[Study on the underlying mechanisms of "" acupuncture for cerebral ischemia-reperfusion injury based on AMPK/mTOR/ULK1 signaling pathway].

Objectives: To observe the effect of "" (regaining consciousness and opening orifice) acupuncture on cerebral ischemia/reperfusion injury(CI/RI), autophagy and adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/UNC-51-like autophagy-activated kinase 1 (ULK1) signaling pathway, so as to explore its underlying mechanisms in improving CI/RI.

Methods: Male Sprague Dowley (SD) rats were randomly divided into sham-operation group, CI/RI model group, and acupuncture group, with 10 rats in each group. The CI/RI model was replicated by occlusion of the middle cerebral artery with intraluminal filament embolization. Two hours after successful modeling, the rats in the acupuncture group received manual acupuncture stimulation of "Shuigou" (GV26), "Neiguan"(PC6), and "Sanyinjiao" (SP6) with "" needling method, and electroacupuncture (EA) stimulation (2 Hz/15 Hz, 1 mA, 20 min) of PC6 and SP6 on the affected side during needle retention. The intervention was conducted twice a day (10:00 and 16:00) for 7 times altogether. The severity of neurological deficits was assessed using Zausinger's six-point method. TTC staining was used to detect the volume of cerebral infarction. H.E. staining was used to observe the histopathological and morphological changes of the infarct brain tissues. A transmission electron microscopy (TEM) was used to observe the neuronal ultrastructure and autophagosome formation in the hippocampal tissue. Western blot was used to detect the protein expression levels of microtubule-associated protein light chain 3-II (LC3-II), AMPK, p-AMPK, mTOR, p-mTOR, ULK1 and p-ULK1 in the hippocampal tissues on the ischemic side.

Results: Compared with the sham-operation group, the model group had a significantly reduced neurological deficit score (<0.001), significantly decreased expression levels of LC3-II, ratios of p-AMPK/AMPK and p-ULK1/ULK1 (<0.001), and significantly increased percentage of cerebral infarct volume and p-mTOR/mTOR ratio (<0.001). In comparison with the model group, the acupuncture group had a significant increase in the neurological deficit score and expression of LC3-II, ratios of p-AMPK/AMPK and p-ULK1/ULK1 (<0.001, <0.05), and a significant decrease in the percentage of cerebral infarct volume and p-mTOR/mTOR ratio (<0.001). Results of H.E. staining showed loose arrangement and reduction in the number of neurons, with pyknotic nucleus, obvious cavities, and appearance of degenerated and necrotic neurons in the ischemic brain tissue, which was relatively milder in the severity of CI/RI of the acupuncture group. Results of TEM showed injured and fractured membrane of neurons, moderate cell edema, mitochondrial swelling and reduction in number, dilation of the endoplasmic reticulum, and appearance of autophagosomes in the hippocampus of the model group, while in the acupuncture group, the nuclear membrane of nerve cells was relatively intact, the numbers of normal mitochondria and endoplasmic reticulum were increased, with appearance of typical autophagosomes.

Conclusions: "" acupuncture can improve neurological behavior and reduce infarction volume in rats with CI/RI, which may be related to its functions in regulating AMPK/mTOR/ULK1 signaling, and promoting cellular autophagy.