Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m6A methylation

doi:10.12122/j.issn.1673-4254.2025.05.04

Abstract

Abstract:

Objective To evaluate the effect of eye acupuncture on neural function and angiogenesis of ischemic cerebral tissue in rats, and explore the roles of METTL3-mediated m⁶A methylation and the HIF-1α/VEGF-A signal axis in mediating this effect. Methods Fifty SD rats were randomized into normal control group, sham-operated group, model group, eye acupuncture group and DMOG (a HIF-1α agonist) group. Rat models of cerebral ischemia/reperfusion injury (CIRI) were established using a modified thread thrombus method, and the changes in neurological deficits of the rats after interventions were evaluated. TTC and Nissl staining were used to examine the changes in infarction size and neuronal injury, and cerebral angiogenesis was detected by double-immunofluorescence staining. m⁶A methylation modification level in the brain tissue was detected by ELISA, and RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of METTL3 and HIF-1α/VEGF-A. Results Compared with the control and sham-operated rats, the CIRI rats had significantly higher neurological deficit scores with larger cerebral infarction area, a greater number of CD31- and EDU-positive new vessels, higher expression levels of HIF-1α and VEGF-A, reduced number of Nissl bodies and m6A methylation level, and lowered METTL3 protein and mRNA expressions. All these changes were significantly improved by interventions with eye acupuncture after modeling or intraperitoneal injections of DMOG for 7 consecutive days prior to modeling, and the effects of the two interventions were similar. Conclusion Eye acupuncture can improve neurological deficits in CIRI rat models possibly by promoting cortical angiogenesis via upregulating METTL3-mediated m⁶A methylation and regulating the HIF-1α/VEGF-A signal axis.

Key words: eye acupuncture, cerebral ischemia reperfusion injury, m⁶A methylation modification, HIF-1α/VEGF-A signaling axis, angiogenesis

Yanpeng PU, Zhen WANG, Haoran CHU. Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m⁶A methylation[J]. Journal of Southern Medical University, 2025, 45(5): 921-928.

Figures/Tables 10

Tab.1 Longa function scoring criteria

Phenotype	Score
There was no neurological impairment	0
When lifting the tail, the contralateral forelimb is adducted and flexed	1
When crawling, rotate to the opposite side	2
When standing or crawling, tip to the opposite side	3
No voluntary movement with disturbance of consciousness	4

Tab.2 Bederson scoring criteria

Inspect	Phenotype	Score
Gently grasp the tail and lift the rat 10 cm above the table top. Normal rats should extend their front PAWS straight.	There was no neurological impairment	0
	Brain lesions flexion of the wrist, elbow and shoulder adduction	1
	The above signs+decreased resistance to push on the paralytic side	2
	Move in a circle towards the paralyzed side (rear-end)	3

Tab.3 Primer sequences used for RT-qPCR

Gene	Primer sequence (5'→3')	Product length (bp)
METTL3	F:TGGGGGTATGAACGGGTAGA R:TCCTTTGACACCAACCAAGC	129
HIF-1α	F:TTACAGGATTCCAGCAGACCCA R:GCTGATGCCTTAGCAGTGGTC	145
VEGF-A	F:TAAATCCTGGAGCGTTCACTGTG R:TTCGTTTAACTCAAGCTGCCTC	145
GAPDH	F:GGCAAGTTCAACGGCACAG R:CGCCAGTAGACTCCACGACAT	150

Fig.1 Neurological function scores of the rats in different groups (Mean±SD, n=10). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

Fig.2 Brain TTC staining showing cerebral infarct sizes in different groups (Mean±SD, n=3). A: Blank group; B: Sham operation group; C: Model group; D: Eye acupunctrue group; E: HIF-1α agonist group. *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

Fig.3 Nissl staining of the cortical tissue in different groups (Scale bar=100 μm; Mean±SD, n=3). A: Blank group; B: Sham operation group; C: Model group; D: Eye acupunctrue group; E: HIF-1α agonist group. *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

Fig.4 CD31 and EDU double-immunofluorescence staining of rat cerebral cortex (Original magnification: ×400).

Fig.5 m6A methylation level in rat cerebral cortex (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

Fig.6 Expression of METTL3 and HIF-1α/VEGF-A in rat cerebral cortex detected by Western blotting (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

Fig.7 mRNA expression of METTL3 and HIF-1α/VEGF-A in rat cerebral cortex detected by RT-qPCR (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

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Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m⁶A methylation

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