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

doi:10.19852/j.cnki.jtcm.20240203.006

Abstract

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.

Key words: acupuncture, ferritinophagy, ferroptosis, ferritin, nuclear receptor coactivator 4, cerebral ischaemia-reperfusion injury

ZHANG Xinchang, HUANG Zheng, HUANG Peiyan, YANG Mengning, ZHANG Zhihui, NI Guangxia. Mechanism of acupuncture in attenuating cerebral ischaemia-reperfusion injury based on nuclear receptor coactivator 4 mediated ferritinophagy[J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 345-352.

Figures/Tables 5

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.

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.

Table 1 Acupuncture reduced the levels of Fe2+ and MDA in rats with CIRI (x? ± s, n = 6)

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

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.

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.

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