocyte neurotoxic polarization and elucidates the underlying signaling pathway. A rat model of embolic stroke with delayed rt-PA thrombolysis was established. The effects of infarct volume, BBB integrity, and neuroinflammation of acupuncture were evaluated. Bulk and single-cell transcriptomic analyses were performed to assess astrocyte-specific transcriptional changes. Western blotting, immunofluorescence, and pharmacological inhibition experiments validated molecular mechanisms. Acupuncture reduced infarct volume, improved neurological function, and restored BBB integrity. Transcriptomic analysis revealed dynamic regulation of astrocyte neurotoxic polarization following acupuncture intervention. Further validation experiments demonstrated that acupuncture suppressed the ERK1/2-Cx43 cascade, thereby attenuating astrocyte-mediated neurotoxicity. Pharmacological modulation of this pathway replicated the protective effects of acupuncture, highlighting the role in mitigating astrocyte dysfunction and promoting BBB recovery. Acupuncture mitigates delayed thrombolysis-induced brain injury by modulating astrocyte polarization via the ERK1/2-Cx43 pathway. These findings highlight acupuncture as a potential strategy to enhance thrombolytic therapy safety in ischemic stroke.

Thrombolytic therapy is the primary treatment for acute ischemic stroke. Extending the therapeutic time window can effectively reduce the harmful side effects associated with thrombolytic therapy. Although electroacupuncture (EA) has been shown to extend this time window, the specific mechanisms remain unclear. We developed an embolic stroke model in rats and administered EA during thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) either 4.5 or 6 h after stroke onset. Neurological deficits were evaluated at 2 and 24 h post-stroke. Brain tissue was collected for analysis using 2,3,5-triphenyl tetrazolium chloride (TTC) staining, water content measurement, blood-brain barrier (BBB) permeability assessment, electron microscopy, and TUNEL assay. Immunofluorescence staining, western blotting, and enzyme-linked immunosorbent assays were employed to quantify the expression of proteins related to BBB integrity and pyroptosis. Neuronal damage and BBB disruption along with increased expression of pyroptosis-related proteins were observed following thrombolytic therapy at the 6-hour mark. EA treatment improved neurological outcomes, reduced infarct volume, and alleviated BBB disruption. EA also inhibited the expression of matrix metalloproteinase 9 (MMP9) and enhanced the expression of tissue inhibitor of metalloproteinases 1 (TIMP1), helping to maintain BBB integrity. Furthermore, EA reduced the expression of pyroptosis-related proteins, including gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18). EA also reduced the co-expression of GSDMD and MMP9 in brain tissues. EA may be a promising therapeutic approach for extending the thrombolytic therapy window by protecting the BBB and inhibiting GSDMD-mediated pyroptosis.

Background: Acute ischemic stroke (AIS) is the second leading cause of death and disability worldwide. Recombinant tissue plasminogen activator (rtPA) was the first FDA‐approved thrombolytic drug for AIS. However, delayed administration of rtPA exacerbates brain injury and increases the risk of hemorrhagic transformation (HT) and mortality. Ferroptosis, an iron‐dependent form of cell death, is closely associated with the pathological process of AIS. Acupuncture, a vital component of traditional Chinese medicine, has been widely used in clinical practice for AIS treatment. This study aims to investigate the protective effects of acupuncture on rats subjected to delayed rtPA thrombolysis in cerebral infarction and its relationship with ferroptosis. Methods: Adult male Sprague‐Dawley rats were used to establish a thromboembolic stroke model and were randomly assigned to different treatment groups. Xingnao Kaiqiao (XNKQ) acupuncture (at Neiguan and Shuigou acupoints) or sham acupuncture was administered in combination with rtPA thrombolysis. Outcome measures included neurological scores, infarct volume, brain water content, blood–brain barrier (BBB) permeability, expression of iron metabolism‐related proteins, lipid peroxidation levels, and mitochondrial ultrastructural changes. Results: XNKQ acupuncture significantly improved neurological deficits following delayed rtPA thrombolysis, reduced infarct volume and brain water content, and decreased the incidence of HT and brain edema. By modulating iron metabolism, inhibiting lipid peroxidation, and preserving mitochondrial integrity, acupuncture attenuated neuronal damage and BBB disruption mediated by ferroptosis. Conclusion: XNKQ acupuncture inhibits neuronal ferroptosis by improving iron metabolism disorders, lipid peroxidation accumulation, and mitochondrial structure, thereby alleviating neuronal damage and BBB disruption, and exerting a protective effect on brain tissue after delayed rtPA thrombolysis.

Background: The clinical application of recombinant tissue plasminogen activator (rt-PA) is significantly constrained by hemorrhagic transformation (HT), a common and severe complication following thrombolysis for ischemic stroke. Notably, the mitochondrial injury-mediated NLRP3 inflammasome plays a crucial role in HT after delayed rt-PA thrombolysis in acute ischemic stroke. Although acupuncture has demonstrated antioxidant and anti-inflammatory effects in acute cerebral infarction, its impact on delayed rt-PA thrombolysis, especially concerning mitophagy and the NLRP3 inflammasome, remains unclear. This study investigates how acupuncture protects against HT resulting from mitochondrial damage and NLRP3 inflammasome activation after delayed rt-PA thrombolysis in acute cerebral stroke.Methods: We selected an embolic stroke model in rats and assessed brain injury after delayed rt-PA in acute ischemic stroke using neurological deficit score, volume of brain infarct, the permeability assay of the blood–brain barrier (BBB), and HT. Then, the levels of proteins and mRNA involved in mitophagy and the NLRP3 inflammasome pathway were measured by western blot and real-time PCR. The levels of interleukin-18 (IL-18) and interleukin-1β (IL-1β) were assessed using enzyme-linked immunosorbent assay (ELISA). Morphological changes in the BBB and mitochondria of neurons were observed via transmission electron microscopy.Results: Acupuncture significantly improved neurological deficit scores, volume of cerebral infarction, BBB destruction, and HT in an embolic stroke model rat. Furthermore, acupuncture induced mitophagy and substantially downregulated the activity of the NLRP3 inflammasome. Additionally, the use of mitochondrial inhibitors significantly reversed the suppressive impact of acupuncture on the NLRP3 inflammasome.Conclusion: Acupuncture can promote mitophagy and suppress NLRP3 inflammasome activation to decrease HT after delayed rt-PA therapy for acute ischemic stroke.