[Acupuncture regulates autophagy in hippocampal neurons of cerebral ischemia/reperfusion injured rats by activating type â ¢ PI3K/Beclin-1 pathway].

OBJECTIVE: To observe the effects of acupuncture on the expression of type Ⅲ phosphatidylinositol 3-hydroxykinase (PI3K) and Beclin-1 in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore the mechanism of acupuncture in regulating type Ⅲ PI3K pathway to activate autophagy in the hippocampal neurons of CI/RI rats. METHODS: SD rats were randomly divided into sham operation group (n=11) and operation group. Then after successful modeling, rats in the operation group were randomly divided into model, acupuncture, model+3-MA and acupuncture+3-MA groups, with 11 rats in each group. The model of CI/RI was established by occlusion of the middle cerebral artery. Rats in the model+3-MA and acupuncture+3-MA groups were injected with 3-MA (400 nmol/ 5 µL) 5 µL into the lateral ventricle 30 min before reperfusion. Rats in the acupuncture and acupuncture+3-MA groups were punctured with filiform needles at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20) and stimulated manually once every 15 min. The acupuncture intervention was conducted for 30 min each time, once every 12 h for a total of 7 times. The degree of neurological impairment was evaluated 2 h after reperfusion and after intervention by Garcia score. After intervention, the percentage of cerebral ischemic area was observed by TTC staining, the protein expression levels of type Ⅲ PI3K, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B), lysosome associated membrane protein 2 (Lamp2) and P62 in ischemic hippocampal tissue were detected by Western blot, the ultrastructure of neurons in ischemic hippocampus was observed by transmission electron microscopy (TEM). RESULTS: Compared with the sham operation group, the Garcia score was decreased (P<0.01), the percentage of cerebral ischemic area was increased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were decreased (P<0.01), and the expression level of P62 protein was increased (P<0.01) in ischemic hippocampal tissue in the model group. Compared with the model group, the Garcia score was increased (P<0.01), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were increased (P<0.01, P<0.05) and the expression level of P62 was decreased (P<0.01) in ischemic hippocampal tissue in the acupuncture group； the percentage of cerebral ischemic area was increased (P<0.05), the expressions of type Ⅲ PI3K and Beclin-1 were decreased (P<0.01) and the expression level of P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the mo-del+3-MA group. Compared with the model +3-MA group, the Garcia score was increased (P<0.05), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ in ischemic hippo-campal tissue were increased (P<0.01, P<0.05) in the acupuncture+3-MA group. Compared with the acupuncture group, the Garcia score was decreased, the percentage of cerebral ischemic area was increased (P<0.05, P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, Lamp2 proteins were decreased (P<0.01, P<0.05) and P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the acupuncture+3-MA group. The results of TEM showed that the edema of neurons was heavier, and few hypolysosomes existed in the model group; there was no obvious damage to neuronal structure, intracellular matrix was abundant, and a few lysosomes existed in the acupuncture group; the neuronal cells had mild edema and primary lysosomes were present in the acupuncture +3-MA group. CONCLUSION: Acupuncture can improve the symptoms of neurological impairment and reduce the percentage of cerebral ischemic area in rats with CI/RI. The mechanism may be related to regulating type Ⅲ PI3K/Beclin-1 pathway, up-regulating the expressions of autophagy related factors LC3B-Ⅱ and Lamp2, and down-regulating the expression of P62.

[Acupuncture mediated miR-34c-5p regulates the autophagy of hippocampal neurons in rats with cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of acupuncture on the expression of miR-34c-5p, autophagy-related proteins and apoptosis rate in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore its mechanism in regulating autophagy in hippocampal neurons in CI/RI rats. METHODS: SD rats were randomly divided into sham operation, model, medication and acupuncture groups, with 20 rats in each group. The rat model of CI/RI was established by occlusion of the middle cerebral artery. In the acupuncture group, "Dazhui" (GV14), "Baihui" (GV20) and "Shuigou" (GV26) were punctured with filiform needles and stimulated manually once every 15 min, for 30 min. The rats of the medication group were intraperito-neally injected with edaravone (5 mg/kg). The treatment was conducted once every 12 h for a total of 7 times. The neurological de-ficit score of all the rats were evaluated according to Garcia's methods, and TTC staining was employed to assess the cerebral ischemic area (percentage of cerebral infarct area, CIA). Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal neurons. The expression of hippocampal miR-34c-5p was measured by real-time PCR, and the protein expressions of hippocampal LC3B, Beclin1 and p62 were measured by Western blot. The apoptosis rate of ischemic brain tissue was observed by TUNEL staining. RESULTS: Compared with the sham operation group, the neurological deficit score, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly decreased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly increased (P<0.01) in the model group. Compared with the model group, the neurological deficit scores, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly increased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly decreased (P<0.01) in the medication and acupuncture groups. Compared with the medication group, the expression of miR-34c-5p was significantly increased (P<0.01). The results of electron microscope showed that the neurons in the acupuncture and medication groups were less damaged than those in the model group, the cells showed mild edema, and the structures were relatively complete. Some normal organelles could be seen, and autophagy bodies, autophagy lysosomes and their encapsulated organelles could still be observed. CONCLUSION: Acupuncture can improve the neurological deficit and reduce the area of cerebral infarction in CI/RI rats, which is closely with its effect in promoting hippocampal neuronal autophagy and anti-apoptosis via up-regulating the expression of miR-34c-5p.