[Effect of ZHU Lian's acupuncture stimulation method on neurological function in rats with ischemic stroke based on the miR-34a-5p/YWHAG pathway].

Ischemic stroke model rats showed significantly increased neurological deficit scores, cerebral infarct volume, neuronal apoptosis, and inflammatory markers compared to sham operation rats.

• Compared with the sham operation group, the model group showed increased Zea-Longa scores, percentage of cerebral infarct volume, and neuronal apoptosis rate in the ischemic cortical tissue (P<0.05).
• Expression levels of IL-6, TNF-α, and miR-34a-5p mRNA were increased in the model group compared to sham operation group (P<0.05).
• YWHAG mRNA and protein expression were decreased in the model group compared to sham operation group (P<0.05).
• The IS model was established using the suture occlusion method in 72 clean-grade male SD rats, with 50 successfully modeled rats used in the study.

ZHU Lian's acupuncture stimulation method reduced neurological deficits, cerebral infarct volume, neuronal apoptosis, and inflammatory markers in IS model rats.

• Compared with the model group, the acupuncture group showed decreased Zea-Longa scores, percentage of cerebral infarct volume, and neuronal apoptosis rate in the ischemic cortical tissue (P<0.05).
• Expression levels of IL-6, TNF-α, and miR-34a-5p mRNA were decreased in the acupuncture group compared to the model group (P<0.05).
• YWHAG mRNA and protein expression were increased in the acupuncture group compared to the model group (P<0.05).
• Acupuncture was applied at GV26 (Shuigou), GV20 (Baihui), GV14 (Dazhui), and bilateral PC6 (Neiguan) and LI4 (Hegu) once daily for 5 days followed by 2 days off, continuing for 21 days.

The positive drug group (nimodipine) also significantly reduced neurological deficits, infarct volume, apoptosis, and inflammatory markers similar to the acupuncture group.

• Compared with the model group, the positive drug group showed decreased Zea-Longa scores, percentage of cerebral infarct volume, and neuronal apoptosis rate (P<0.05).
• IL-6, TNF-α, and miR-34a-5p mRNA expression levels were decreased in the positive drug group compared to the model group (P<0.05).
• YWHAG mRNA and protein expression were increased in the positive drug group compared to the model group (P<0.05).
• Nimodipine was administered at 10.8 mg/kg via gavage once daily for the same 21-day treatment period.

Intracerebroventricular injection of miR-34a-5p agomir reversed the beneficial effects of acupuncture in IS rats.

• Compared with the acupuncture+agonist control group, the acupuncture+agonist group showed increased Zea-Longa scores, percentage of cerebral infarct volume, and neuronal apoptosis rate in the ischemic cortical tissue (P<0.05).
• IL-6, TNF-α, and miR-34a-5p mRNA expression levels were increased in the acupuncture+agonist group compared to the acupuncture+agonist control group (P<0.05).
• YWHAG mRNA and protein expression were decreased in the acupuncture+agonist group compared to the acupuncture+agonist control group (P<0.05).
• The miR-34a-5p agomir and negative control (agomir NC) were administered by intracerebroventricular injection at a dose of 5 μL.

miR-34a-5p and YWHAG have a targeted regulatory relationship confirmed by dual-luciferase assay.

• The dual-luciferase assay verified the relationship between miR-34a-5p and YWHAG (P<0.05).
• miR-34a-5p was upregulated in IS model rats while YWHAG was downregulated, indicating an inverse regulatory relationship.
• Acupuncture treatment downregulated miR-34a-5p and upregulated YWHAG, consistent with miR-34a-5p acting as a negative regulator of YWHAG.
• YWHAG refers to tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein γ.

The acupuncture+agonist control group (receiving agomir NC plus acupuncture) showed similar beneficial effects to the acupuncture group alone.

• Compared with the model group, the acupuncture+agonist control group showed decreased Zea-Longa scores, percentage of cerebral infarct volume, neuronal apoptosis rate, IL-6, TNF-α, and miR-34a-5p mRNA expression (P<0.05).
• YWHAG mRNA and protein expression were increased in the acupuncture+agonist control group compared to the model group (P<0.05).
• This group received intracerebroventricular injection of negative control (agomir NC) at 5 μL followed by the same acupuncture protocol.
• These results confirm that the negative control injection did not interfere with the acupuncture effects, validating the agomir reversal findings.