2.1 Animals

Adult male Sprague-Dawley rats (300 ± 20 g) and maintained at the SPF level, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; License No. SCXK (Jing): 2021-0006). The rats were maintained in a controlled environment with a humidity of 55 ± 5% and a temperature of 23 ± 3°C under a 12 h light-dark cycle and were provided with food and water according to a regular schedule. The animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine (Approval No. 202404A014).

2.2 Animal Grouping

In the first part of the study, the rats were randomly divided into 6 groups: the Sham, Model, rtPA (4.5, 6 h), 6 h rtPA+Acu, and 6 h rtPA+Sham Acu groups. In another part of the study, the rats were randomly divided into 6 groups: the Sham, Model, 6 h rtPA, 6 h rtPA+Acu, 6 h rtPA+Iron-dextran, and 6 h rtPA+Iron-dextran+Acu groups. The rats that were injected with the ferroptosis inducer were intraperitoneally injected with Iron-dextran (0.5g/kg; MedChemExpress, Shanghai, China) 6 h before modeling. Specifically, the model group represented a state of cerebral infarction without rtPA thrombolytic therapy or acupuncture. The sham group did not receive a thrombus injection into the middle cerebral artery (MCA), which was intended to exclude the impact of nonpathological factors during the procedure. The rtPA (4.5, 6 h) group refers to the group receiving rtPA thrombolytic therapy after 4.5 or 6 h of infarction.

2.3 Establishment of the Thromboembolic Stroke Model

Blood from the inner canthus of each rat was collected to generate a thrombus, and then the thrombus was transferred to a syringe filled with normal saline. The rats were anesthetized and immobilized, and the blood vessels in the neck were exposed. The syringe containing the thrombus is pushed through the PE-50 catheter to the beginning of the MCA on the right side, and the MCA blood flow is blocked. The catheter is removed after 5 s (L. Zhang et al. 2015). Laser speckle blood flow imaging (RFLSI ZW, RWD, Shenzhen, China) was employed to monitor cerebral blood flow obstruction and validate the model's effectiveness. A substantial reduction in blood perfusion on the right side indicated successful model construction (H. Wang et al. 2021).

2.4 Treatment With Acupuncture and Sham Acupuncture

The rats in the acupuncture group and the sham acupuncture group received corresponding acupuncture treatment or sham acupuncture intervention along with thrombolysis treatment. The acupoints selected were bilateral Neiguan (PC6) and Shuigou (GV26), with the locations of the acupoints referring to the Names and Locations of Common Acupoints in Laboratory Animals Part 2: Rats established by the Chinese Acupuncture Society.

The acupuncture treatment tool used was a stainless steel needle (disposable; diameter: 0.18 mm; depth: 13 mm; Suzhou Hwato Medical Instrument). The acupuncture needles were inserted into points GV26 and PC6, with depths ranging from 2–3 mm. The thumb was used to apply force to the acupuncture needle, resulting in lifting, stabbing, twisting, shrinking, and other techniques, each time it was applied for 1 min, after which the needles were retained for a duration of 30 s.

The sham acupuncture scheme was the same as that used in the acupuncture group except that the treatment tools used were different from those used in the acupuncture group. A specialized adhesive foam pad was placed over the GV26 and bilateral PC6 acupoints, and then a specially designed blunt needle was inserted into the foam pad at the designated acupoint location, ensuring that the blunt tip of the needle did not penetrate the skin at the acupoint (Figure 1).

2.5 Laser Speckle Flow Imaging

Laser speckle flow imaging (RWD, Shenzhen, China) was used to monitor changes in cerebral ischemic blood perfusion 24 h before surgery and 24 h after surgery. The rats were anesthetized and immobilized, and their skulls were fully exposed. Cranial research was used to sharpen the skull, exposing the right side of the brain. The laser wavelength was set to 785 ± 10 nm, the working height of the laser speckle lens fiber was adjusted to below 10 cm, and the spatial algorithm sliding mode was used, with a spatial filtering constant of 15 s, a frame rate of 0.33 fps, and a recording time of 20 ms. Dynamic perfusion images of blood vessels were collected. The right cerebral cortex blood perfusion (PU) was calculated via RFLSI analysis software.

2.6 Assessment of Neurobehavior

After the successful establishment of the model in rats for 24 h, neurological function was assessed via the modified Bederson score (0–5 points), the corner turn test, and the adhesive-removal test. The modified Bederson score (0–5 points) scoring criteria are as follows: 0, no obvious functional deficits; 1, the rat's left forelimb is mildly flexed; 2, when the tail is pulled, the rat's left forelimb grip strength is significantly reduced, and lifting the rat reveals the left forelimb adhering to the chest wall; 3, the rat's spontaneous activity is nondirectional, and it only turns to the left when the tail is pulled; 4, the rat spontaneously turns to the left and leans to the left when walking; 5, death. In the corner turn test, the rat is placed between a specially designed device consisting of two wooden boards set at a 30° angle. The rat is positioned with its head facing the vertex of the angle. As the rat walks forward and reaches the vertex, it turns at the corner. This process is repeated 10 times, and the test result is indicated by the ratio of the number of pathological turns to the total number of turns. In the adhesive-removal test, a small piece of adhesive tape is attached to the rat's forelimb, and the time it takes for the rat to remove the tape with its teeth is observed and recorded.

2.7 Measurement of the Infarct Volume

Twenty-four hours after the successful establishment of the rat model, the infarct volume of the brain tissue was measured via TTC staining. The anesthetized rat brain tissue was stored at −20°C for 20 s, after which the brain tissue was cut into 2 mm slices. The brain sections were immersed in 2% triphenyltetrazepine ammonium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA) at room temperature and incubated in darkness for 10 s. Image J software (version 1.5.4) was used to analyze the brain slice images. The formula for calculating cerebral infarction volume (%) was as follows: sum of the ischemic area of each section/sum of the brain section area of each section×100%.

2.8 Measurement of Brain Water Content

Twenty-four hours after the successful establishment of the rat model, the wet weight of the brain tissue was measured by weighing. The brain tissue was then placed in an oven at 100°C for 24 h and reweighed to determine the dry weight. The water content (%) was calculated via the following formula: (wet weight‒dry weight)/wet weight×100%.

2.9 Assessment of Blood–Brain Barrier (BBB) Permeability

BBB permeability was evaluated via the use of a 2% Evans blue (EB) dye solution (Sigma-Aldrich, St. Louis, USA). Two hours prior to euthanasia, the rats were administered a tail vein injection of 2% EB dye (0.4 mL/100 g). Twenty-four hours postsurgery, following systemic perfusion with 0.9% saline, the right brain tissues were excised, weighed, and then submerged in formamide (Macklin, Shanghai, China) at a concentration of 500 µL per 100 mg of tissue for homogenization. The tissues were incubated at 60°C for 24 h, followed by centrifugation for 20 s. The supernatant was harvested, and its absorbance at 620 nm was determined with a spectrophotometer. The concentration of EB (µg/g) was then calculated via the following equation: (EB concentration in µg/mL) × (volume of formamide in mL)/(mass of brain tissue in g).

2.10 Measurement of Hemorrhagic Transformation

HT was assessed via a hemoglobin colorimetric assay kit (Beyotime Biotechnology, Shanghai, China). Twenty-four hours after surgery, brain tissues were extracted from the rats in each group. The side of the right brain tissue was homogenized in 2 mL of phosphate-buffered saline (PBS). After centrifugation for 30 s, the supernatant was collected, and the absorbance at 410 nm was measured via a spectrophotometer.

2.11 Nissl Staining

Nissl staining was utilized to evaluate neuronal injury. Twenty-four hours after surgery, the rats from each group were anesthetized and perfused systemically with a solution containing 0.9% saline and 4% paraformaldehyde. The brain tissues were subsequently removed and fixed in a paraformaldehyde solution for 24 h. Following dehydration, paraffin sections were prepared. These sections were stained with Nissl staining solution (Solarbio, Beijing, China) for 15 s, and subsequently examined under an optical microscope for any pathological alterations in the brain tissue. The quantity of Nissl-stained positive cells within the brain sections was quantified via Image J software (version 1.5.4).

2.12 Prussian Blue Iron Staining

Iron deposition in the brain tissue was observed via a Prussian blue iron stain kit (enhanced with DAB) (Solarbio, Beijing, China). Twenty-four hours after surgery, anesthetized rats from each group were perfused systemically with 0.9% saline and 4% paraformaldehyde. The brain tissues were extracted and made into paraffin sections with a thickness of 4 µm. The paraffin sections were stained with Perls working solution at 37°C for 20 s; then, the sections were rinsed with distilled water 3 times, each for 10 s. Stain The sections were incubated with a working solution at 37°C for 20 s and then rinsed with PBS 3 times, each for 10 s. The sections were incubated with an enhanced working solution at 37°C for 20 s, and then rinsed with PBS 3 times, each for 10 s. The samples were stained with a redyeing solution for 5 s and then rinsed with distilled water for 10 s. Iron deposition in the right-side brain tissues of the rats in each group was observed under an optical microscope, with positive cells containing brown–yellow particles. The area of iron ion (Fe²⁺) deposition in the brain sections was quantified using Image J software.

2.13 Ultrastructure by Transmission Electron Microscopy

Transmission electron microscopy (TEM) was used to examine the ultrastructural alterations in the BBB and morphological changes associated with ferroptosis, including changes in the mitochondrial ultrastructure. Following perfusion with PBS and 2.5% glutaraldehyde, the rat brain tissues were promptly excised. Right cortical tissue blocks measuring 1 mm³ were placed in a 2.5% glutaraldehyde solution at 4°C and subsequently fixed with a 1% osmium tetroxide solution for 2 h. After undergoing graded dehydration and infiltration with acetone, the tissues were embedded in 618 epoxy resin. Ultrathin sections approximately 90 nm in thickness were prepared via an ultramicrotome and then sequentially stained with uranyl acetate and lead citrate.

2.14 Measurement of Brain Iron, Glutathione, and Malondialdehyde

The level of iron ions (Fe²⁺) was detected via an iron assay kit (Dojindo, Kumamoto, Japan). Lipid peroxidation levels were measured with a GSH assay kit and MDA assay kits (Beyotime Biotechnology, Shanghai, China). The right side of the rat cerebral cortex tissue was homogenized and then centrifuged and aliquoted according to the instructions provided with each assay kit. The absorbance of each sample was measured at the corresponding wavelengths (593/405/532 nm) via a spectrophotometer. The contents of Fe²⁺, GSH, and MDA were calculated on the basis of the instructions provided with the assay kits.

2.15 Immunofluorescence

The brain tissues were excised and processed into 12 µm-thick frozen sections. These sections were rinsed three times with PBST and then blocked with PBS containing 0.3% Triton X-100 and 5% goat serum for 1 h. They were subsequently incubated overnight with primary antibodies, which included Ferritin (1:1000, ab75973, Abcam, Cambridge, UK) and NeuN (1:500, ab104224, Abcam, Cambridge, UK). Afterward, the sections were washed three times with PBST and incubated with an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (1:500, ab150115, Abcam, Cambridge, UK) and an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:500, ab150077, Abcam, Cambridge, UK). Finally, all the sections were mounted with an anti-fade mounting medium containing DAPI. The images were observed via a fluorescence microscope (Leica Thunder, Wetzlar, Germany) and analyzed via Image J software.

2.16 Western Blot Analysis

Proteins were extracted from the right cerebral cortex and quantified via a quantitative BCA assay kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by 10% SDS‒PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated overnight at 4°C with the following primary antibodies: Claudin5 (1:1000, 29767-1-AP, Proteintech, Wuhan, China), ZO-1 (1:1000, 21773-1-AP, Proteintech, Wuhan, China), Ferritin (1:1000, ab75973, Abcam, Cambridge, UK), GPX4 (1:1000, ab125066, Abcam, Cambridge, UK), FPN1 (1:500, DF13561, Affinity, Jiangsu, China), TFR1 (1:500, AF5343, Affinity, Jiangsu, China), Solute Carrier Family 7 Member 11 (SLC7A11) (1:500, DF12509, Proteintech, Jiangsu, China), and GAPDH (1:2000, UM4002, Utibody, Tianjin, China). The samples were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG(H+L) or goat anti-mouse IgG(H+L) (1:10,000, Affinity, Jiangsu, China). Following chemiluminescence detection with ECL reagents and a Fusion Edge Multi-Function Imaging System (Vilber, Collégien, France), the relative optical densities of the protein bands were quantified via Image J software.

2.17 Statistical Analysis

The data were processed via SPSS software (version 23.0) and are expressed as the mean±standard deviations. The Shapiro‒Wilk normality test was used to examine whether the data were normally distributed. When the data were normally distributed and had equal variances, differences among multiple groups were analyzed via one-way ANOVA, with post-hoc multiple comparisons performed via the least significant difference (LSD) test for pairwise comparisons between groups. If the data did not conform to a normal distribution, nonparametric tests were used for analysis. If the data had unequal variances, Dunn's test was applied. GraphPad software (version 9.5.0) was used to create statistical graphs. A difference was considered statistically significant if p < 0.05.

3.1 Effect of Acupuncture on Brain Injury After Delayed rtPA Thrombolysis in Rats With Cerebral Infarction

To verify the neuroprotective effect of acupuncture after delayed rtPA thrombolysis in rats with cerebral infarction, we first assessed neurological function via the Bederson score, corner turn test, and adhesive-removal test. The results shown in Figure 2A–C indicate that compared with the Model group, the rats treated with 4.5 h of rtPA thrombolysis presented lower Bederson scores, fewer corner turns, and shorter adhesive-removal times. In contrast, the rats treated with 6 h of rtPA thrombolysis presented significantly higher Bederson scores, greater corner turn counts, and longer adhesive-removal times, suggesting that thrombolysis within the 4.5 h time window can salvage neurological function in rats, whereas delayed thrombolysis outside this window at 6 h can lead to severe neurological deficits. Compared with those in the 6 h rtPA group, the Bederson scores, corner turn counts, and adhesive-removal times of the rats in the 6 h rtPA+Acu group tended to normalize, whereas those in the 6 h rtPA+Sham Acu group tended to change little, indicating that acupuncture can mitigate the neurological deficits caused by 6 h delayed rtPA thrombolysis in cerebral infarction.

TTC staining and laser speckle flow imaging were used to observe cerebral infarct volume and blood flow perfusion. As shown in Figure 2D–G, compared with the Model group, the rats in the 4.5 h rtPA group presented reduced infarct volumes and increased blood flow perfusion in the affected cerebral cortex, whereas the rats in the 6 h rtPA group presented increased infarct volumes and significantly lower blood flow perfusion in the affected cerebral cortex. This finding indicates that after cerebral infarction, the infarct volume in the rats did not significantly expand under thrombolysis treatment within the 4.5 h time window and that blood flow perfusion was restored. However, delayed thrombolysis outside this window at 6 h led to a significant increase in infarct volume and blood flow perfusion. Compared with those in the 6 h rtPA group, the infarct volume and blood flow perfusion in the affected cerebral cortex were lower in the 6 h rtPA+Acu group, whereas the infarct volume and blood flow perfusion in the 6 h rtPA+Sham Acu group were not significantly different. These findings suggest that acupuncture can salvage the increased infarct volume and blood flow perfusion caused by delayed rtPA thrombolysis.

Overall, these data indicate that acupuncture significantly mitigates neurological deficits, infarct volume, and blood flow perfusion caused by delayed rtPA thrombolysis in rats with cerebral infarction.

3.2 Influence of Acupuncture on Complications Caused by Delayed rtPA Thrombolysis in Rats With Cerebral Infarction

Cerebral infarction with delayed rtPA thrombolysis is prone to complications such as intracranial hemorrhage and brain edema. A hemoglobin quantitative detection kit was used to assess intracranial hemorrhage. The results shown in Figure 3 indicate that we evaluated the degree of brain edema through brain water content detection. As shown in Figure 3, compared with the Model group, the 6 h rtPA group presented increased hemoglobin and brain water contents. Compared with those in the 6 h rtPA group, there was a notable reduction in both hemoglobin and brain water content in the 6 h rtPA+Acu group, whereas the 6 h rtPA+Sham Acu group presented no significant alterations. These findings suggest that delayed rtPA thrombolysis in cerebral infarction rats can cause severe intracranial hemorrhage and brain edema, whereas acupuncture treatment can improve the degree of bleeding and edema. These findings indicate that acupuncture can reduce complications such as intracranial hemorrhage and brain edema caused by delayed rtPA thrombolysis in rats with cerebral infarction.

3.3 Influence of Acupuncture on the Blood‒Brain Barrier After Delayed rtPA Thrombolysis in Cerebral Infarction Rats

BBB disruption is a direct cause of complications such as HT and brain edema following thrombolysis (Jiang et al. 2018; Dharmasaroja 2016), and its normal function is closely related to permeability and structural integrity. First, EB staining was used to observe changes in the BBB permeability. As shown in Figure 4A–B, EB dye leaked into the brain parenchyma through the BBB in the brain tissue of the rats in the Model group. Compared with the Model group, the 4.5 h rtPA group had less EB leakage, but the 6 h rtPA group had significantly more EB leakage. These findings indicate that delayed rtPA thrombolysis increases BBB permeability. Compared with the 6 h rtPA group, the 6 h rtPA+Acu group presented a significant reduction in EB leakage, whereas the 6 h rtPA+Sham Acu group did not significantly decrease EB leakage. These findings suggest that acupuncture can significantly reduce EB leakage in the brain tissue of rats subjected to delayed rtPA thrombolysis, thereby improving BBB permeability.

Second, we observed changes in the expression of the TJs proteins Claudin5 and ZO-1 to understand the effects of acupuncture on the structural integrity of the BBB after delayed rtPA thrombolysis. As shown in Figure 4C–E, compared with those in the Model group, the protein levels of Claudin5 and ZO-1 were lower in the 6 h rtPA group, indicating that delayed rtPA thrombolysis exacerbates the abnormal expression of TJ-related proteins, leading to disruption of BBB structural integrity. Compared with those in the 6 h rtPA group, there was a marked increase in the protein expression levels of Claudin5 and ZO-1 in the 6 h rtPA+Acu group, whereas the levels in the 6 h rtPA+Sham Acu group were not significantly different. These results indicate that acupuncture can regulate the expression of TJ-related proteins after delayed rtPA thrombolysis, modulating the opening and closing of TJs and thereby improving BBB integrity.

Third, we observed changes in the ultrastructures of TJs in the BBB via TEM. As shown in Figure 4F, the morphology of the BBB in the sham group was normal, with a smooth and intact basement membrane (BM) surface and no swelling observed, and the structure of the TJs was dense and complete. In the Model group, the endothelial cells and BM were swollen and deformed, the connections between the endothelial cell layers were loose, and the TJs were damaged. In the 6 h rtPA group, the swelling and deformation of the endothelial cells and BM in the BBB were further exacerbated, the TJs were disrupted, and the ultrastructure of the BBB was severely damaged. In the 6 h rtPA+Acu group, the degree of ultrastructural damage to the BBB was reduced, with less swelling of the endothelial cells and BM, the morphology was essentially intact, and the disruption of the TJs was significantly reduced. The disruption of TJs in the 6 h rtPA+Sham Acu group did not significantly improve.

In summary, after delayed rtPA thrombolysis in cerebral infarction, EB leakage, increased BBB permeability, abnormal expression of TJ-related proteins, and severe morphological damage to TJs increase, leading to significant disruption of BBB structural integrity. However, acupuncture treatment can significantly reduce EB leakage in the brain tissue of rats with delayed rtPA thrombolysis, improve BBB permeability, regulate the expression of TJ-related proteins, alleviate morphological damage to TJs, and maintain the integrity of the BBB structure.

3.4 Effect of Acupuncture on Neurons After Delayed rtPA Thrombolysis in Rats With Cerebral Infarction

After cerebral infarction, the BBB is disrupted, and neurons are damaged. We first used Nissl staining to observe the extent of neuronal damage. As shown in Figure 5A,B, compared with those in the Model group, the neurons in the 6 h rtPA group presented a shrunken morphology, fewer Nissl bodies, and blurred contours, indicating that delayed rtPA thrombolysis in cerebral infarction led to morphological damage to neurons, a decrease in Nissl bodies, and increased neuronal damage. Subsequently, we artificially exacerbated ferroptosis using an exogenous ferroptosis inducer (Iron-dextran) to verify whether delayed thrombolysis-induced neuronal damage is mediated through the ferroptosis pathway. Compared with those in the 6 h rtPA group, the neurons in the 6 h rtPA+Iron-dextran group presented severe morphological damage, a further reduction in Nissl bodies, and the disappearance of contours. After acupuncture treatment, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the morphology of neurons in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran+Acu group improved, and the number of Nissl body-positive cells increased accordingly, indicating that acupuncture intervention may improve neuronal morphology and increase the number of Nissl body-positive cells by regulating ferroptosis-related pathways.

Next, we used immunofluorescence staining to observe neuronal damage, marking neurons with the neuron-specific nuclear protein NeuN. As shown in Figure 5C,D, compared with that in the Model group, the relative fluorescence intensity of NeuN was lower in the 6 h rtPA group, indicating that delayed rtPA thrombolysis in cerebral infarction causes neuronal damage. After iron-induced ferroptosis, the relative fluorescence intensity of NeuN was even lower in the 6 h rtPA+Iron-dextran group than in the 6 h rtPA group. After acupuncture intervention, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the relative fluorescence intensity of NeuN increased in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran+Acu group, indicating that acupuncture intervention may alleviate neuronal damage by modulating ferroptosis-related pathways.

3.5 Influence of Acupuncture on Iron Metabolism After Delayed rtPA Thrombolysis in Rats With Cerebral Infarction

Iron metabolism disorder is a typical characteristic that distinguishes ferroptosis from other forms of programmed cell death. To investigate the changes in iron metabolism in neurons following delayed rtPA thrombolysis in cerebral infarction, we first used immunofluorescence colocalization to visually observe the distribution and changes in Ferritin in NeuN. As shown in Figure 6A,B, compared with those in the Model group, the colocalization levels of NeuN (red) and Ferritin (green) were greater in the 6 h rtPA group; compared with those in the 6 h rtPA group, the colocalization levels of NeuN and Ferritin were even greater in the 6 h rtPA+Iron-dextran group. These findings indicate that Ferritin is expressed and accumulates in neurons after delayed rtPA thrombolysis in cerebral infarction and that the degradation of Ferritin may exacerbate neuronal damage. After acupuncture treatment, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the colocalization levels of NeuN and Ferritin were correspondingly reduced in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran+Acu group. These findings suggest that acupuncture may exert neuroprotective effects by regulating iron metabolism processes and inhibiting excessive neuronal ferritin deposition, potentially through suppression of ferroptosis pathways.

Next, we used Fe²⁺ content detection and Prussian blue staining to observe the degree of iron deposition. As shown in Figure 6C–E, compared with the 6 h rtPA group, the 6 h rtPA+Iron-dextran group presented increased Fe²⁺ content and a greater number of brown granules (representing Fe²⁺ deposition), indicating that delayed rtPA thrombolysis in cerebral infarction leads to the accumulation of free Fe²⁺ and more severe iron deposition. The severe accumulation of free Fe²⁺ may be related to ferroptosis. After acupuncture treatment, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the accumulation of free Fe²⁺ and the number of brown granules were reduced in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran+Acu group, indicating that acupuncture can alleviate the accumulation of free Fe²⁺ caused by delayed rtPA thrombolysis and reduce iron deposition.

Western blot analysis was employed to quantify the expression levels of proteins involved in iron metabolism, specifically Ferritin, Ferroportin 1 (FPN1), and Transferrin Receptor 1 (TFR1). As depicted in Figure 6F–I, compared with that in the 6 h rtPA group, the expression of Ferritin and FPN1 was significantly lower, whereas TFR1 expression was notably elevated in the 6 h rtPA+Iron-dextran group. Following acupuncture treatment, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the expression levels of Ferritin and FPN1 were restored, and TFR1 expression was reduced in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran group. These findings indicate that acupuncture effectively modulates the aberrant expression of iron metabolism-related proteins following delayed rtPA thrombolysis for 6 h in rats with cerebral infarction.

Overall, following delayed rtPA thrombolysis in cerebral ischemia, there is an accumulation of Ferritin in neurons, increased degradation of ferritin-releasing Fe²⁺, abnormal expression of iron metabolism-related proteins, and disordered iron ion metabolism, with free Fe²⁺ accumulation in neurons and severe iron deposition. However, acupuncture treatment can regulate the abnormal expression of iron metabolism-related proteins, reduce iron deposition in neurons, and maintain iron metabolism levels, thereby inhibiting ferroptosis and reducing neuronal damage.

3.6 Effect of Acupuncture on Lipid Peroxidation Levels After Delayed rtPA Thrombolysis in Rats With Cerebral Infarction

Evaluating lipid peroxidation levels is a hallmark feature that differentiates ferroptosis from other modes of programmed cell death (Kenny et al. 2019). Western blot analysis was used to quantify the expression levels of the antioxidant-associated proteins SLC7A11 and GPX4, while the content of GSH and the lipid peroxidation product MDA were assessed to determine the extent of lipid peroxidation. As shown in Figure 7, at 6 h after rtPA thrombolysis in cerebral infarction, the levels of the negative regulators GSH, GPX4, and SLC7A11 were reduced, and the level of the lipid peroxidation product MDA was increased. These changes were exacerbated by Iron-dextran-induced ferroptosis. However, after acupuncture treatment, compared with both the 6 h rtPA and 6 h rtPA+Iron-dextran groups, the levels of the negative regulators GSH, GPX4, and SLC7A11 were significantly increased, and the level of the lipid peroxidation product MDA was decreased in both the 6 h rtPA+Acu group and the 6 h rtPA+Iron-dextran+Acu group. These findings demonstrate that acupuncture can effectively counteract the antioxidant defense system impairment induced by delayed rtPA thrombolysis and ferroptosis. By upregulating key antioxidant proteins (GPX4 and SLC7A11), restoring GSH content, and reducing MDA levels, acupuncture successfully reestablishes cellular redox homeostasis. This confirms that acupuncture alleviates neuronal damage following delayed thrombolysis in cerebral infarction by modulating ferroptosis-associated lipid peroxidation pathways, thereby providing molecular-level mechanistic insights into its neuroprotective effects.

3.7 Changes in the Mitochondrial Structure of Ferroptosis After Delayed rtPA Thrombolysis in Rats With Cerebral Infarction Induced by Acupuncture

Mitochondria are important intracellular storage sites for iron. Ferroptosis is characterized by the destruction of the mitochondrial structure at the morphological level (Y. Zhang et al. 2021). We observed the ultrastructure of mitochondria in neurons via TEM. In the Sham group, the double membrane and cristae of the mitochondria were intact, with no abnormalities. In the Model group, the mitochondria were shrunken, with increased membrane density, reduced cristae, and ruptured outer membranes. In the 6 h rtPA group, the mitochondria further shrank, with significantly reduced or absent cristae, vacuolization, and outer membrane rupture. Compared with those in the 6 h rtPA group, the mitochondrial structure and shape in the 6 h rtPA+Acu group were essentially normal, with visible cristae. Damage to the cells in the 6 h rtPA+Iron-dextran group was further exacerbated, with severe mitochondrial shrinkage, even fewer cristae, and increased rupture of the outer membranes. Compared with that in the 6 h rtPA+Iron-dextran group, the mitochondrial morphology in the 6 h rtPA+Iron-dextran+Acu group was improved (Figure 8). These results indicate that delayed rtPA thrombolysis post-cerebral infarction induces characteristic ferroptosis-associated mitochondrial damage including ultrastructural alterations such as shrinkage, cristae reduction, and outer membrane rupture. Acupuncture treatment significantly ameliorates these pathological changes, not only restoring near-normal mitochondrial morphology in the 6 h rtPA group but also reversing Iron-dextran-aggravated mitochondrial injury. This confirms that acupuncture counteracts ferroptosis progression by preserving mitochondrial structural and functional integrity, providing direct ultrastructural evidence for its neuroprotective effects against neuronal damage.

In summary, delayed rtPA thrombolysis after cerebral infarction can disrupt the BBB and damage neurons. This leads to changes in neuronal morphology, a reduction in the number of Nissl bodies, and a decrease in the relative fluorescence intensity of NeuN. Meanwhile, it triggers iron metabolism disorders, manifested as the accumulation of Ferritin, an increase in free Fe²⁺, and abnormal expression of iron metabolism-related proteins. It also causes an elevation in the level of lipid peroxidation and damage to the mitochondrial structure. These changes are consistent with the activation of ferroptosis, as they are significantly exacerbated by the stimulation of Iron-dextran. However, XNKQ acupuncture intervention effectively reverses these pathological changes. By means of ferroptosis-related mechanisms such as restoring iron homeostasis, inhibiting lipid peroxidation, and protecting mitochondria, it simultaneously salvages the neuronal damage caused by 6 h rtPA thrombolysis and that aggravated by Iron-dextran. This strongly demonstrates that ferroptosis is a key factor contributing to neuronal injury induced by delayed thrombolysis.