Mechanism of acupuncture in attenuating cerebral ischaemia-reperfusion injury based on nuclear receptor coactivator 4 mediated ferritinophagy

Abstract

OBJECTIVE:

To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy.

METHODS:

Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe²⁺) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining.

RESULTS:

Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe²⁺ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe²⁺.

CONCLUSIONS:

Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.

1. INTRODUCTION

Stroke is a devastating neurological disease that affects the global population.¹ Ischaemic stroke represents 87% of all types of strokes in the US² , and accounts for more than 80% of stroke cases in China.³ The key to treating acute ischaemic stroke is to restore cerebral blood flow perfusion in the ischaemic area as soon as possible.⁴ However, it should be noted that cerebral blood flow reperfusion can cause cerebral ischaemia-reperfusion injury (CIRI), which results in further damage to some ischaemic brain tissue and the nervous system. Multiple mechanisms are involved in CIRI, such as various forms of programmed cell death.⁵

A newly iron-dependent type of cell death, ferroptosis, was proposed in 2012.⁶ It is characterized by intracellular iron ion accumulation and elevated lipid peroxidation. Studies have reported that the administration of a ferroptosis inhibitor (ferrostatin-1) after reperfusion can reduce the infarct size and improve brain ischaemic injury.⁷ It has been suggested that ferroptosis plays an important role in CIRI.^8,9 However, its upstream regulatory targets are obscure and require further study.

Recent research has discovered that ferroptosis can be initiated by ferritinophagy. Ferritinophagy was first proposed by Mancias et al ¹⁰ and is regulated by nuclear receptor coactivator 4 (NCOA4). NCOA4 can directly interact with ferritin and then assist ferritin breakdown and release into lysosomes. This process has been termed ferritinophagy.¹¹ In the cytosol, ferritin consists of ferritin heavy chain subunits (FTH1) and ferritin light chain subunits (FTL). NCOA4 selectively recognizes the FTH1 subunit in ferritin.¹² Mancias et al ¹³ showed that FTH1 is an essential structure for the binding of ferritin to NCOA4. Animal experiments have confirmed that the deletion of NCOA4 significantly abolishes the ferritinophagy induced by CIRI.¹⁴ Therefore, regulating ferritinophagy is crucial in preventing the build-up of cellular labile iron and further alleviating CIRI after ischaemic stroke.

Acupuncture is an effective treatment for ischaemic stroke that acts by regulating a sequence of pathological responses.¹⁵ Acupuncture has been reported to inhibit ferroptosis and regulate the expression level of FTH1.¹⁶ Whether acupuncture can ameliorate CIRI by regulating ferritinophagy is still unknown. Inspired by these findings, we aimed to observe and gain a better understanding of the potential mechanism of acupuncture treatment for CIRI, by focusing on NCOA4-mediated ferritinophagy.

2. MATERIALS AND METHODS

2.1. Animals model

A total of 136 healthy adult male Sprague-Dawley rats of specific pathogen free grade, seven-week-old, weighing 240-280 g, were fed in the experimental animal centre at Nanjing University of Chinese Medicine and allocated to four groups by random number table method: sham group, model group, acupuncture group, and sham acupuncture group. Experimental procedures were performed under the approval of the Animal Ethics Committee of Nanjing University of Chinese Medicine (Ethics Permit No. 202210A075).

2.2. MCAO model

Anaesthesia was induced and maintained with isoflurane (1.5%-2%). The rats were placed in the supine position. Following temporary ligation of the right common carotid artery (CCA), an incision in the external carotid artery (ECA) was made using microscopic scissors. A 0.26-mm-diameter nylon monofilament was first inserted into the right ECA through the incision, passed distally through the internal carotid artery (ICA), and then advanced approximately 2 cm from the carotid bifurcation along the ICA to block the middle cerebral artery. After occlusion for 2 h, the nylon monofilament was gently withdrawn to permit reperfusion for 24 h.

For the sham group, the rats were subjected to the same surgery except that the middle cerebral artery was occluded.

2.3. Acupuncture method

At 2 h after ischaemia, the rats in the acupuncture and sham acupuncture groups received one session of treatment.^17,⇓-19 And the treatment duration of acupuncture was 30 min. In this study, Neiguan (PC6) and Shuigou (GV26) acupoints were selected for treatment. Selection and location of animal acupoints refer to the names and locations of common acupoints in Laboratory Animals Part 2: Rats.²⁰ We used Disposable sterile acupuncture needles (0.18 mm × 13 mm, Huatuo, Suzhou, China) for the acupuncture group, and disposable purpose-built blunt needles (0.18 mm × 13 mm, Huatuo, Suzhou, China) for the sham acupuncture group. The specific operations of the acupuncture group were as follows: bilateral PC6 was perpendicularly inserted through the pad at a depth of approximately 3 mm while GV26 was inserted towards the nasal septum through the pad at a depth of 2-3 mm. Manipulations involving lifting, thrusting and twirling were performed on each needle for 1 min. And the specific operations of the sham acupuncture group differed from those of the acupuncture group, adhesive pads were attached to the skin at the PC6 and GV26 acupoints. The blunt needles only contact the skin surface of the PC6 and GV26 through the adhesive pads without penetrating the skin. No manipulation was performed on the needles.

2.4. Neurological function assessment

The Bederson (0-5) score^21,22 was applied to evaluate neurological function at 2 h after cerebral ischaemia and 24 h after reperfusion. The Bederson scores ranged from 0 to 5. Grade 0: no observable deficit. Grade 1: forelimb flexion. Grade 2: decreased resistance to lateral push (and forelimb flexion) without circling. Grade 3: same behaviour as grade 2 but with circling. Grade 4: turn spontaneously to the opposite side. Grade 5: death.

2.5. Measurement of the infarct volume

The infarct volume was assessed at 24 h after reperfusion by triphenyltetrazolium chloride (TTC) staining. The brain sections were incubated in 2% TTC (Sigma, St. Louis, MO, USA) at 37 ℃ for 10 min. The infarct volume was quantified using computerized image analysis systems.

2.6. Western blotting

The rats’ right ischaemic cerebral cortex was separated. Bicinchoninic acid assay kits (Beyotime, Nanjing, China) were used to quantify the concentrations of the extracted proteins. Equal amounts (30 μg) of total protein from each sample were separated by using a 5%-12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Following electrophoresis, the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.22 μm). Then, 5% bovine serum albumin containing tris-buffered saline was used to block the membranes for 2 h. Next, we incubated the membranes overnight at 4 ℃ with the following specific primary antibodies: anti-NCOA4 (sc-373739, Santa Cruz Biotechnology, Dallas, TEX, USA), anti-ferritin heavy chain (ab183781, Abcam, Cambridge, UK), and β-tubulin (10094-1-AP, Proteintech, Wuhan, China). The membranes were washed three times with TBST and then incubated with appropriate secondary antibodies (goat anti-mouse IgG, SA00002-1, Proteintech, Wuhan, China and goat anti-rabbit IgG, 33101ES60, Yeasen, Shanghai, China) at 4 ℃ for 1 h. After being washed, the membranes were treated with enhanced ECL luminescent working solution. Lastly, the immunoreactive bands were detected with a Bioshine ChemiQ4800 imaging system.

2.7. Immunofluorescence staining

OCT-embedded frozen brains were cut into 12-µm-thick slices for immunofluorescence staining. Tissue slices were blocked for 1 h with 5% goat serum in phosphate-buffered saline and 0.3% Triton X-100. After that, the slices were stained overnight at 4 ℃ using the following primary antibodies: anti-ferritin (ab75973, Abcam, Cambridge, UK), anti-NCOA4 (Santa Cruz Biotechnology, Dallas, TEX, USA), anti-NeuN (ab104224, Abcam, Cambridge, UK), and anti-Microtubule-Associated Protein 1 Light Chain 3 (LC3, ab192890, Abcam, Cambridge, UK). Then, the slices were incubated with Alexa Fluor 488- or 674-labelled antibodies followed by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, Nanjing, China) solution and were analysed by fluorescence microscopy (Zeiss Axio Scope A1, Oberkochen, Germany).

2.8. Iron measurements and Malondialdehyde (MDA) assay

The rats were euthanized at 24 h after reperfusion, and the right ischaemic cerebral cortex was separated. The levels of Fe²⁺ in the rat brains were detected using an Iron Assay Kit (I291, Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. The absorbance of the wells was measured on a microplate reader at a wavelength of 593 nm. An MDA assay kit (Beyotime, Nanjing, China) was used to measure the MDA contents of the rat brains in accordance with the instructions.

2.9. Statistical analysis

Normally distributed data are expressed as the means ± standard deviation. One-way analysis of variance (ANOVA) was used for comparisons across multiple groups, and pairwise comparisons between groups were performed using the least significant difference test. All statistical analyses were performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). If the variance was not homogeneous, a nonparametric test was used. Statistical significance was set at ^aP < 0.05 or ^bP < 0.01.

3. RESULTS

3.1. Acupuncture Improved Neurological Function in Rats with CIRI

To observe the neuroprotective effect of acupuncture on CIRI, we evaluated rat neurological function by taking their Bederson (0-5) scores at 2 h after cerebral ischaemia and 24 h after reperfusion.

At 24 h after reperfusion, the neurological deficit score of the model group was higher than that of the sham group. However, compared with the model and sham acupuncture groups, the neurological deficit score was significantly decreased in the acupuncture group (P < 0.05, Figure 1A, 1B). The results indicated that acupuncture can improve neurological function in rats after CIRI.

Figure 1. Acupuncture improved neurological function in rats with CIRI.

A: the neurological deficit score of rats 2 h after cerebral ischaemia in each group (n = 10); B: the neurological deficit score of rats 24 h after reperfusion in each group (n = 10). We took the tissue of the ischemic area of the right cerebral cortex from each group of rats at the 24-h ischemic time point and detected it using relevant reagent kits. At the time point of 2-h of ischemia, we performed acupuncture and sham acupuncture operations. We evaluated the neurobehavioral scores (Bederson 0-5 scores) of each group of rats at the time points of ischemia for 2-h and 24-h, respectively. CIRI: cerebral ischaemia-reperfusion injury. h: hour. Data were statistically analysed by least significant difference test. Data were expressed as mean ± standard deviation. ^aP < 0.05, compared with the acupuncture group.

3.2. Acupuncture decreased infarct volume in rats with CIRI

The cerebral infarct volume in each group was analysed by TTC staining at 24 h after reperfusion. The cerebral infarction volume of the model group was higher than that of the sham group (P < 0.01, Figure 2). However, the cerebral infarction volume in the acupuncture group was markedly decreased compared with that in the model and sham acupuncture groups (P < 0.01, Figure 2). The results demonstrated that acupuncture can reduce the cerebral infarct volume in rats after CIRI.

Figure 2. Acupuncture decreased infarct volume in rats with CIRI.

A: representative images of brain slices stained with TTC; B: comparison of the percentages of cerebral infarction volumes in rats from each group. We took the tissue of the ischemic area of the right cerebral cortex from each group of rats at the 24-h ischemic time point and detected it using relevant reagent kits. At the time point of 2-hour of ischemia, we performed acupuncture and sham acupuncture operations. TTC staining (8 g TTC, 40 mL PBS). CIRI: cerebral ischaemia-reperfusion injury; TTC: triphenyltetrazolium chloride. Data were statistically analysed by least significant difference test. Data were expressed as mean ± standard deviation (n = 6). ^aP < 0.01, compared with the sham group; ^bP < 0.01, compared with the acupuncture group.

3.3. Acupuncture reduced the levels of Fe²⁺ and MDA in rats with CIRI

Ferroptosis is characterized by iron-dependent lipid peroxidation. MDA is a product of lipid peroxidation that can indicate the degree of lipid peroxidation. Therefore, we measured the levels of Fe²⁺ and MDA in the right ischaemic cerebral cortex from rats in each group. At 24 h after reperfusion, the Fe²⁺ and MDA levels in the model group were significantly higher than those in the sham group (P < 0.01, Table 1). This finding suggested that ferroptosis occurred in cells after cerebral ischaemia-reperfusion. However, acupuncture significantly reduced the Fe²⁺ and MDA levels compared to the model group and the sham acupuncture group (P < 0.01, Table 1). The results showed that ferroptosis was involved in CIRI and that acupuncture decreased the degree of ferroptosis after cerebral ischaemia-reperfusion.

Table 1.

Acupuncture reduced the levels of Fe²⁺ and MDA in rats with CIRI (x̅ ± s, n = 6)

Item	Sham group	Model group	Acupuncture group	Sham acupuncture group
Fe2+	4.0±0.9a	10.3±1.0b	6.4±1.3	9.9±2.0b
MDA	2.6±0.9a	8.6±0.8b	6.4±0.5	8.2±0.9b

Notes: We took the tissue of the ischemic area of the right cerebral cortex from each group of rats at the 24-h ischemic time point and detected it using relevant reagent kits. At the time point of 2-h of ischemia, we performed acupuncture and sham acupuncture operations. Ferrous ion: Fe²⁺(mmol/ml); MDA: malondialdehyde (μmol/mg); CIRI: cerebral ischemia reperfusion injury. Data were statistically analyzed by least significant difference test. Data were expressed as mean ± standard deviation. ^aP < 0.01, compared with the Model group; ^bP < 0.01, compared with the acupuncture group.

3.4. Acupuncture Inhibited the Level of Ferritinophagy in Rats with CIRI

To verify acupuncture can regulate the level of ferritinophagy in rats with CIRI, we conducted immunofluorescence staining to detect the expression and colocalization of ferritinophagy-associated proteins (ferritin, NCOA4 and LC3) at 24 h after reperfusion.

First, we examined the colocalization of ferritin and neurons in the different groups. Compared with that in the sham group, the colocalization of ferritin with neurons in the model group was significantly higher (P < 0.01). However, the colocalization of ferritin with neurons in the acupuncture group was markedly lower than that in both the model and sham acupuncture groups (P < 0.01) (Figure 3A, 3B). Then, we examined the colocalization of NCOA4 and LC3 in different groups. Compared with that in the sham group, the colocalization of NCOA4 with LC3 in the model group was significantly higher (P < 0.01). However, the colocalization of NCOA4 with LC3 in the acupuncture group was notably lower than that in both the model and sham acupuncture groups (P < 0.01, Figure 3C, 3D).

Figure 3. Acupuncture inhibited the level of ferritinophagy in rats with CIRI.

A: representative immunofluorescence images of ferritin increase in neurons (NeuN) (scale bar = 20 μm, n = 6, use of immunofluorescence staining); B: bar graphs showed quantitative evaluation of A; C: representative images show the colocalization of NCOA4 (red) with LC3 (green) (scale bar = 20 μm, n = 6, use of immunofluorescence staining); D: bar graphs showed quantitative evaluation of B. A1: ferritin of sham group. A2: ferritin of model group. A3: ferritin of acupuncture group. A4: ferritin of sham acupuncture group. A5: NeuN of sham group. A6: NeuN of model group. A7: NeuN of acupuncture group. A8: NeuN of sham acupuncture group. A9: DAPI of sham group. A10: DAPI of model group. A11: DAPI of acupuncture group. A12: DAPI of sham acupuncture group. A13: merge of sham group. A14: merge of model group. A15: merge of acupuncture group. A16: merge of sham acupuncture group. C1: NCOA4 of sham group. C2: NCOA4 of model group. C3: NCOA4 of acupuncture group. C4: NCOA4 of sham acupuncture group. C5: LC3 of sham group. C6: LC3 of model group. C7: LC3 of acupuncture group. C8: LC3 of sham acupuncture group. C9: DAPI of sham group. C10: DAPI of model group. C11: DAPI of acupuncture group. C12: DAPI of sham acupuncture group. C13: merge of sham group. C14: merge of model group. C15: merge of acupuncture group. C16: merge of sham acupuncture group. We took the tissue of the ischemic area of the right cerebral cortex from each group of rats at the 24-h ischemic time point and detected it using relevant reagent kits. At the time point of 2-h of ischemia, we performed acupuncture and sham acupuncture operations. NCOA4: nuclear receptor coactivator 4; LC3: microtubule-associated protein 1 light chain 3; NeuN: neuron; merge: NCOA4 with LC3; CIRI: cerebral ischemia reperfusion injury. Statistical significance between multiple groups was determined in this study primarily using one-way or two-way analysis of variance. All data are expressed as mean ± standard deviation (n = 6). ^aP < 0.01, compared with the sham group; ^bP < 0.01, compared with the acupuncture group; ^cP < 0.01, compared with the model group.

These results visually verified the upregulation of ferritin at 24 h after cerebral ischaemia-reperfusion and indicated that acupuncture can decrease the colocalization of ferritin in neurons. Moreover, acupuncture can reduce the level of ferritinophagy by regulating the expression of ferritin, NCOA4 and LC3.

3.5. Acupuncture inhibited NCOA4-medicated ferritinophagy in rats with CIRI

NCOA4 has been identified as a selective cargo receptor for ferritinophagy. We next sought to further elucidate the key targets of acupuncture in regulating ferritinophagy after cerebral ischaemia-reperfusion.

First, we detected the NCOA4 and FTH1 protein expression by Western blotting 24 h after cerebral ischaemia-reperfusion. Compared with that in the sham group, the expression of NCOA4 and FTH1 in the model group was significantly increased (P < 0.01). However, the expression levels of NCOA4 and FTH1 in the acupuncture group were markedly decreased compared with those in the model (P < 0.01) and sham acupuncture groups (P < 0.05) (Figure 4A-4D). In addition, we observed the colocalization of NCOA4 and ferritin at 24 h after cerebral ischaemia-reperfusion by immunofluorescence staining. Compared with that in the sham group, the colocalization of NCOA4 and ferritin in the model group was significantly higher. However, the colocalization of NCOA4 and ferritin in the acupuncture group was markedly lower than that in both the model and sham acupuncture groups (P < 0.01, Figure 4E, 4F).

Figure 4. Acupuncture inhibited NCOA4-medicated ferritinophagy in rats with CIRI.

A: Western blot showing NCOA4 expression in CIRI rats (n = 6). B: bar graphs showed quantitative evaluation of A. C: Western blot showing FTH1 expression in CIRI rats (n = 6). D: bar graphs showed quantitative evaluation of C. E: Representative images show the colocalization of NCOA4 (red) with ferritin (green) (scale bar = 20 μm, n = 6, use of immunofluorescence staining). F: bar graphs showed quantitative evaluation of E. E1: NCOA4 of sham group. E2: NCOA4 of model group. E3: NCOA4 of acupuncture group. E4: NCOA4 of sham acupuncture group. E5: ferritin of sham group. E6: ferritin of model group. E7: ferritin of acupuncture group. E8: ferritin of sham acupuncture group. E9: DAPI of sham group. E10: DAPI of model group. E11: DAPI of acupuncture group. E12: DAPI of sham acupuncture group. E13: merge of sham group. E14: merge of model group. E15: merge of acupuncture group. E16: merge of sham acupuncture group. We took the tissue of the ischemic area of the right cerebral cortex from each group of rats at the 24-h ischemic time point and detected it using relevant reagent kits. At the time point of 2-h of ischemia, we performed acupuncture and sham acupuncture operations. NCOA4: nuclear receptor coactivator 4; NeuN: neuron; merge: NCOA4 with ferritin; CIRI: cerebral ischemia reperfusion injury. Statistical significance between multiple groups was determined in this study primarily using one-way or two-way analysis of variance. All data are expressed as mean ± standard deviation (n = 6). ^aP < 0.01, compared with the sham group; ^bP < 0.01, compared with the acupuncture group; ^cP < 0.05, compared with the model group; ^dP < 0.01, compared with the model group.

These results demonstrated that the levels of NCOA4 and FTH1 in CIRI rats were significantly increased and that acupuncture inhibited ferritinophagy by decreasing the NCOA4 and FTH1 levels.

4. DISCUSSION

This study aimed to investigate whether acupuncture inhibits ferritinophagy by downregulating NCOA4 expression after CIRI. We confirmed that acupuncture can reduce the colocalization of NCOA4 with LC3 and NCOA4 with ferritin to inhibit NCOA4-mediated ferritinophagy and then decrease the Fe²⁺ and MDA levels to protect neurons from CIRI. Our findings show that acupuncture exerts a beneficial effect on CIRI through the modulation of NCOA4-mediated ferritinophagy.

Iron homeostasis is crucial for the normal function of the brain.²³ Under normal physiological conditions, extracellular ferric iron (Fe³⁺) can bind to transferrin (Tf), which is recognized by high-affinity transferrin receptor 1 (TfR1) on the cell membrane. Afterwards, the Fe³⁺-Tf-TfR1 complex is imported into cells by endocytosis to form endosomes.²⁴ In endosomes, Fe³⁺ is released from Tf and catalysed by ferrous reductase to form Fe²⁺, which is subsequently transported into the cytoplasm.²⁵ The majority of Fe²⁺ released from cells is eventually used in a variety of physiological processes, such as the Fenton reaction. However, iron homeostasis in the brain is destroyed after ischaemic stroke. Abnormal iron overload in specific areas of the brain after ischaemic stroke is an important cause of cell death.²⁶ On the one hand, excessive Fe²⁺ produces a large number of ROS through the Fenton reaction. On the other hand, Fe²⁺ participates in the synthesis of lipoxygenase, which catalyses lipid peroxidation. This peroxidation can lead to ferroptosis^27-29 and aggravate brain injury. Our study also confirmed that the levels of Fe²⁺ following cerebral ischaemia-reperfusion were significantly increased. In addition, ferroptosis is accompanied by massive lipid peroxidation, which can further damage the cell membrane and impair brain function. MDA is a lipid peroxidation product that can indicate the degree of lipid peroxidation. We found that brain tissue produced abundant MDA following cerebral ischaemia and reperfusion, which can eventually lead to severe ferroptosis. However, a recent study determined that acupuncture can inhibit ferroptosis by regulating oxidative stress and iron-related proteins.¹⁶ Similarly, our research also showed that acupuncture can decrease the Fe²⁺ and MDA levels, regulate FTH1 expression, and attenuate CIRI induced by ferroptosis.

Ferritinophagy plays a crucial role in iron homeostasis and ferroptosis conduction.³⁰ NCOA4, a protein on the surface of autophagosomes that targets ferritin for destruction, has been identified as a selective cargo receptor for ferritinophagy.¹³ Mancias et al ¹⁰ also demonstrated that a C-terminal element in NCOA4 and a key surface arginine in FTH1 are required for the delivery of ferritin to the lysosome via autophagosomes. The ferritinophagic flux is determined by the NCOA4 levels, which are in turn regulated by the iron levels. When iron levels are high in cells, NCOA4 is ubiquitinated and degraded by the proteasome, thereby promoting ferritin accumulation and iron capture.³¹ NCOA4-mediated ferritinophagy can lead to ferritin degradation and increase the cellular labile iron level, thus triggering cell ferroptosis.^9,12 NCOA4 inhibition can reduce iron levels, thereby inhibiting lipid peroxidation during ferroptosis.¹⁴ Currently, several studies have demonstrated that ferritinophagy plays an important role in CIRI. During ischaemic stroke, iron homeostasis disruption is accompanied by autophagy, and abnormal autophagy promotes ferritin degradation, resulting in iron overload and ferroptosis.³² Both in vivo and in vitro experiments have demonstrated that ferritinophagy is involved in ischaemic neuronal injury by increasing the levels of cytoplastic NCOA4. During the later phase of CIRI, ferritin levels were increased to a higher level, and Fe²⁺ was released from ferritin, thereby contributing to brain reperfusion injury. Based on our results, we also found that NCOA4 and LC3 expression was increased following cerebral ischaemia-reperfusion, which suggested that NCOA4-mediated ferritinophagy resulted in CIRI. NCOA4 inhibition can reduce Fe²⁺ levels and decrease ferroptosis to protect neurons from CIRI.^33,34

According to the theory of traditional Chinese medicine, ischaemic stroke is the result of an imbalance between Yin and Yang, along with an obstruction of Qi and blood. Acupuncture is considered an effective treatment for ischaemic strokes. In this study, we selected PC6 and GV26 as acupuncture points. GV26 is the acupoint of the governor meridian that travels through the brain and governs the Qi in the body.^35,36 PC6 is the acupoint of the pericardium meridian, which is present in a close physiological and pathological setting of Qi and blood. An animal study found that electroacupuncture can inhibit ferroptosis by regulating iron-related proteins; for example, it increased the FTH1 level in a rat model, thus conferring protection against MCAO.¹⁶ Another study found that electroacupuncture preconditioning can reduce oxidative stress after cerebral ischaemia-reperfusion by regulating iron overload.³⁷

To assess the true effects of acupuncture, we chose to apply non-penetrating acupuncture at acupoints with a blunt needle as the sham acupuncture.³⁸ By analysing the results of each experimental data point in our study, we found that the effect of the acupuncture group was clearly better than that of the sham acupuncture group and the model group, which indicated that acupuncture can alleviate CIRI. For the non-penetrating acupuncture, a blunt needle is used that only contacts the skin surface of the right acupoints through the adhesive pads without penetrating the skin, which is the most common type of sham acupuncture.^39,40 In addition, variations in acupuncture manipulation, timing, duration, acupoints, and frequency all influence the efficacy of acupuncture.⁴¹ More studies are needed to explore the deeper mechanisms underlying the therapeutic effects of acupuncture in CIRI.

In conclusion, acupuncture significantly improved neurological function and reduced the infarct volume in rats with CIRI. Moreover, these effects were probably related to the inhibition of ferritinophagy by attenuating the expression of NCOA4 and FTH1. Our findings provide a new idea for revealing the mechanism by which acupuncture attenuates CIRI.