Abstract: BACKGROUND: Research has found that cellular autophagy is an important target for treating cerebral hemorrhage. Acupuncture improves cerebral blood flow, alleviates inflammatory responses, and promotes nerve regeneration by regulating neuronal apoptosis, pyroptosis, autophagy, and other processes, thereby protecting against nerve injury induced by cerebral hemorrhage.
OBJECTIVE: To explore the potential regulatory mechanism of acupuncture at both Baihui and Xuanli on autophagy response caused by cerebral hemorrhage in rats and the recovery of neural function after cerebral hemorrhage. 
METHODS: Sixty-three male Sprague-Dawley rats were randomly divided into seven groups. (1) Except for the sham operation group, a cerebral hemorrhage model was prepared based on autologous blood injection. (2) In the acupuncture group, acupuncture at both Baihui and Xuanli was initiated on the second day post-modeling, administered twice daily. In the 3-methyladenine group, the autophagy inhibitor 3-methyladenine was injected into the lateral ventricle 15 minutes prior to modeling. (4) In the 3-methyladenine+acupuncture group, the same acupuncture treatment as the acupuncture group was given based on the treatment in the 3-methyladenine group. (5) In the rapamycin group, the autophagy activator rapamycin was injected into the lateral ventricle 15 minutes before modeling. (6) In the rapamycin+acupuncture group, based on the treatment in the rapamycin group, the same acupuncture treatment as the acupuncture group was performed. Neurological dysfunction in rats was assessed using the Longa score at 1, 3, and 7 days after acupuncture. Hematoxylin-eosin staining was used to observe pathological changes in rat brain tissue. Immunohistochemistry was used to detect the expression of the autophagy protein LC3. RT-qPCR was used to detect the expression of autophagy genes. Western blot was used to detect the expression of related proteins in rat brain tissue.
RESULTS AND CONCLUSION: (1) Longa score results showed that acupuncture reduced nerve injury caused by cerebral hemorrhage in rats. (2) In the sham operation group, the structural layers of the rat brain were clear, and cells were arranged regularly; in the model group, there was infiltration of inflammatory cells and microglia; in the acupuncture group, inflammatory injury and cellular swelling decreased over time; in the 3-methyladenine group, inflammatory cell infiltration increased over time, and microglia proliferated freely; in the 3-methyladenine+acupuncture group and the rapamycin group, inflammatory cell infiltration gradually decreased, and neuronal cells gradually arranged in an orderly manner; in the rapamycin+acupuncture group, inflammatory cell infiltration decreased with increasing acupuncture time, and neurons remained normal and arranged regularly. (3) On day 7 of acupuncture, compared with the sham operation group, the model group showed significantly increased LC3 expression (P < 0.05), significantly decreased p62 expression (P < 0.05), and significantly increased p-mTOR and p-p70S6K expression (P < 0.05). Compared with the model group, the acupuncture group showed significantly increased LC3 expression, while p62, p-mTOR, and p-p70S6K expressions were significantly reduced (P < 0.05). Compared with the 3-methyladenine group, LC3 expression was significantly increased, and p62, p-mTOR, and p-p70S6K expressions were significantly reduced (P < 0.05) in the 3-methyladenine+acupuncture group. Compared with the rapamycin group, the rapamycin+acupuncture group showed significantly reduced LC3 expression (P < 0.05), significantly increased p62 expression (P < 0.05), and no significant difference in p-mTOR and p-p70S6K expression (P > 0.05). In conclusion, acupuncture at both Baihui and Xuanli can promote the recovery of neurological function in rats with cerebral hemorrhage and promote autophagy by activating the mTOR/p70S6K signaling pathway.

Key words: acupuncture, intracerebral hemorrhage, autophagy, mTOR/p70 S6K signaling pathway, 3-methyladenine