Mechanism of Electroacupuncture at Baihui and Shenting Alleviating Cerebral Ischemia-reperfusion Injury via Regulating BNIP3L-Mediated Mitophagy

Abstract

<sec><title>Objective</title> The purpose of this study is to explore the mechanism of electroacupuncture at Baihui and Shenting acupoints to alleviate cerebral ischemia-reperfusion injury by regulating BNIP3L-mediated mitophagy. </sec><sec><title>Methods</title> A total of 60 SD rats were randomly divided into sham operation group (<italic>n</italic>=15) and operation group (<italic>n</italic>=45) by using the random number table method. Only the blood vessels were separated and exposed in the sham operation group, without ligation and no nylon thread inserted. The rats in the operation group were treated with the middle cerebral artery occlusion (MCAO) method to prepare the focal cerebral ischemia-reperfusion (I/R) model, and the Zea Longa method was used to score the neurological function 2h after the operation. Finally, 30 rats were included in the operation group. The included operation group rats were further randomly divided into the model group, electroacupuncture group, and electroacupuncture+3-MA group, with 10 rats in each group. After grouping, each group was treated with different methods such as electroacupuncture for 7 days, and the Zea Longa method was used to evaluate the neurological function again on the 1, 3, 5, and 7 days. After 7 days of intervention, the left cerebral cortex tissue of each group was obtained, hematoxylin-eosin (HE) staining was used to observe the pathological changes of the brain tissue, the expression level of LC3-Ⅱ/LC3-Ⅰ protein was detected by Western blot, and the tissue immunofluorescence technology was applied to detect the expression and colocalization of mitophagy-related protein BNIP3L and SQSTM1 labeled. The TUNEL method detected the apoptosis rate of rat nerve cells in each group. </sec><sec><title>Results</title> 1) The neurological scores of the rats in the operation group were significantly increased 2 hours after the MCAO, and the rats in the sham-operation group did not show symptoms of neurological deficit. On the 7th day after the intervention, the neurological scores of the rats in the model group and the electroacupuncture+3-MA group still increased significantly, which were significantly different from those in the sham operation group (<italic>P</italic><0.05). However, the neurological scores of rats in the electroacupuncture group reduced after electroacupuncture intervention, which was significantly different from the model group and electroacupuncture+3-MA group (<italic>P</italic><0.05). 2) The results of HE staining showed that the cell body of neurons in the sham operation group was large, polygonal (multiple protrusions), the nucleus was lightly colored, and the characteristic structure of Nissl body could be seen in the cytoplasm. In the model group, neurons shrank, the cytoplasmic structure was unclear, cytoplasmic eosinophilic staining was enhanced, Nissl bodies gathered or disappeared, nuclear pyknosis, and some cell bodies deformed and shrunk to triangles and the spaces around the cells widened. The electroacupuncture group could reduce the pathological damage caused by ischemia-reperfusion to a certain extent. 3) Western blot results showed that the autophagy level (LC3-Ⅱ/LC3-Ⅰ) of the model group decreased on the 7th day after MCAO, there wass a significant difference compared with the sham operation group (<italic>P</italic><0.05), while the electroacupuncture group could activate the level of autophagy, which was significantly different from the model group (<italic>P</italic><0.05). 4) In the model group, the level of mitophagy in the cortical infarction area was absent, as evidenced by the weakened red fluorescence and the intensity of green fluorescence. However, the intervention of electroacupuncture could activate the mitophagy of nerve cells in the infarct area, which was manifested by increased expression of red fluorescence and green fluorescence and enhanced colocalization (orange). 5) TUNEL test results indicated the apoptotic rate of rats in the model group rising, and there was a significant difference compared with the sham operation group (<italic>P</italic><0.05). However, the apoptotic rate of the electroacupuncture group decreased, which was significantly different from that of the model group (<italic>P</italic><0.05). The results of the electroacupuncture+3-MA group showed that the autophagy inhibitor 3-MA could reverse the neuroprotective effect of electroacupuncture in MCAO model rats. </sec><sec><title>Conclusion</title> Electroacupuncture at Baihui and Shenting acupoints can alleviate neurological damage in rats with cerebral ischemia-reperfusion. The specific mechanism is related to regulating BNIP3L-mediated mitophagy to reduce cerebral ischemia-reperfusion injury and cell apoptosis. </sec>