刘良明 卢儒权 胡德耀 陈惠孙 陈艳 但飞君
【摘要】 目的 探讨μ,δ和κ阿片受体与创伤失血性休克大鼠心血管功能抑制的关系。方法 用大鼠创伤失血性休克模型,观察创伤失血性休克后大鼠心脏和脑μ,δ和κ阿片受体变化及其与血流动力学指标的变化的关系;观察δ和κ阿片受体特异性拮抗剂对创伤失血性休克大鼠血流动力学指标的影响。结果 创伤失血性休克后,大鼠心脏和脑δ和κ阿片受体数目明显升高,亲和力无明显变化,心脏和脑的δ和κ阿片受体数目升高与创伤失血性休克后大鼠血流动力学指标下降呈显著负相关。δ和κ阿片受体特异性拮抗剂可明显逆转创伤失血性休克大鼠血流动力学指标的下降。结论 δ和κ阿片受体在创伤失血性休克心血管功能抑制中起重要作用,参与了创伤失血性休克的发病过程。
【关键词】 创伤失血性休克 阿片受体 心血管功能 大鼠
Importance of μ,δ and κ Opioid Receptors in Cardiovascular Depression Following Traumatic Hemorrhagic Shock in Rats LIU Liang-ming, LU Ru-quan, HU De-yao, et al. Research Institute of Surgery, Third Military Medical University, Chongqing 400042
【Abstract】 Aim To investigate the importance of μ, δ and κ opioid receptors in the cardiovascular depression following traumatic hemorrhagic shock, elucidate the facts that the subclass of these opioid receptors is involved in the pathogenesis of traumatic hemorrhagic shock. Methods With a model of traumatic hemorrhagic shock rat, the relationship between the changes of myocardial and brain μ, δ and κ opioid receptors and cardiovascular functions and effects of δ and κ opioid receptor antagonists on hemodynamic parameters of traumatic hemorrhagic shock rats were observed. Results The number of myocardial and brain δ and κ opioid receptors significantly increased, and they were significantly associated with the decreased hemodynamic parameters such as mean arterial pressure (MAP), left intraventricular systolic pressure (LVSP), the maximal rate of the change of intraventricular pressure (±dp/dtmax) and the area of p-dp/dt vector loop (Lo). However, μ opioid receptor of heart and brain did not obviously alter and δ opioid receptor antagonist ICI78, 864 and κ opioid receptor antagonist Nor-binaltorphimine (50μg, icv) could significantly reverse those decreased hemodynamic parameters of traumatic hemorrhagic shock rats. Conclusion These results suggest that opioid receptors, especially δ and κ opioid receptors, are closely related to the pathogenesis of traumatic hemorrhagic shock, and they play an important role in the depression of cardiovascular function following traumatic hemorrhagic shock.
【Key words】 Traumatic hemorrhagic shock Opioid receptors Cardiovascular function Rat
大量文献资料报道阿片受体特异性拮抗剂如纳洛酮(Naloxone, NAL)、纳曲酮(Natrexone, NTX)和纳叮啡(Nalbuphine, NBP)等对多种循环性休克如失血性休克、内毒素休克、脊髓损伤性休克等均有很好的抗休克作用［1，2］。虽然如此,但由于他们的阿片受体亚型选择性不高,在抗休克的同时可阻断μ阿片受体而降低休克患者的痛阈,因而不宜用于创伤休克的治疗［3］。因此针对创伤休克病人,若能首先弄清与创伤休克发病过程有关的亚型阿片受体,然后针对选择不作用于μ阿片受体,与创伤休克发病过程有关的亚型阿片受体的特异性拮抗剂来治疗创伤休克,将会比NAL等非选择性阿片受体拮抗剂有更好的应用前景。
材料与方法
1.药品及试剂:δ-阿片受体特异性拮抗剂ICI174 864和κ阿片受体特异性拮抗剂Norbinaltorphimine(Nor-BNI)由Jhon Holaday博士提供,［3H］双氢埃托啡(［3H］DHE,比放1.83PBq/mol)和埃托啡(Eto)购自军事医学科学院。［3H］D-丙2,D-亮5脑啡肽(［3H］DADLE,比放为1.21pBq/mol)和［3H］D Norbinaltorphimine(［3H］Nor-BNI,0.88Bq/mol)由中国原子能研究院同位素研究所合成。2.实验方法:(1)创伤失血性休克后心、脑组织μ、δ、κ阿片受体变化及与心血管功能变化的关系。动物实验:Wistar大鼠36只,(220±35)g,雌雄不拘,戊巴比妥钠40mg/kg腹腔麻醉。6只作正常对照。余30只股动脉插管放血和监测血压(MAP),经右颈总动脉作左心室插管连接血流动力学分析仪监测左室内压(LVSP),左室内压最大变化速度(±dp/dtmax)和心肌收缩向量环面积(Lo)等血流动力学指标。插管毕,动物致右侧股骨中段粉碎性骨折,骨折后5分钟按20ml/kg经股动脉插管放血,在10分钟内放毕复制创伤失血性休克模型,动物分别于放血后即刻、1、2、3、4小时五个时相点测定上述血流动力学指标,并活杀(每个时间点6只动物)取心脏和脑,置液氮中保存,用于μ、δ、κ阿片受体数目(Bmax)和解离常数(KD)测定。
心、脑组织匀浆制备:心肌细胞膜和脑匀浆制备参照Romano和本实验室以前的方法［4～6］进行。膜蛋白用双缩脲法定量,心肌细胞膜蛋白稀释成2mg/ml备用,脑匀浆膜蛋白稀释成5mg/ml备用。
阿片受体结合实验:心脏μ、δ、κ阿片受体测定每管含膜蛋白0.4mg,脑组织μ、δ、κ阿片受体测定每管含膜蛋1mg。均分别加入0.5～13nmol/L的放射配体,μ阿片受体加［3H］-DHE,δ受体加［3H］DADLE,κ受体加［3H］Nor-BNI,非特异结合管中加入Eto(1μmol/L,各管以缓冲液补至总体积500μl,摇匀,37℃,温浴30分钟,立即置冰浴终止反应,按常规抽滤,冲洗后液闪测定放射性计数,根据Scatchard作图,利用直线回归方程求出受体Bmax和KD。
(2)δ、κ阿片受体拮抗剂对创伤失血性休克大鼠血流动力学指标的影响。Wistar大鼠18只,(220±40)g,雌雄不拘,均分三组,戊巴比妥钠麻醉,左心室插管监测LVSP、±dp/dtmax、Lo等血流动力学指标,股动脉插管监测MAP、放血。侧脑室插管给药。术毕,作右侧股骨中段粉碎性骨折,5分钟后从股动脉插管放血,10分钟内放血至MAP5.3kPa(40mmHg),维持60分钟,然后从侧脑室给予ICI174 864(50μg,20μl),Nor-BNI(50μg,20μl)或等量等渗盐水,观察给药后10、30、60、120分钟各血流动力学指标的变化。
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