“醒脑开窍”针刺法调节ATP敏感性钾通道开放对抗脑缺血再灌注损伤的机制研究
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摘要
目的:观察"醒脑开窍"针刺法调节ATP敏感性钾通道开放对抗脑缺血再灌注损伤的作用机制。方法:将84只大鼠随机分为假手术组、模型组、电针组、电针+三磷酸腺苷敏感性钾通道(K_(ATP))阻滞剂组(电针+K_(ATP)阻滞剂组),每组21只。电针+K_(ATP)阻滞剂组大鼠给予K_(ATP)通道阻断剂格列吡嗪(1μmol/5μL)10μL脑室内注入。模型组、电针组、电针+K_(ATP)阻滞剂组采用Zea Longa线栓法复制大脑中动脉缺血再灌注模型,假手术组仅分离颈总动脉及颈外动脉,不插入尼龙线栓。电针组、电针+K_(ATP)阻滞剂组给予针刺"内关""水沟""三阴交"治疗,留针20 min,留针期间将患侧"内关""三阴交"穴分别连接神经穴位刺激仪,施以疏密波,频率2 Hz/15 Hz,电流强度1 mA。首次针刺在动物造模成功90 min后进行,每天10:00、16:00各针刺1次,共3 d。假手术组、模型组大鼠同样抓取,但不予针刺。采用Zausinger六分法评价神经功能,2,3,5-氯化三苯基四氮唑(TTC)染色检测脑梗死体积,流式细胞技术检测海马神经细胞凋亡率,Western-blot技术检测B淋巴细胞瘤-2基因(Bcl-2)、B细胞淋巴瘤因子相关X蛋白(Bax)表达。结果:干预后,电针组神经功能评分明显高于模型组(P<0.01),脑梗死体积及海马神经细胞总凋亡率明显低于模型组(均P<0.05),电针组Bcl-2蛋白表达、Bcl-2/Bax比值较模型组均明显升高(P<0.05,P<0.01),Bax蛋白表达较模型组明显降低(P<0.01);与电针组比较,电针+K_(ATP)阻滞剂组大鼠神经功能评分明显降低(P<0.05),海马神经细胞总凋亡率明显升高(P<0.05),海马组织Bcl-2蛋白表达明显降低(P<0.05),Bax蛋白表达明显升高(P<0.05)。结论:"醒脑开窍"针刺法对脑缺血再灌注损伤大鼠有明显的脑保护作用,其机制可能与调节脑组织K_(ATP)通道开放、减轻神经细胞凋亡密切相关。
Objective To observe the mechanism of "Xingnao Kaiqiao" acupuncture for the opening of ATP sensitive potassium channel(K_(ATP)) against cerebral ischemia reperfusion injury in rats. Methods Eighty-four rats were randomly divided into a sham-operation group, a model group, an electroacupuncture(EA) group, an EA+K_(ATP) blocker group, 21 rats in each group. 10 μL intracerebral injection with glipizide(1 μmol/5 μL) was used in the EA+K_(ATP) blocker group. The cerebral ischemia reperfusion model was established by Zea Longa's suture method in the model group, the EA group and the EA+K_(ATP) blocker group. Rats in the sham-operation group were received the same surgery but without nylon filament insertion. Acupuncture(20 min a time) was performed at "Neiguan"(PC 6), "Shuigou"(GV 26) and "Sanyinjiao"(SP 6) in the EA group and the EA+K_(ATP) blocker group at 10:00 and 16:00 for 3 days, firstly 90 min after model establishment. EA(2 Hz/15 Hz, 1 mA) was connected at the affected "Neiguan"(PC 6) and "Sanyinjiao"(SP 6). The same fixation was used in the sham-operation group and the model group, without EA. Neurological function was assessed by Zausinger's neurologic assessment scale. 2, 3, 5-triphenyltetrazolium chloride(TTC) staining was used to detect infarct volume. Neurocyte apoptosis in the hippocampus was detected by flow cytometry and the protein expressions of B lymphocytoma-2 gene(Bcl-2) and B cell lymphoma factor-associated X protein(Bax) were measured by Western-blot. Results In comparison with the model group, the neurological score of the EA group increased(P<0.01); the infarction volume and the hippocampal neuron's total apoptosis rate of the EA group decreased(both P<0.05); the protein expression of Bcl-2 and Bcl-2/Bax of the EA group increased(P<0.05, P<0.01); and the protein expression of Bax of the EA group decreased(P<0.01). Compared with the EA group, the neurological score of the EA+K_(ATP) blocker group decreased(P<0.05); the total apoptosis rate of hippocampus neurons of the EA+K_(ATP) blocker group increased(P<0.05); the expression of Bcl-2 protein of the EA+K_(ATP) blocker group reduced(P<0.05); the expression of Bax protein of the EA+K_(ATP) blocker group increased(P<0.05). Conclusion "Xingnao Kaiqiao" acupuncture has brain protective effect on rats with focal cerebral ischemia reperfusion injury. The mechanism may be related to regulating the opening of K_(ATP) channels and decreasing the apoptosis of neurons.
Objective To observe the mechanism of "Xingnao Kaiqiao" acupuncture for the opening of ATP sensitive potassium channel(K_(ATP)) against cerebral ischemia reperfusion injury in rats. Methods Eighty-four rats were randomly divided into a sham-operation group, a model group, an electroacupuncture(EA) group, an EA+K_(ATP) blocker group, 21 rats in each group. 10 μL intracerebral injection with glipizide(1 μmol/5 μL) was used in the EA+K_(ATP) blocker group. The cerebral ischemia reperfusion model was established by Zea Longa's suture method in the model group, the EA group and the EA+K_(ATP) blocker group. Rats in the sham-operation group were received the same surgery but without nylon filament insertion. Acupuncture(20 min a time) was performed at "Neiguan"(PC 6), "Shuigou"(GV 26) and "Sanyinjiao"(SP 6) in the EA group and the EA+K_(ATP) blocker group at 10:00 and 16:00 for 3 days, firstly 90 min after model establishment. EA(2 Hz/15 Hz, 1 mA) was connected at the affected "Neiguan"(PC 6) and "Sanyinjiao"(SP 6). The same fixation was used in the sham-operation group and the model group, without EA. Neurological function was assessed by Zausinger's neurologic assessment scale. 2, 3, 5-triphenyltetrazolium chloride(TTC) staining was used to detect infarct volume. Neurocyte apoptosis in the hippocampus was detected by flow cytometry and the protein expressions of B lymphocytoma-2 gene(Bcl-2) and B cell lymphoma factor-associated X protein(Bax) were measured by Western-blot. Results In comparison with the model group, the neurological score of the EA group increased(P<0.01); the infarction volume and the hippocampal neuron's total apoptosis rate of the EA group decreased(both P<0.05); the protein expression of Bcl-2 and Bcl-2/Bax of the EA group increased(P<0.05, P<0.01); and the protein expression of Bax of the EA group decreased(P<0.01). Compared with the EA group, the neurological score of the EA+K_(ATP) blocker group decreased(P<0.05); the total apoptosis rate of hippocampus neurons of the EA+K_(ATP) blocker group increased(P<0.05); the expression of Bcl-2 protein of the EA+K_(ATP) blocker group reduced(P<0.05); the expression of Bax protein of the EA+K_(ATP) blocker group increased(P<0.05). Conclusion "Xingnao Kaiqiao" acupuncture has brain protective effect on rats with focal cerebral ischemia reperfusion injury. The mechanism may be related to regulating the opening of K_(ATP) channels and decreasing the apoptosis of neurons.
引文
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[12]孔晶晶,曹云鹏.ATP敏感性钾通道开放剂神经保护作用的研究进展[J].中华神经医学杂志,2013,12(6):639-641.
[13]Sun HS,Feng ZP.Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia[J].Acta Pharmacol Sin,2013,34(1):24-32.
[14]张生玉,刘哨兵,谢洪武,等.醒脑开窍针刺法结合高压氧对缺血缺氧性脑病患者血液流变学的影响[J].针刺研究,2017,42(6):518-521.
[15]李钦潘,王伟,韩永升,等.“醒脑开窍”针刺法对脑缺血再灌注大鼠模型早期运动功能恢复及SYN表达影响的研究[J].中国中医急症,2015,24(1):19-23.
[16]Jiang KW,Zhao ZY,Shui QX,et al.Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide,an ATP-sensitive potassium channel blocker[J].Brain Res,2004,998(1):13-19.
[17]许静,王光义.针刺对大鼠脑缺血再灌注损伤皮层线粒体ATP敏感性钾通道及凋亡相关基因表达的影响[C]//中国针灸学会.中国针灸学会2009学术年会论文集(上集).北京:中国针灸学会,2009.
[18]付宏亮,张刚利,张汉伟,等.线栓法制备大鼠局灶性脑缺血模型研究[J].中国药物与临床,2010,10(1):64-65.
[19]孔伟,吴硕琳,毛淑静,等.亚低温对大鼠脑缺血再灌注后出血转化及超微结构的影响[J].中国卒中杂志,2012,7(2):103-108.
[20]姜恩平,王帅群,王卓,等.北五味子总木脂素对脑缺血模型大鼠神经细胞凋亡及p-AKT表达的影响[J].中国中药杂志,2014,39(9):1680-1684.
[2]Dong H,Wang S,Zhang Z,et al.The effect of mitochondrial calcium uniporter opener spermine on diazoxide against focal cerebral ischemia--reperfusion injury in rats[J].J Stroke Cerebrovasc Dis,2014,23(2):303-309.
[3]吴永奇,马超,程卯生.ATP敏感性钾通道在缺血性脑血管疾病中的作用[J].沈阳药科大学学报,2015,32(1):70-76.
[4]Ran YH,Wang H.Iptakalim,an ATP-sensitive potassium channel opener,confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells[J].J Zhejiang Univ Sci B,2011,12(10):835-845.
[5]Yuan F,Fu H,Sun K,et al.Effect of dexmedetomidine on cerebral ischemia-reperfusion rats by activating mitochondrial ATP-sensitive potassium channel[J].Metab Brain Dis,2017,32(2):539-546.
[6]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[7]张俊清,孟智宏,樊小农.大鼠神经行为缺损评分与脑梗死率相关性研究分析[J].辽宁中医杂志,2012(11):2140-2142.
[8]闫明茹,郑宇,孙洁,等.不同时机介入"醒脑开窍"针刺法对脑缺血再灌注大鼠SOD活性和MDA含量的影响[J].现代生物医学进展,2013,13(10):1887-1891.
[9]靳兰洁,李莎莎,周璇,等.电针脑缺血大鼠水沟穴调节微小RNA-328促进血管新生的机制研究[J].中华老年心脑血管病杂志,2017,19(3):308-311.
[10]凌锋.脑血管病理论与实践[M].北京:人民卫生出版社,2007:77-80.
[11]张中元.醒脑开窍针刺法治疗缺血性脑血管病急性期的临床研究[J].天津中医药,2010,27(4):348-350.
[12]孔晶晶,曹云鹏.ATP敏感性钾通道开放剂神经保护作用的研究进展[J].中华神经医学杂志,2013,12(6):639-641.
[13]Sun HS,Feng ZP.Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia[J].Acta Pharmacol Sin,2013,34(1):24-32.
[14]张生玉,刘哨兵,谢洪武,等.醒脑开窍针刺法结合高压氧对缺血缺氧性脑病患者血液流变学的影响[J].针刺研究,2017,42(6):518-521.
[15]李钦潘,王伟,韩永升,等.“醒脑开窍”针刺法对脑缺血再灌注大鼠模型早期运动功能恢复及SYN表达影响的研究[J].中国中医急症,2015,24(1):19-23.
[16]Jiang KW,Zhao ZY,Shui QX,et al.Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide,an ATP-sensitive potassium channel blocker[J].Brain Res,2004,998(1):13-19.
[17]许静,王光义.针刺对大鼠脑缺血再灌注损伤皮层线粒体ATP敏感性钾通道及凋亡相关基因表达的影响[C]//中国针灸学会.中国针灸学会2009学术年会论文集(上集).北京:中国针灸学会,2009.
[18]付宏亮,张刚利,张汉伟,等.线栓法制备大鼠局灶性脑缺血模型研究[J].中国药物与临床,2010,10(1):64-65.
[19]孔伟,吴硕琳,毛淑静,等.亚低温对大鼠脑缺血再灌注后出血转化及超微结构的影响[J].中国卒中杂志,2012,7(2):103-108.
[20]姜恩平,王帅群,王卓,等.北五味子总木脂素对脑缺血模型大鼠神经细胞凋亡及p-AKT表达的影响[J].中国中药杂志,2014,39(9):1680-1684.