CB1拮抗剂利莫那班联合CB2激动剂JWH133对脑缺血再灌注病理损伤保护作用的研究
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摘要
目的探讨大麻系统(ECS)药物CB1拮抗剂利莫那班联合CB2激动剂JWH133预处理对大鼠脑缺血再灌注损伤的神经保护作用机制。方法将健康成年雄性SD大鼠随机分为假手术组、模型组、JWH133组和LHGY组(利莫那班+JWH133)。造模前1 h用二甲基亚砜(DMSO)溶解药物,预处理腹腔注射给药;其他两组注射等体积生理盐水,改良ZEA-LONGA线栓法建立大鼠右脑中动脉缺血再灌注模型。采用Longa评分法进行神经功能评分,TTC染色测量脑梗死体积,测定缺血侧脑组织及周围血清中和白细胞介素-6(IL-6)和白细胞介素-10(IL-10)含量,比色法检测诱导型一氧化氮合酶(i NOS)活性的表达。结果模型组、JWH133组和LHGY组均出现不同程度的神经行为功能异常,两组神经行为功能恢复明显好于其他两组(P<0.05),LHGY组表现略优于JWH133组(P>0.05)。其中,脑切片TTC染色显示JWH133组和LHGY组均较模型组白色梗死灶缩小(P<0.05),后者梗死面积程度小于前者(P>0.05)。模型组大鼠脑组织及血清中IL-6含量比假手术组含量有较显著的升高,IL-10则相反;而JWH133组和LHGY组治疗后两种炎性因子呈现不同程度的反相变化(P<0.05)。与假手术组相比,模型组脑组织中i NOS活性明显升高;JWH133组和LHGY组脑组织中i NOS活力均明显降低(P<0.05)。结论 JWH133组和LHGY组预处理后通过抑制脑缺血再灌注损伤过程中的炎性反应而发挥神经保护作用,其机制可能与调节IL-6、IL-10和i NOS等因子水平有关。而CB1拮抗剂利莫那班联合CB2激动剂JWH133给药方案产生一定程度的叠加效果,这也提示ECS对脑缺血再灌注损伤产生神经保护作用机制尚待进一步探讨。
【Objective】To observe the neural protection of Endocannabinoid system(ECS) drugs CB1 antagonist rimonabant combined with CB2 agonist JWH133 in pretreating rats with ischemia-reperfusion injury.【Methods】Healthy adult male SD rats were randomly divided into sham group,model group,JWH133 group and LHGY group(rimonabant & JWH133).One hour before modeling the drug was dissolved by DMSO,and rats were pretreated with abdominal injection.The other two groups were injected with equivalent normal saline.The model rats with right middle cerebral artery ischemia-reperfusion were established using modified ZEA-LONGA method.In order to prove the effect of CB1 antagonist rimonabant combined with CB2 agonist JWH133 on rat MCAO model,nerve function was scored with Longa method; lateral ischemic brain tissue was determined after TTC staining; interleukin 6(IL-6)and interleukin 10(IL-10) contents in the surrounding serum were detected; the expression of induced nitric oxide synthase(i NOS) in brain tissue was detected by colorimetric method.【Results】Abnormal nerve function with different degrees was found in the model group,JWH133 group and LHGY group.Recovery of nerve function in JWH133 group and LHGY group was significantly superior to that in model group and sham group(P < 0.05),and the condition of LHGY group was slightly better than JWH133 group(P > 0.05).TTC staining of cerebral slice showed that white infarction area in JWH133 group and LHGY group was decreased than in model group(P < 0.05),and the infarction area in LHGY group was smaller than in JWH133 group(P > 0.05).IL-6 content in rat cerebral tissue and serum in model group was significantly increased than in sham group,while Il-10 content was the opposite.Two inflammatory factors appeared opposite changes in JWH133 group and LHGY group(P < 0.05).Compared with sham group,the activation of i NOS in cerebral tissue in model group was increased significantly; the activation of i NOS in cerebral tissue in JWH133 group and LHGY group was reduced significantly(P < 0.05).【Conclusions】Pretreatment with CB1 antagonist rimonabant combined with CB2 agonist JWH133 can inhibit inflammatory reaction and play an important role in neural protection.The mechanism may be related to the regulation of the IL-6,IL-10 and i NOS level.The protective effects of ECS on focal cerebral ischemia-reperfusion injury and its action mechanism remains to be investigated.
【Objective】To observe the neural protection of Endocannabinoid system(ECS) drugs CB1 antagonist rimonabant combined with CB2 agonist JWH133 in pretreating rats with ischemia-reperfusion injury.【Methods】Healthy adult male SD rats were randomly divided into sham group,model group,JWH133 group and LHGY group(rimonabant & JWH133).One hour before modeling the drug was dissolved by DMSO,and rats were pretreated with abdominal injection.The other two groups were injected with equivalent normal saline.The model rats with right middle cerebral artery ischemia-reperfusion were established using modified ZEA-LONGA method.In order to prove the effect of CB1 antagonist rimonabant combined with CB2 agonist JWH133 on rat MCAO model,nerve function was scored with Longa method; lateral ischemic brain tissue was determined after TTC staining; interleukin 6(IL-6)and interleukin 10(IL-10) contents in the surrounding serum were detected; the expression of induced nitric oxide synthase(i NOS) in brain tissue was detected by colorimetric method.【Results】Abnormal nerve function with different degrees was found in the model group,JWH133 group and LHGY group.Recovery of nerve function in JWH133 group and LHGY group was significantly superior to that in model group and sham group(P < 0.05),and the condition of LHGY group was slightly better than JWH133 group(P > 0.05).TTC staining of cerebral slice showed that white infarction area in JWH133 group and LHGY group was decreased than in model group(P < 0.05),and the infarction area in LHGY group was smaller than in JWH133 group(P > 0.05).IL-6 content in rat cerebral tissue and serum in model group was significantly increased than in sham group,while Il-10 content was the opposite.Two inflammatory factors appeared opposite changes in JWH133 group and LHGY group(P < 0.05).Compared with sham group,the activation of i NOS in cerebral tissue in model group was increased significantly; the activation of i NOS in cerebral tissue in JWH133 group and LHGY group was reduced significantly(P < 0.05).【Conclusions】Pretreatment with CB1 antagonist rimonabant combined with CB2 agonist JWH133 can inhibit inflammatory reaction and play an important role in neural protection.The mechanism may be related to the regulation of the IL-6,IL-10 and i NOS level.The protective effects of ECS on focal cerebral ischemia-reperfusion injury and its action mechanism remains to be investigated.
引文
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[8]李晶,乐乐乐,董志.脑缺血/再灌注对大鼠脑内花生四烯酸乙醇胺浓度的影响及其机理初步研究[J].激光杂志,2010,3(31):93-96.
[9]MEHTA SL.Molecular targets in cerebral ischemia for developing novel therapeutics[J].Brain Res Rev,2007,54(13):34-66.
[2]BEDYSHV E,SCHMID PC,KREBSBACH RJ,et al.Canabinoid-receptor-independent cell signalling by N-acylethanolamines[J].Biochem,2001,360(Pt 1):67-75.
[3]WHITE BC,SULLIVAN JM,DEGRACIA DJ,et al.Brain ischemia and reperfusion:molecular mechanisms of neuronal injury[J].Neurological Sciences,2000,179(81):1-33.
[4]ZAUSINGER S,HUNGERHUBER E,BAETHMANN A,et al.Neurological impairment in rats after transient middle cerebral artery occlusion:a comparative study under various treatment paradigms[J].Brain Res,2010,863(97):94-105.
[5]MARIO V,VINCENZO D.Cannabinoid receptors and their role in neuroprotection[J].Neuro Molecular Medicine,2005,5(7):37-50.
[6]CORREA F.Activation of cannabinoid CB2 receptor negatively regulates IL-12p40 production in murine macrophages[J].Br J Pharmacol,2005,145(4):441-448.
[7]乐乐乐,董志.利莫那班对局灶性脑缺血/再灌注损伤大鼠的保护作用[J].中国老年学杂志,2011,31(12):55-59.
[8]李晶,乐乐乐,董志.脑缺血/再灌注对大鼠脑内花生四烯酸乙醇胺浓度的影响及其机理初步研究[J].激光杂志,2010,3(31):93-96.
[9]MEHTA SL.Molecular targets in cerebral ischemia for developing novel therapeutics[J].Brain Res Rev,2007,54(13):34-66.