A型肉毒毒素对大鼠离体十二指肠平滑肌自发性收缩的影响

目的 观察A型肉毒毒素(botulinum toxin A,BTX-A)对离体十二指肠平滑肌的自发性收缩是否存在抑制作用,这种抑制作用的时效特点与胆碱能M受体抑制剂作用的异同,BTX-A是否可抑制外源性乙酰胆碱(acetylcholine,ACh)增强的十二指肠收缩,BTX-A预处理平滑肌后外原性ACh是否可增强十二指肠平滑肌的收缩.旨在为临床应用BTX-A治疗小肠收缩功能紊乱所致的疾病提供理论和实验依据.方法 选取Sprague-Dawley大鼠,实验时击头部致昏后,距幽门0.5 cm处取1.0～1.5 cm的肠管,置于37℃Krebs液的恒温平滑肌槽中,肌条的一端固定在肌槽底部的塑料弯钩上,另一端固定在张力传感器上.肌条在1 g的前负荷下孵育,随机分为BTX-A组(n=12),阿托品组(n=12),ACh+ BTX-A素组(n=12),ACh+阿托品组(n=12),BTX-A+ ACh组(n=12).在自发性收缩平稳20分钟后,根据研究方案,分别加入BTX-A( 10 U/ml)或阿托品(1μmol/L)、ACh(100 μmol/L).Biolap 420 E生物机能实验系统记录十二指肠纵形平滑肌条在不同给药条件下的收缩变化.结果 BTX-A抑制了十二指肠平滑肌自发性收缩的频率、张力及振幅(P＜0.01),这种抑制作用持续＞1小时.阿托品抑制了十二指肠平滑肌自发性收缩的频率、张力及振幅(P ＜0.01),但用药5分钟后,十二指肠平滑肌的收缩开始恢复,用药10分钟后,十二指肠平滑肌的收缩已基本恢复；十二指肠平滑肌自发性收缩的频率、张力及振幅与用药前比较,差异无统计学意义(P＞0.05).ACh增强了十二指肠平滑肌自发性收缩的频率、张力及振幅(P＜0.01),BTX-A抑制了ACh增强的十二指肠平滑肌收缩的频率、张力及振幅(P ＜0.01).ACh增强了十二指肠自发性收缩的频率、张力及振幅(P＜0.01),阿托品抑制了ACh增强十二指肠平滑肌收缩的频率、张力及振幅(P＜0.01).BTX-A抑制了十二指肠平滑肌自发性收缩的频率、张力(P＜0.01)及振幅(P ＜0.05),加入外源性ACh后不能增强十二指肠平滑肌收缩的频率、张力及振幅.结论 BTX-A抑制离体十二指肠平滑肌自发性收缩,BTX-A的抑制作用与阿托品不同,表现出不完全抑制十二指肠的收缩频率、张力和收缩幅度,这种作用为逐渐而持续性抑制.BTX-A抑制了外源性ACh增强的十二指肠平滑肌的收缩.BTX-A抑制了十二指肠平滑肌自发性收缩后,外源性ACh不能增强十二指肠平滑肌的收缩,提示BTX-A可作用于突触后膜M受体,从而抑制了ACh与M受体结合,具有类似“阿托品样效应”.

Inhibition of botulinum toxin A on rat duodenal smooth muscle in vitro

Objective To investigate whether botulinum toxin type A(BTX-A) inhibits spontaneous contractility of isolated duodenal smooth muscle and acetylcholine(ACh)-induced contractility,whether the inhibitory characteristics of BTX-A are the same manner with antagonists of muscarinic receptor.Pretreatment of duodenal smooth muscle with BTX-A,ACh administration still enhances the contractile response or not.The aim of this study is to demonstrate the efficiency of BTX-A to treat the disorder of intestinal function.Methods Sprague-Dawley rats were used in this study.Duodenal smooth muscle strip of 1.0 to 1.5 cm was quickly removed from distal pylori after the unconsciousness in the rats by hitting head.The smooth muscle strip was rinsed with Krebs bicarbonate buffer.One end of the strip was fixed to a hook on the bottom of the bath,the other end was connected to an isometric force transducer.Muscle strips were prepared to 1 g loading tension,and subdivided randomly into BTX-A group(n=12),atropine group(n=12),ACh + BTX-A group(n=12),ACh+atropine group(n=12),BTX-A + ACh group(n=12).The contractile graph in motility of the muscle strips was simultaneously recorded with physiological experimental system.Results BTX-A(10 U/ml) reduced the frequency,tension and amplitude of spontaneous contractility of duodenal smooth muscle compared to incubation in Kreb solution as control(P<0.01).The inhibitory effect of BTX-A persisted more than 1 hour.The administration of atropine(1 μmol/L) might completely suppress the frequency,tension and amplitude of spontaneous contractility of duodenal smooth muscle compared to control(P<0.01),but the inhibitory effect of atropine persisted only 5 minutes,and then the duodenal spontaneous contractility gradually recovered as compared with that of control with significance(P<0.05).The administration of ACh(100 μmol/L) enhanced the frequency,tension and amplitude of spontaneous contractility of duodenal smooth muscle compared to control(P<0.01).Subsequently,the respective addition of BTX-A(10 U/ml) or atropine(1 μmol/L) inhibited the contractile response to ACh(P<0.01).The administration of ACh(100 μmol/L) did not induce the duodenal smooth muscle contractility after the inhibition with BTX-A(10 U/ml).Conclusion The inhibitory effect of BTX-A on spontaneous contractility of duodenal smooth muscle in vitro suggests the inhibitory efficient time of BTX-A was longer than that of atropine.BTX-A also reduced duodenal muscle contractile response to external addition of ACh.After BTX-A treatment,ACh did no longer agitate duodenal muscle contractile response.These data demonstrate that BTX-A inhibits duodenal smooth muscle contractility suggesting not only inhibition of ACh release from cholinergic nerves but also inhibition of cholinergic muscarinic muscular transmission.