异丙酚对豚鼠离体气管平滑肌张力作用的研究
摘要
目的
全麻诱导、气管插管常可引起支气管痉挛，对哮喘等气道高反应性(airway hyperresponsiveness，AHR)疾病患者实施麻醉诱导时，或对COPD、ARDS等重症患者实施急救气管插管治疗时，此种潜在的威胁可能是致命的，由麻醉药引起的组胺释放或交感神经活性降低、副交感神经活性增高常常增加此种危险。在临床麻醉中，选择适宜的静脉麻醉药将对该类患者的麻醉安全和预后产生重要影响，但目前临床上选择何种静脉麻醉药用于AHR疾病患者的麻醉诱导和维持仍存在争议。
异丙酚(Propofol，PF)作为一种新型的静脉麻醉药，具有起效快、作用时间短、消除迅速、苏醒快而完全、术后恶心呕吐发生率低等优点，已广泛应用于临床麻醉和ICU镇静。近年来国外关于PF对气道平滑肌(airway smooth muscle，ASM)张力作用的报道较多，大多显示PF对在体、离体的正常动物和人的ASM有剂量依赖的舒张作用，但国内罕见报道，并且PF对哮喘动物ASM的作用及对ASM细胞Ryanodine受体(RyR)的作用尚未见报道。本研究通过建立哮喘豚鼠模型，探讨PF对正常及哮喘豚鼠离体ASM的药理作用及作用机制，为临床麻醉工作中静脉麻醉药的选择提供理论依据。
方法
一、哮喘豚鼠模型的建立
将健康豚鼠68只(沈阳市药品检验所提供)，雌雄不拘，体重150～200g，随机分成正常组和哮喘组。哮喘组以10％卵白蛋白(ovalbumin，OA)生理盐水溶液1ml腹腔注射致敏，饲养14天后，
每天置于 3 L玻璃钟罩内雾化吸人 1％OA生理盐水溶液诱发哮喘
发作，以豚鼠逐渐出现呼吸加深加快，直至出现明显腹肌收缩为模
型建立成功的标志，每日激发哮喘一次，持续两周建立迟发型哮喘、。
模型。正常组以生理盐水 IInl代替 OA腹腔注射，饲养 14天后每
日雾化吸人生理盐水。
二、豚鼠高体气管环制备
击头处死豚鼠，迅速取其主气管约 2－3 cm，置于通有混合气
体阳5％O卜5％CO＊冰浴的 K－H营养液中剪除气管周围结缔组
织及脂肪组织，制成长3－srnm左右的气管环，悬挂于盛有K—H
营养液的夹层肌槽中，保持温度37＊0．5℃，持续通人混合气体。
三、豚鼠离体气管平滑肌张力测定
豚鼠离体气管平滑肌（racheal smooth mucle，TIM）的张力变
化可通过与气管环上端相连的力一位移换能器描记在台式平衡记
录仪上。标本初始负荷Zg，每20min换一次营养液，平衡Zh达到
理想初长度后向浴管中加人实验药物。所有药物浓度以标本孵育
液中的终浓度表示，量效曲线的实验采用累积加药法。
四、豚鼠离体TSM依内钙性收缩和依外钙性收缩的测定
标本平衡Zh后，用无钙K－H营养液冲洗三次，稳定后第
smin分别加人不同浓度的 PF或加人 100pmol／L的 Ryanodine作
用 10min后加人不同浓度的 PF，20ndn后加人 0．lpmol／L的乙酸
胆碱（ACh人此时出现的气管环收缩为ACh引起细胞内钙释放所
致，即依内钙性收缩；待收缩达最大效应后，向浴管内加人CaCI。
（终浓度为2．smxnol／L人出现的气管环收缩，为Ach引起细胞外
钙内流所致，即依外钙性收缩。
五、统计分析
实验数据采用 SPSS for windows 10．0软件进行单因素方差分
析和独立样本的 t检验，P＜0．05为差异有显著性的意义。
·2·
结 果
1．PF对静息状态TIM的作用
N对正常豚鼠静息状态下的nM无明显作用（尸＞0．05人
10％Intralopd有轻微收缩 ’ISM的作用，但与对照组比较无显著差
异。与对照组比较不同浓度的PF剂量依赖性地舒张哮喘豚鼠静
息邹M，mo卜OOVL，300pmOVL Pq的舒张作用有显著差异（83．
33 t ZI．60，161．67 t38．69，P＜0．05）。
2．PF对ACh所致TIM收缩的作用
正常组和哮喘组中不同浓度 PF对 0．lpmol／L ACh所致的
TIM收缩都呈剂量依赖性的舒张作用。正常组：100卜mol／L PF使
ACh所致 ’I’SM收缩反应降为拍．36。7．58）％，与对照亚组比较
差异显著；哮喘组：30 pm。VLN使油h所致BM收缩反应降为
（7．88。6．03）％，与对照亚组比较差异显著；且与正常组同浓度
PF的作用比较有显著差异；10％Intralopd不影响 PF舒张5M的
作用。
3．PF预适应对ACh所致的’I’SM收缩量效曲线的影响
给予 100psmoUL，300pmol／L的 PF 20ndn后可剂量依赖性地
抑制ACh引起的’ISM收缩，使其量效曲线右移，但最大反应变化
很小；10％Intralwid不影响 PF对 Ach所致 TSM收缩的抑制作用。
4．普索洛尔（e）对PF抑制ACh所致TIM收缩量效曲线作
用的影响
以ic卜mo＊L PrO作用b 阻断p。受体后，得到1mpmo＊L
PF预适应后ACh致TIM收缩的量效曲线。与无Pro作用比较，
100pmol／L PF抑制Ach所致TIM收缩的量效曲线无明显变化。
5．PF预适应对ACh所致的h M两种收缩组分的影响
PF预适应可剂量依赖性地抑制Ach引起的TIM收缩，正常
组：与对照亚组比较，100pmoVL PF对 0．lpeVL ACh所致 TIM
·3·
依内钙性收缩、依外钙性收缩?
Objective
The potential bronchial spasm is usually lethal during the induction of general anaesthesia in patients with airway hyperresponsiveness (AHR) disease, such as COPD, ARDS and so on. The increased risk of bronchial spasm is due to the drug - induced histamine release , decreased sympathetic nerve activity or enhanced parasympathetic activity. For that reason, the choice of the appropriate venous anesthesic is very important for the safety and prognosis of the patients. However , no consensus has been reached in the choice of the proper venous anesthetic that exerts protective effect on airway.
Characterized with rapid effect, short duration, immediate elimination, complete recovery, and low incidence of postoperative vomiting, Propofol (PF) , as a new generation of venous anesthetic, has been widely used in clinical anesthesia and ICU sedation Compared with research abroad, the effect of Propofol on airway smooth muscle ( ASM ) tone , especially on that of asthma and on Ryanodine receptor ( RyR) of ASM cell, is poorly reported in our country. Aiming to provide theoretical basis for the choice of the most appropriate anaesthe-sic , our study focused on the pharmacological effects and mechanism of PF on isolated guinea pigs ASM by means of guinea pigs asthma model establishment.
Methods
1. Establishment of guinea pigs asthma model
68 guinea pigs with weight ranging from 150 to 200 g (which provided by Institute of Drug Control, Shenyang) were divided into the normal and asthma group randomly. Sex was not under consideration nor recorded. According to the previous report, our guinea pigs asthma model was established in the way as follows: firstly, the asthma group was sensitized by intraperitoneal injection of 10% ovalbumin ( OA) normal saline; then, after 14 days interval, were given the 1% OA normal saline aerosol inhalation in the 3L glass bell jar once a day for 2 weeks. The success of model establishment was indicated by quickened and deepened constricted obviously breath until obvious contraction of abdominal muscles. For the normal group, 1ml normal saline was given instead of OA normal saline, as well as normal saline aerosol inhalation was adopted instead of OA normal saline administration.
2. Preparation of isolated guinea pigs tracheal ring
The guinea pigs were sacrificed by stunning. The tracheas about 2-3 cm were exercised immediately and placed in cold Krebs - Hens-ekeit (KH) solution supplemented with mixed gases (95%O2, 5% CO2). After overlying fatty and connective tissues were dissected , the tracheas were cut into rings of 3 ~ 5 mm width, which were then hung vertically in an organ bath containing 10 ml continuously gassed K-H solution at 37 +0.5C.
3. Force measurement of isolated guinea pigs tracheal smooth muscle (TSM)
The tension of each tracheal ring was recorded by using a force displacement transducer with a pen recorder attached to the upper end of it. An initial resting load of 2g was applied to each tracheal ring hung in bath that solution was renewed every 20 minutes. The drugs
were administrated after 2 hours equilibrium.
In our study, the drug concentration here referred to "that of the end stage and cumulative method was adopted in making the dose -effect curve.
4. Respective measurement of intracellular and extracellular Ca2 + dependent contraction of guinea pigs TSM
After equilibrium for 2 hours, the trachea! ring was rinsed by calcium - free K - H solution for 3 times, and then with the interval of 5 minutes to restore the equilibrium, PF at different concentrations was added directly or after preconditioning with 100umol/L Ryanodine for 10 minutes. 20 minutes after PF administration, 0.1umol/L acetyl-choline ( Ach) was added. The contraction of tracheal ring at this moment was the result of intracellular calcium release, so it was also called intracellular calcium dependent contraction. After the maximum contraction appeared, calcium chloride was added into the bath (the end concentration, 2. 5 mmol/L) , and the recontraction of trachea
全麻诱导、气管插管常可引起支气管痉挛，对哮喘等气道高反应性(airway hyperresponsiveness，AHR)疾病患者实施麻醉诱导时，或对COPD、ARDS等重症患者实施急救气管插管治疗时，此种潜在的威胁可能是致命的，由麻醉药引起的组胺释放或交感神经活性降低、副交感神经活性增高常常增加此种危险。在临床麻醉中，选择适宜的静脉麻醉药将对该类患者的麻醉安全和预后产生重要影响，但目前临床上选择何种静脉麻醉药用于AHR疾病患者的麻醉诱导和维持仍存在争议。
异丙酚(Propofol，PF)作为一种新型的静脉麻醉药，具有起效快、作用时间短、消除迅速、苏醒快而完全、术后恶心呕吐发生率低等优点，已广泛应用于临床麻醉和ICU镇静。近年来国外关于PF对气道平滑肌(airway smooth muscle，ASM)张力作用的报道较多，大多显示PF对在体、离体的正常动物和人的ASM有剂量依赖的舒张作用，但国内罕见报道，并且PF对哮喘动物ASM的作用及对ASM细胞Ryanodine受体(RyR)的作用尚未见报道。本研究通过建立哮喘豚鼠模型，探讨PF对正常及哮喘豚鼠离体ASM的药理作用及作用机制，为临床麻醉工作中静脉麻醉药的选择提供理论依据。
方法
一、哮喘豚鼠模型的建立
将健康豚鼠68只(沈阳市药品检验所提供)，雌雄不拘，体重150～200g，随机分成正常组和哮喘组。哮喘组以10％卵白蛋白(ovalbumin，OA)生理盐水溶液1ml腹腔注射致敏，饲养14天后，
每天置于 3 L玻璃钟罩内雾化吸人 1％OA生理盐水溶液诱发哮喘
发作，以豚鼠逐渐出现呼吸加深加快，直至出现明显腹肌收缩为模
型建立成功的标志，每日激发哮喘一次，持续两周建立迟发型哮喘、。
模型。正常组以生理盐水 IInl代替 OA腹腔注射，饲养 14天后每
日雾化吸人生理盐水。
二、豚鼠高体气管环制备
击头处死豚鼠，迅速取其主气管约 2－3 cm，置于通有混合气
体阳5％O卜5％CO＊冰浴的 K－H营养液中剪除气管周围结缔组
织及脂肪组织，制成长3－srnm左右的气管环，悬挂于盛有K—H
营养液的夹层肌槽中，保持温度37＊0．5℃，持续通人混合气体。
三、豚鼠离体气管平滑肌张力测定
豚鼠离体气管平滑肌（racheal smooth mucle，TIM）的张力变
化可通过与气管环上端相连的力一位移换能器描记在台式平衡记
录仪上。标本初始负荷Zg，每20min换一次营养液，平衡Zh达到
理想初长度后向浴管中加人实验药物。所有药物浓度以标本孵育
液中的终浓度表示，量效曲线的实验采用累积加药法。
四、豚鼠离体TSM依内钙性收缩和依外钙性收缩的测定
标本平衡Zh后，用无钙K－H营养液冲洗三次，稳定后第
smin分别加人不同浓度的 PF或加人 100pmol／L的 Ryanodine作
用 10min后加人不同浓度的 PF，20ndn后加人 0．lpmol／L的乙酸
胆碱（ACh人此时出现的气管环收缩为ACh引起细胞内钙释放所
致，即依内钙性收缩；待收缩达最大效应后，向浴管内加人CaCI。
（终浓度为2．smxnol／L人出现的气管环收缩，为Ach引起细胞外
钙内流所致，即依外钙性收缩。
五、统计分析
实验数据采用 SPSS for windows 10．0软件进行单因素方差分
析和独立样本的 t检验，P＜0．05为差异有显著性的意义。
·2·
结 果
1．PF对静息状态TIM的作用
N对正常豚鼠静息状态下的nM无明显作用（尸＞0．05人
10％Intralopd有轻微收缩 ’ISM的作用，但与对照组比较无显著差
异。与对照组比较不同浓度的PF剂量依赖性地舒张哮喘豚鼠静
息邹M，mo卜OOVL，300pmOVL Pq的舒张作用有显著差异（83．
33 t ZI．60，161．67 t38．69，P＜0．05）。
2．PF对ACh所致TIM收缩的作用
正常组和哮喘组中不同浓度 PF对 0．lpmol／L ACh所致的
TIM收缩都呈剂量依赖性的舒张作用。正常组：100卜mol／L PF使
ACh所致 ’I’SM收缩反应降为拍．36。7．58）％，与对照亚组比较
差异显著；哮喘组：30 pm。VLN使油h所致BM收缩反应降为
（7．88。6．03）％，与对照亚组比较差异显著；且与正常组同浓度
PF的作用比较有显著差异；10％Intralopd不影响 PF舒张5M的
作用。
3．PF预适应对ACh所致的’I’SM收缩量效曲线的影响
给予 100psmoUL，300pmol／L的 PF 20ndn后可剂量依赖性地
抑制ACh引起的’ISM收缩，使其量效曲线右移，但最大反应变化
很小；10％Intralwid不影响 PF对 Ach所致 TSM收缩的抑制作用。
4．普索洛尔（e）对PF抑制ACh所致TIM收缩量效曲线作
用的影响
以ic卜mo＊L PrO作用b 阻断p。受体后，得到1mpmo＊L
PF预适应后ACh致TIM收缩的量效曲线。与无Pro作用比较，
100pmol／L PF抑制Ach所致TIM收缩的量效曲线无明显变化。
5．PF预适应对ACh所致的h M两种收缩组分的影响
PF预适应可剂量依赖性地抑制Ach引起的TIM收缩，正常
组：与对照亚组比较，100pmoVL PF对 0．lpeVL ACh所致 TIM
·3·
依内钙性收缩、依外钙性收缩?
Objective
The potential bronchial spasm is usually lethal during the induction of general anaesthesia in patients with airway hyperresponsiveness (AHR) disease, such as COPD, ARDS and so on. The increased risk of bronchial spasm is due to the drug - induced histamine release , decreased sympathetic nerve activity or enhanced parasympathetic activity. For that reason, the choice of the appropriate venous anesthesic is very important for the safety and prognosis of the patients. However , no consensus has been reached in the choice of the proper venous anesthetic that exerts protective effect on airway.
Characterized with rapid effect, short duration, immediate elimination, complete recovery, and low incidence of postoperative vomiting, Propofol (PF) , as a new generation of venous anesthetic, has been widely used in clinical anesthesia and ICU sedation Compared with research abroad, the effect of Propofol on airway smooth muscle ( ASM ) tone , especially on that of asthma and on Ryanodine receptor ( RyR) of ASM cell, is poorly reported in our country. Aiming to provide theoretical basis for the choice of the most appropriate anaesthe-sic , our study focused on the pharmacological effects and mechanism of PF on isolated guinea pigs ASM by means of guinea pigs asthma model establishment.
Methods
1. Establishment of guinea pigs asthma model
68 guinea pigs with weight ranging from 150 to 200 g (which provided by Institute of Drug Control, Shenyang) were divided into the normal and asthma group randomly. Sex was not under consideration nor recorded. According to the previous report, our guinea pigs asthma model was established in the way as follows: firstly, the asthma group was sensitized by intraperitoneal injection of 10% ovalbumin ( OA) normal saline; then, after 14 days interval, were given the 1% OA normal saline aerosol inhalation in the 3L glass bell jar once a day for 2 weeks. The success of model establishment was indicated by quickened and deepened constricted obviously breath until obvious contraction of abdominal muscles. For the normal group, 1ml normal saline was given instead of OA normal saline, as well as normal saline aerosol inhalation was adopted instead of OA normal saline administration.
2. Preparation of isolated guinea pigs tracheal ring
The guinea pigs were sacrificed by stunning. The tracheas about 2-3 cm were exercised immediately and placed in cold Krebs - Hens-ekeit (KH) solution supplemented with mixed gases (95%O2, 5% CO2). After overlying fatty and connective tissues were dissected , the tracheas were cut into rings of 3 ~ 5 mm width, which were then hung vertically in an organ bath containing 10 ml continuously gassed K-H solution at 37 +0.5C.
3. Force measurement of isolated guinea pigs tracheal smooth muscle (TSM)
The tension of each tracheal ring was recorded by using a force displacement transducer with a pen recorder attached to the upper end of it. An initial resting load of 2g was applied to each tracheal ring hung in bath that solution was renewed every 20 minutes. The drugs
were administrated after 2 hours equilibrium.
In our study, the drug concentration here referred to "that of the end stage and cumulative method was adopted in making the dose -effect curve.
4. Respective measurement of intracellular and extracellular Ca2 + dependent contraction of guinea pigs TSM
After equilibrium for 2 hours, the trachea! ring was rinsed by calcium - free K - H solution for 3 times, and then with the interval of 5 minutes to restore the equilibrium, PF at different concentrations was added directly or after preconditioning with 100umol/L Ryanodine for 10 minutes. 20 minutes after PF administration, 0.1umol/L acetyl-choline ( Ach) was added. The contraction of tracheal ring at this moment was the result of intracellular calcium release, so it was also called intracellular calcium dependent contraction. After the maximum contraction appeared, calcium chloride was added into the bath (the end concentration, 2. 5 mmol/L) , and the recontraction of trachea
引文
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