内源性大麻素系统参与电针预处理诱导的脑缺血耐受
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摘要
缺血性脑损伤是近年来导致残疾和死亡的最常见原因之一,而现在临床上针对这种致残性疾病的药物治疗效果还十分有限。寻找一种有效的、对人体伤害小的、易被患者接受的预处理方法无疑具有十分重要的意义。电针不仅继承了传统针刺激治疗的优点,而且还融合了电刺激的生理效应,具有简单易行、安全可靠、患者易接受等优点,其对脑缺血的治疗作用已被较多的临床和动物实验所证实。我们以往的研究证实,重复电针刺激百会穴预处理具有神经保护作用,能够减轻随后的缺血性脑损伤,明显减少大脑梗死容积,显著改善大鼠的神经功能损害程度,具有较明显的脑缺血损伤保护作用[1]。这种作用是由脑啡肽的释放并作用于δ和μ受体介导的[2]。研究发现,大麻素与阿片类物质的神经传递之间存在交互作用,并具有相似的药理学作用:都能够诱导产生镇痛、镇静、低体温和低血压以及免疫抑制等效应[3, 4]。此外,心脏研究中发现,缺血前给予内源性大麻素能够模拟缺血预处理的神经保护作用[5],并且,在活体和离体试验中都已证实内源性和外源性大麻素都能够对抗多种类型的急性神经元损伤,具有神经保护作用[6-9]。这些结果表明,内源性大麻素系统参与了电针预处理诱导的脑保护作用。因此,本研究拟探讨内源性大麻素系统是否参与了电针预处理诱导的脑缺血耐受。
第一部分电针刺激百会穴预处理对内源性大麻素系统的影响
方法
1.单次电针预处理对大鼠脑内内源性大麻素系统的影响健康雄性SD大鼠(280～320g),随机分为3组:Sham组、SP组和EA组。Sham组未行任何处理,SP组仅腹腔注射(ip)戊巴比妥钠40 mg/kg。EA组在腹腔注射戊巴比妥钠40 mg/kg麻醉下电针刺激百会穴30 min。电针预处理后2 h进行CB1受体与NeuN免疫双重荧光染色及内源性大麻素含量测定;电针预处理后30 min、1 h和2 h行CB1受体RT-PCR和Western blot检测。
2.重复电针预处理对大鼠脑内内源性大麻素的影响
健康雄性SD大鼠(280～320g),随机分为4组:Sham-2组、EA-2组、Sham-24组和EA-24组。Sham-2组和Sham-24组仅每天腹腔注射戊巴比妥钠40 mg/kg,连续5 d。EA-2组和EA-24组每天在戊巴比妥钠40 mg/kg(ip)麻醉下电针刺激百会穴30 min,连续5 d。Sham-2组和EA-2组大鼠在最后一次电针预处理2 h后,Sham-24组和EA-24组在最后一次电针预处理24 h后深麻醉断头处死,检测内源性大麻素含量。
结果
1.单次电针预处理对大鼠脑内内源性大麻素系统的影响
1.1 CB1受体与NeuN免疫双重荧光染色
与Sham组和SP组相比,电针预处理显著上调了右侧半脑CB1受体的表达。CB1受体与NeuN免疫双重荧光染色表明CB1受体主要表达于神经元。电针预处理后2 h,CB1受体表达增强,并与NeuN表达相重合。
1.2 CB1受体mRNA表达
与Sham组和SP组相比,电针预处理结束后30 min和60 min,CB1受体mRNA表达均显著升高,而电针结束后120 min则回降至Sham组水平。
1.3 CB1受体蛋白含量
Western blot条带分析结果显示,与Sham组和SP组相比,电针预处理显著提高了电针后120 min时CB1受体蛋白含量。
1.4内源性大麻素含量
与Sham组和SP组相比,单次电针预处理显著上调了右侧半脑内源性大麻素AEA和2-AG的含量。
2.重复电针预处理对大鼠脑内内源性大麻素的影响
重复电针预处理显著上调了右侧半脑内源性大麻素的含量。AEA和2-AG含量在电针预处理后2 h均显著升高,在24 h时AEA含量仍维持在较高水平,而2-AG含量下降至对照组水平。
第二部分内源性大麻素系统在电针预处理诱导脑缺血耐受中的作用
方法
1. CB1受体参与单次电针预处理诱导的大鼠脑缺血耐受健康雄性SD大鼠(280～320g),均在MCAO前2.5 h腹腔注射戊巴比妥钠40 mg/kg麻醉,并随机分为5组:Con组、EA-MCAO组、AM-EA-MCAO组、v-EA-MCAO组和AM-MCAO组。Con组仅行颈内动脉尼龙线线栓法致MCAO模型;EA-MCAO组在电针刺激百会穴30 min后2 h时行MCAO;AM-MCAO组在MCAO前3 h腹腔注射AM251 1 mg/kg;v-EA-MCAO组和AM-EA-MCAO组在电针预处理前30 min分别腹腔注射溶剂3 ml/kg和AM251 1 mg/kg,并在电针结束后2 h行MCAO。再灌注24 h和7 d后,进行神经功能学评分并测量大脑梗死容积百分比。
2. CB1受体参与重复电针预处理诱导的大鼠脑缺血耐受
健康雄性SD大鼠(280～320g),随机分为6组:Sham组、Con组、EA-MCAO组、AM-EA-MCAO组、v-EA-MCAO组和AM-MCAO组。除Sham组外其余各组动物每天腹腔注射戊巴比妥钠40 mg/kg麻醉,连续5 d。Sham组仅行假手术;Con组仅缺血再灌注;EA-MCAO组接受电针刺激百会穴30 min/d,连续5 d;AM-MCAO组每天腹腔注射AM251 1 mg/kg,连续5 d;v-EA-MCAO组和AM-EA-MCAO组每天在电针预处理前30 min分别给予溶剂3 ml/kg(ip)和AM251 1 mg/kg(ip),连续5 d。除Sham组外其余各组均在最后一次预处理24 h后行MCAO。再灌注24 h后,进行神经功能学评分并测量大脑梗死容积百分比。
3.重复电针预处理对脑缺血再灌注后内源性大麻素的影响
健康雄性SD大鼠(280～320g),随机分为5组:Sham组、Con-2组、EA-MCAO-2组、Con-24组和EA-MCAO-24组。Sham组、Con-2组和Con-24组每天腹腔注射戊巴比妥钠40 mg/kg,连续5 d,EA-MCAO-2组和EA-MCAO-24组每天在戊巴比妥钠40 mg/kg(ip)麻醉下电针刺激百会穴30 min,连续5 d。除Sham组外其余各组均在最后一次预处理24 h后行MCAO。Sham组在最后一次处理24 h后,Con-2组和EA-MCAO-2组在再灌注2 h后,Con-24组和EA-MCAO-24组在再灌注24 h后深麻醉断头处死,检测内源性大麻素含量。
结果
1. CB1受体参与单次电针预处理诱导的大鼠脑缺血耐受
1.1神经功能学评分
EA-MCAO组大鼠再灌注24 h和7 d后神经功能学评分显著高于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的神经功能学评分没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
1.2脑梗死容积百分比
EA-MCAO组大鼠再灌注24 h和7 d后脑梗死容积百分比显著小于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的脑梗死容积百分比没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
2. CB1受体参与重复电针预处理诱导的大鼠脑缺血耐受
2.1神经功能学评分
Sham组神经功能学评分正常,EA-MCAO组神经功能学评分显著高于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的神经功能学评分没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
2.2脑梗死容积百分比
Sham组未见脑梗死灶。EA-MCAO组大鼠脑梗死容积百分比显著小于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的脑梗死容积百分比没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
3.重复电针预处理对脑缺血再灌注后内源性大麻素的影响
与Sham组相比Con-2组AEA含量显著升高,而Con-24组则显著下降。EA-MCAO-2组与Sham组及Con-2组相比AEA含量均显著升高,EA-MCAO-24组与Con-24组相比AEA含量显著升高。EA-MCAO-2组2-AG含量较Sham组和Con-2组均显著提高。
小结
1、单次及重复电针刺激大鼠百会穴能够上调脑内内源性大麻素AEA和2-AG的含量及CB1受体的表达,提示电针预处理激活了内源性大麻素系统。
2、电针预处理上调了缺血再灌注后脑内内源性大麻素AEA和2-AG的含量,且电针预处理诱导的神经保护作用被CB1受体拮抗剂所逆转,表明电针预处理激活了内源性大麻素系统诱导了脑缺血耐受。
Cerebral Ischemia is one of the most common causes of disability and death. Clinical treatment of this debilitating disorder is inadequate. To explore an effective, less harmful and more acceptable pretreatment is of great significance. As a product of integration of traditional acupuncture and modern electrical stimulation technique, electroacupuncture possesses a number of advantages such as simple operation, safety, reliability and acceptability. Its therapeutic and protective effect of cerebral ischemic injury has been proved by clinical and animal experiments. Our previous study has demonstrated that pretreatment with electroacupuncture (EA) at the Baihui acupoint has significant neuroprotective effect on cerebral ischemic injury. It can protect the brain from the subsequent cerebral ischemic injury, significantly reduce brain infarct volumes and greatly improve the neurological function scores of rats, and this neuroprotective effect is possibly mediated by the release of enkephalins, which may bindδ- andμ-opioid receptors to induce the tolerance against focal cerebral ischemia. The interaction between endocannabinoid and opioid neurotransmission has been reported. They share a similar pharmacological profile: both induce analgesia, hypothermia, hypotension, immunosuppression and so on. Besides, previous studies demonstrated that pretreatment with either 2-AG or AEA before ischemia mimicked preconditioning inasmuch as it protected the ischemic insults in a similar fashion in rat isolated hearts. Synthetic and endocannabinoids exert neuroprotective effects in animals as well as in vitro models of various forms of acute neuronal injury. These findings indicate that the endocannabinoid system participates in the ischemic tolerance which induced by electroacupuncture. Thus, the present study is designed to explore whether the endocannabinoid system are involved in the ischemic tolerance induced by EA pretreatment at the Baihui acupoint.
Part 1 The effect of EA pretreatment at the Baihui acupoint on the endocannabinoid system
Methods
1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system Male SD rats weighing 280-320 g were randomly assigned to 3 groups: Sham, SP and EA groups. The rats in Sham group did not receive any treatment. The rats in SP group only received anesthesia with intraperitoneal injection (ip) of 40 mg/kg sodium pentobarbital; rats in EA group were anesthetized with 40 mg/kg sodium pentobarbital (ip) and received EA stimuli for 30 min. At 2 h after the end of EA pretreatment, CB1 receptor expression was examined and the endocannabinoid content was determined; at 30 min, 1 h and 2 h after EA pretreatment, expression of both CB1 receptor mRNA and protein was determined.
2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system
Male SD rats weighing 280-320 g were randomly assigned to 4 groups: Sham-2, EA-2, Sham-24 and EA-24 groups. Rats in Sham-2 group and Sham-24 group were anesthetized with sodium pentobarbital (ip) once a day for 5 consecutive days, rats in EA-2 group and EA-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. Rats in Sham-2 group and EA-2 group were decapitated 2 h after last pretreatment; rats in Sham-24 group and EA-24 group were decapitated 24 h after last pretreatment. The endocannabinoid content was determined.
Results
1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system
1.1. Double immunofluorescence for CB1 receptor and NeuN
The CB1 receptor-like immunoreactivity in the ipsilateral hemisphere was significantly increased by EA pretreatment. Double immunofluorescence for CB1 receptor and NeuN demonstrated that CB1 receptor was mainly localized in neurons. The CB1 receptor immunoreactivities were increased and colocalized with the neuronal immunoreactivities at 2 h after EA pretreatment in the ipsilateral hemisphere.
1.2 CB1 receptor mRNA expression
The expression of CB1 receptor mRNA significantly increased at 30 and 60 min after EA pretreatment compared with that in Sham or SP group while it was similar at 120 min after EA pretreatment.
1.3 CB1 receptor protein expression
The analysis of Western blot bands indicated that EA pretreatment significantly increased CB1 receptor protein expression level compared with that in Sham or SP group at 120 min after EA pretreatment.
1.4. The content of endocannabinoid
Pretreatment with EA increased the brain tissue content of the endocannabinoid 2-AG and AEA.
2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system
Pretreatment with repeated electroacupuncture increased the brain tissue content of the endocannabinoid 2-AG and AEA. The content of AEA was significantly increased in the EA-2 group and EA-24 group. The content of 2-AG was increased in the EA-2 group but decreased in the EA-24 group.
Part 2
The effect of the endocannabinoid system on the ischemic tolerance induced by EA pretreatment
Methods
1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture Male SD rats weighing 280-320 g were anesthetized with sodium pentobarbital (ip) at 2.5 h before induction of focal cerebral ischemia, and were randomly assigned to 5 groups: Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. The rats in Con group only received MCAO and the rats in EA-MCAO group received MCAO at 2 h after EA pretreatment for 30 min. Rats in AM-MCAO group were intraperitoneally injected with 1 mg/kg AM251 at 3 h before MCAO. Rats in v-EA-MCAO group and AM251-EA-MCAO group were administered with 3 ml/kg vehicle and 1 mg/kg AM251 at 30 min prior to the onset of EA pretreatment respectively, and then subjected to MCAO at 2 h after the end of EA pretreatment. At 24 h or 7 d after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.
2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupuncture
Male SD rats weighing 280-320 g were randomly assigned to 6 groups: Sham, Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. All of the rats were anesthetized with intraperitoneal injection of 40 mg/kg sodium pentobarbital once a day for 5 consecutive days except those in Sham group. Rats in Sham group only received sham operation. Rats in Con group were only subjected to ischemia and reperfusion; rats in AM-MCAO group received 1 mg/kg AM251 (ip) once a day for 5 consecutive days; rats in EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min a day for 5 consecutive days; rats in v-EA-MCAO group and AM-EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min, and were respectively injected with 3 ml/kg vehicle and 1 mg/kg AM251 (ip) 30 min prior to the onset of electroacupuncture once a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion. At 24 h after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.
3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injury
Male SD rats weighing 280-320 g were randomly assigned to 5 groups: Sham, Con-2, EA-MCAO-2, Con-24 and EA-MCAO-24 groups. Rats in Sham group, Con-2 group and Con-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) for 5 consecutive days; rats in EA-MCAO-2 group and EA-MCAO-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and then received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion with 3-0 nylon monofilament. The rat were decapitated at 24 h after last treatment in Sham group, at 2 h after reperfusion in Con-2 group and EA-MCAO-2 group, 24 h after reperfusion in Con-24 group and EA-MCAO-24 group. The endocannabinoid content was determined.
Results
1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture
1.1. Neurologic Outcome
24 h and 7 d after reperfusion, pretreatment with EA significantly improved the neurological function scores compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
1.2. Infarct Volume
EA-MCAO group showed a smaller brain infarct volume at 24 h and 7 d after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupuncture
2.1. Neurologic Outcome
In sham group, no neurological deficits was observed. Pretreatment with EA significantly improved the neurological function scores at 24 h after reperfusion compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
2.2. Infarct Volume
In sham group, no infarct volume was observed. EA-MCAO group showed a smaller brain infarct volume at 24 h after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injury
The brain tissue content of AEA was significantly increased in Con-2 group compared with that in Sham group, but decreased at 24 h after reperfusion. Compared with that in Sham group and Con-2 group, AEA content was significantly increased in EA-MCAO-2 group. In EA-MCAO-24 group, the AEA content was also significantly increased compared with that in Con-24 group. In EA-MCAO-2 group, the 2-AG content was significantly increased compared with that in Sham group and Con-2 group.
Summary
1. Pretreatment with single or repeated electroacupuncture at the Baihui acupoint increases the expression of CB1 receptor and the production of the endocannabinoid 2-AG and AEA. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system.
2. Pretreatment with electroacupuncture increases the production of the endocannabinoid 2-AG and AEA in the brain after ischemia/reperfusion injury and the protective effect of EA pretreatment is reversed by CB1 receptor antagonist. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system which is involved in the ischemic tolerance induced by pretreatment with electroacupuncture.
第一部分电针刺激百会穴预处理对内源性大麻素系统的影响
方法
1.单次电针预处理对大鼠脑内内源性大麻素系统的影响健康雄性SD大鼠(280～320g),随机分为3组:Sham组、SP组和EA组。Sham组未行任何处理,SP组仅腹腔注射(ip)戊巴比妥钠40 mg/kg。EA组在腹腔注射戊巴比妥钠40 mg/kg麻醉下电针刺激百会穴30 min。电针预处理后2 h进行CB1受体与NeuN免疫双重荧光染色及内源性大麻素含量测定;电针预处理后30 min、1 h和2 h行CB1受体RT-PCR和Western blot检测。
2.重复电针预处理对大鼠脑内内源性大麻素的影响
健康雄性SD大鼠(280～320g),随机分为4组:Sham-2组、EA-2组、Sham-24组和EA-24组。Sham-2组和Sham-24组仅每天腹腔注射戊巴比妥钠40 mg/kg,连续5 d。EA-2组和EA-24组每天在戊巴比妥钠40 mg/kg(ip)麻醉下电针刺激百会穴30 min,连续5 d。Sham-2组和EA-2组大鼠在最后一次电针预处理2 h后,Sham-24组和EA-24组在最后一次电针预处理24 h后深麻醉断头处死,检测内源性大麻素含量。
结果
1.单次电针预处理对大鼠脑内内源性大麻素系统的影响
1.1 CB1受体与NeuN免疫双重荧光染色
与Sham组和SP组相比,电针预处理显著上调了右侧半脑CB1受体的表达。CB1受体与NeuN免疫双重荧光染色表明CB1受体主要表达于神经元。电针预处理后2 h,CB1受体表达增强,并与NeuN表达相重合。
1.2 CB1受体mRNA表达
与Sham组和SP组相比,电针预处理结束后30 min和60 min,CB1受体mRNA表达均显著升高,而电针结束后120 min则回降至Sham组水平。
1.3 CB1受体蛋白含量
Western blot条带分析结果显示,与Sham组和SP组相比,电针预处理显著提高了电针后120 min时CB1受体蛋白含量。
1.4内源性大麻素含量
与Sham组和SP组相比,单次电针预处理显著上调了右侧半脑内源性大麻素AEA和2-AG的含量。
2.重复电针预处理对大鼠脑内内源性大麻素的影响
重复电针预处理显著上调了右侧半脑内源性大麻素的含量。AEA和2-AG含量在电针预处理后2 h均显著升高,在24 h时AEA含量仍维持在较高水平,而2-AG含量下降至对照组水平。
第二部分内源性大麻素系统在电针预处理诱导脑缺血耐受中的作用
方法
1. CB1受体参与单次电针预处理诱导的大鼠脑缺血耐受健康雄性SD大鼠(280～320g),均在MCAO前2.5 h腹腔注射戊巴比妥钠40 mg/kg麻醉,并随机分为5组:Con组、EA-MCAO组、AM-EA-MCAO组、v-EA-MCAO组和AM-MCAO组。Con组仅行颈内动脉尼龙线线栓法致MCAO模型;EA-MCAO组在电针刺激百会穴30 min后2 h时行MCAO;AM-MCAO组在MCAO前3 h腹腔注射AM251 1 mg/kg;v-EA-MCAO组和AM-EA-MCAO组在电针预处理前30 min分别腹腔注射溶剂3 ml/kg和AM251 1 mg/kg,并在电针结束后2 h行MCAO。再灌注24 h和7 d后,进行神经功能学评分并测量大脑梗死容积百分比。
2. CB1受体参与重复电针预处理诱导的大鼠脑缺血耐受
健康雄性SD大鼠(280～320g),随机分为6组:Sham组、Con组、EA-MCAO组、AM-EA-MCAO组、v-EA-MCAO组和AM-MCAO组。除Sham组外其余各组动物每天腹腔注射戊巴比妥钠40 mg/kg麻醉,连续5 d。Sham组仅行假手术;Con组仅缺血再灌注;EA-MCAO组接受电针刺激百会穴30 min/d,连续5 d;AM-MCAO组每天腹腔注射AM251 1 mg/kg,连续5 d;v-EA-MCAO组和AM-EA-MCAO组每天在电针预处理前30 min分别给予溶剂3 ml/kg(ip)和AM251 1 mg/kg(ip),连续5 d。除Sham组外其余各组均在最后一次预处理24 h后行MCAO。再灌注24 h后,进行神经功能学评分并测量大脑梗死容积百分比。
3.重复电针预处理对脑缺血再灌注后内源性大麻素的影响
健康雄性SD大鼠(280～320g),随机分为5组:Sham组、Con-2组、EA-MCAO-2组、Con-24组和EA-MCAO-24组。Sham组、Con-2组和Con-24组每天腹腔注射戊巴比妥钠40 mg/kg,连续5 d,EA-MCAO-2组和EA-MCAO-24组每天在戊巴比妥钠40 mg/kg(ip)麻醉下电针刺激百会穴30 min,连续5 d。除Sham组外其余各组均在最后一次预处理24 h后行MCAO。Sham组在最后一次处理24 h后,Con-2组和EA-MCAO-2组在再灌注2 h后,Con-24组和EA-MCAO-24组在再灌注24 h后深麻醉断头处死,检测内源性大麻素含量。
结果
1. CB1受体参与单次电针预处理诱导的大鼠脑缺血耐受
1.1神经功能学评分
EA-MCAO组大鼠再灌注24 h和7 d后神经功能学评分显著高于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的神经功能学评分没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
1.2脑梗死容积百分比
EA-MCAO组大鼠再灌注24 h和7 d后脑梗死容积百分比显著小于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的脑梗死容积百分比没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
2. CB1受体参与重复电针预处理诱导的大鼠脑缺血耐受
2.1神经功能学评分
Sham组神经功能学评分正常,EA-MCAO组神经功能学评分显著高于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的神经功能学评分没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
2.2脑梗死容积百分比
Sham组未见脑梗死灶。EA-MCAO组大鼠脑梗死容积百分比显著小于Con组。单独给予CB1受体拮抗剂AM251对于MCAO大鼠的脑梗死容积百分比没有影响,但在电针前30 min给予AM251则能够逆转电针预处理的神经保护作用。
3.重复电针预处理对脑缺血再灌注后内源性大麻素的影响
与Sham组相比Con-2组AEA含量显著升高,而Con-24组则显著下降。EA-MCAO-2组与Sham组及Con-2组相比AEA含量均显著升高,EA-MCAO-24组与Con-24组相比AEA含量显著升高。EA-MCAO-2组2-AG含量较Sham组和Con-2组均显著提高。
小结
1、单次及重复电针刺激大鼠百会穴能够上调脑内内源性大麻素AEA和2-AG的含量及CB1受体的表达,提示电针预处理激活了内源性大麻素系统。
2、电针预处理上调了缺血再灌注后脑内内源性大麻素AEA和2-AG的含量,且电针预处理诱导的神经保护作用被CB1受体拮抗剂所逆转,表明电针预处理激活了内源性大麻素系统诱导了脑缺血耐受。
Cerebral Ischemia is one of the most common causes of disability and death. Clinical treatment of this debilitating disorder is inadequate. To explore an effective, less harmful and more acceptable pretreatment is of great significance. As a product of integration of traditional acupuncture and modern electrical stimulation technique, electroacupuncture possesses a number of advantages such as simple operation, safety, reliability and acceptability. Its therapeutic and protective effect of cerebral ischemic injury has been proved by clinical and animal experiments. Our previous study has demonstrated that pretreatment with electroacupuncture (EA) at the Baihui acupoint has significant neuroprotective effect on cerebral ischemic injury. It can protect the brain from the subsequent cerebral ischemic injury, significantly reduce brain infarct volumes and greatly improve the neurological function scores of rats, and this neuroprotective effect is possibly mediated by the release of enkephalins, which may bindδ- andμ-opioid receptors to induce the tolerance against focal cerebral ischemia. The interaction between endocannabinoid and opioid neurotransmission has been reported. They share a similar pharmacological profile: both induce analgesia, hypothermia, hypotension, immunosuppression and so on. Besides, previous studies demonstrated that pretreatment with either 2-AG or AEA before ischemia mimicked preconditioning inasmuch as it protected the ischemic insults in a similar fashion in rat isolated hearts. Synthetic and endocannabinoids exert neuroprotective effects in animals as well as in vitro models of various forms of acute neuronal injury. These findings indicate that the endocannabinoid system participates in the ischemic tolerance which induced by electroacupuncture. Thus, the present study is designed to explore whether the endocannabinoid system are involved in the ischemic tolerance induced by EA pretreatment at the Baihui acupoint.
Part 1 The effect of EA pretreatment at the Baihui acupoint on the endocannabinoid system
Methods
1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system Male SD rats weighing 280-320 g were randomly assigned to 3 groups: Sham, SP and EA groups. The rats in Sham group did not receive any treatment. The rats in SP group only received anesthesia with intraperitoneal injection (ip) of 40 mg/kg sodium pentobarbital; rats in EA group were anesthetized with 40 mg/kg sodium pentobarbital (ip) and received EA stimuli for 30 min. At 2 h after the end of EA pretreatment, CB1 receptor expression was examined and the endocannabinoid content was determined; at 30 min, 1 h and 2 h after EA pretreatment, expression of both CB1 receptor mRNA and protein was determined.
2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system
Male SD rats weighing 280-320 g were randomly assigned to 4 groups: Sham-2, EA-2, Sham-24 and EA-24 groups. Rats in Sham-2 group and Sham-24 group were anesthetized with sodium pentobarbital (ip) once a day for 5 consecutive days, rats in EA-2 group and EA-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. Rats in Sham-2 group and EA-2 group were decapitated 2 h after last pretreatment; rats in Sham-24 group and EA-24 group were decapitated 24 h after last pretreatment. The endocannabinoid content was determined.
Results
1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system
1.1. Double immunofluorescence for CB1 receptor and NeuN
The CB1 receptor-like immunoreactivity in the ipsilateral hemisphere was significantly increased by EA pretreatment. Double immunofluorescence for CB1 receptor and NeuN demonstrated that CB1 receptor was mainly localized in neurons. The CB1 receptor immunoreactivities were increased and colocalized with the neuronal immunoreactivities at 2 h after EA pretreatment in the ipsilateral hemisphere.
1.2 CB1 receptor mRNA expression
The expression of CB1 receptor mRNA significantly increased at 30 and 60 min after EA pretreatment compared with that in Sham or SP group while it was similar at 120 min after EA pretreatment.
1.3 CB1 receptor protein expression
The analysis of Western blot bands indicated that EA pretreatment significantly increased CB1 receptor protein expression level compared with that in Sham or SP group at 120 min after EA pretreatment.
1.4. The content of endocannabinoid
Pretreatment with EA increased the brain tissue content of the endocannabinoid 2-AG and AEA.
2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system
Pretreatment with repeated electroacupuncture increased the brain tissue content of the endocannabinoid 2-AG and AEA. The content of AEA was significantly increased in the EA-2 group and EA-24 group. The content of 2-AG was increased in the EA-2 group but decreased in the EA-24 group.
Part 2
The effect of the endocannabinoid system on the ischemic tolerance induced by EA pretreatment
Methods
1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture Male SD rats weighing 280-320 g were anesthetized with sodium pentobarbital (ip) at 2.5 h before induction of focal cerebral ischemia, and were randomly assigned to 5 groups: Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. The rats in Con group only received MCAO and the rats in EA-MCAO group received MCAO at 2 h after EA pretreatment for 30 min. Rats in AM-MCAO group were intraperitoneally injected with 1 mg/kg AM251 at 3 h before MCAO. Rats in v-EA-MCAO group and AM251-EA-MCAO group were administered with 3 ml/kg vehicle and 1 mg/kg AM251 at 30 min prior to the onset of EA pretreatment respectively, and then subjected to MCAO at 2 h after the end of EA pretreatment. At 24 h or 7 d after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.
2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupuncture
Male SD rats weighing 280-320 g were randomly assigned to 6 groups: Sham, Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. All of the rats were anesthetized with intraperitoneal injection of 40 mg/kg sodium pentobarbital once a day for 5 consecutive days except those in Sham group. Rats in Sham group only received sham operation. Rats in Con group were only subjected to ischemia and reperfusion; rats in AM-MCAO group received 1 mg/kg AM251 (ip) once a day for 5 consecutive days; rats in EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min a day for 5 consecutive days; rats in v-EA-MCAO group and AM-EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min, and were respectively injected with 3 ml/kg vehicle and 1 mg/kg AM251 (ip) 30 min prior to the onset of electroacupuncture once a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion. At 24 h after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.
3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injury
Male SD rats weighing 280-320 g were randomly assigned to 5 groups: Sham, Con-2, EA-MCAO-2, Con-24 and EA-MCAO-24 groups. Rats in Sham group, Con-2 group and Con-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) for 5 consecutive days; rats in EA-MCAO-2 group and EA-MCAO-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and then received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion with 3-0 nylon monofilament. The rat were decapitated at 24 h after last treatment in Sham group, at 2 h after reperfusion in Con-2 group and EA-MCAO-2 group, 24 h after reperfusion in Con-24 group and EA-MCAO-24 group. The endocannabinoid content was determined.
Results
1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture
1.1. Neurologic Outcome
24 h and 7 d after reperfusion, pretreatment with EA significantly improved the neurological function scores compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
1.2. Infarct Volume
EA-MCAO group showed a smaller brain infarct volume at 24 h and 7 d after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupuncture
2.1. Neurologic Outcome
In sham group, no neurological deficits was observed. Pretreatment with EA significantly improved the neurological function scores at 24 h after reperfusion compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
2.2. Infarct Volume
In sham group, no infarct volume was observed. EA-MCAO group showed a smaller brain infarct volume at 24 h after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.
3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injury
The brain tissue content of AEA was significantly increased in Con-2 group compared with that in Sham group, but decreased at 24 h after reperfusion. Compared with that in Sham group and Con-2 group, AEA content was significantly increased in EA-MCAO-2 group. In EA-MCAO-24 group, the AEA content was also significantly increased compared with that in Con-24 group. In EA-MCAO-2 group, the 2-AG content was significantly increased compared with that in Sham group and Con-2 group.
Summary
1. Pretreatment with single or repeated electroacupuncture at the Baihui acupoint increases the expression of CB1 receptor and the production of the endocannabinoid 2-AG and AEA. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system.
2. Pretreatment with electroacupuncture increases the production of the endocannabinoid 2-AG and AEA in the brain after ischemia/reperfusion injury and the protective effect of EA pretreatment is reversed by CB1 receptor antagonist. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system which is involved in the ischemic tolerance induced by pretreatment with electroacupuncture.
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