电针百会、神庭调控BNIP3L介导的线粒体自噬和减轻脑缺血再灌注损伤的机制研究CSCD

目的:探讨电针百会、神庭调节线粒体自噬减轻脑缺血再灌注损伤的作用机制。方法:采用随机数字表法将60只SD大鼠随机分为假手术组15只和手术组45只。假手术组只分离、暴露血管,不结扎,不插入尼龙线。手术组大鼠采用大脑中动脉线栓阻塞法(MCAO)制备大鼠局灶性脑缺血-再灌注(I/R)模型,术后2 h采用Zea Longa法进行神经功能评分,最终纳入30只手术组大鼠。进一步将纳入的手术组大鼠随机分为模型组、电针组和电针+3-MA组,每组10只。造模分组成功后,各组给予电针等相应方式干预7 d,采用Zea Longa法于第1天、第3天、第5天、第7天再次进行神经功能评估。干预7 d后获取各组大鼠左侧大脑皮层组织,苏木精-伊红(HE)染色后观察脑组织病理学变化,Western blot法检测LC3-Ⅱ/LC3-Ⅰ蛋白表达水平,组织免疫荧光技术检测线粒体自噬相关蛋白表达共定位情况(BNIP3L和SQSTM1标记),TUNEL法检测各组大鼠神经细胞凋亡水平。结果:(1)造模2 h后手术组大鼠神经功能评分均显著升高,假手术组大鼠未表现出神经功能缺损症状,评分均为0分。干预后第7天模型组及电针+3-MA组大鼠神经功能评分仍显著升高,与假手术组比较,差异有统计学意义(P<0.05)。而电针组大鼠在电针干预后神经功能评分显著降低,与模型组及电针+3-MA组比较,差异均有统计学意义(P<0.05)。(2)HE染色结果可见,假手术组神经元胞体较大,多角形(多个突起),核着色浅,胞质可见特征性结构尼氏体;模型组神经元皱缩,胞质结构不清,胞浆嗜依红染色增强,尼氏体边聚或消失,核固缩,有的胞体变形缩小,呈三角形,细胞周围间隙增宽;电针组可以在一定程度上减轻缺血再灌注所致的病理损伤。(3)Western blot结果表明,MCAO后第7天模型组自噬水平(LC3-Ⅱ/LC3-Ⅰ)下降,与假手术组比较,差异有统计学意义(P<0.05);电针组可以激活自噬水平,与模型组比较,差异有统计学意义(P<0.05)。(4)模型组中皮质梗死区域线粒体自噬水平缺失,表现为红色荧光和绿色荧光强度减弱,而电针干预可以激活梗死区域神经细胞的线粒体自噬,表现为红色荧光和绿色荧光的表达增高及共定位增强(橘黄色)。(5)TUNEL检测结果表明,模型组大鼠凋亡率升高,与假手术组比较,差异有统计学意义(P<0.05);电针组的凋亡率降低,与模型组比较,差异有统计学意义(P<0.05)。电针+3-MA组结果表明,自噬抑制剂3-MA可以逆转电针对MCAO模型大鼠的神经保护作用。结论:电针百会、神庭可以减轻脑缺血再灌注模型大鼠神经功能损伤,其具体机制和调控BNIP3L介导的线粒体自噬、减轻脑缺血再灌注损伤和细胞凋亡有关。

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

Objective: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 BNIP3 L-mediated mitophagy.Methods:A total of 60 SD rats were randomly divided into sham operation group(n=15)and operation group(n=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 2 h 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 BNIP3 L and SQSTM1 labeled.The TUNEL method detected the apoptosis rate of rat nerve cells in each group.Results: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 7 th 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(P<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(P<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 7 th day after MCAO,there wass a significant difference compared with the sham operation group(P<0.05),while the electroacupuncture group could activate the level of autophagy,which was significantly different from the model group(P<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(P<0.05).However,the apoptotic rate of the electroacupuncture group decreased,which was significantly different from that of the model group(P<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.Conclusion:Electroacupuncture at Baihui and Shenting acupoints can alleviate neurological damage in rats with cerebral ischemia-reperfusion.The specific mechanism is related to regulating BNIP3 L-mediated mitophagy to reduce cerebral ischemia-reperfusion injury and cell apoptosis.