祛痰治呛方穴位敷贴脑缺血再灌注损伤后吞咽障碍大鼠的作用机制

Acupoint dressing therapy using Expectorant Choking Formula for swallowing disorders in rats after cerebral ischemia-reperfusion injury

背景:前期临床研究发现祛痰治呛方穴位敷贴对于改善脑卒中后吞咽障碍患者的吞咽功能有一定疗效,但作用机制不明确。在脑干吞咽中枢,疑核是腹侧区吞咽中枢的主要构成部分,并且能够诱发吞咽动作的出现和吞咽时间的产生,而疑核内谷氨酸受体、γ氨基丁酸受体、c-fos蛋白等递质与吞咽密切相关。目的:观察祛痰治呛方穴位敷贴对缺血再灌注损伤大鼠疑核内神经递质谷氨酸受体、γ氨基丁酸受体及c-fos蛋白表达的影响,探究祛痰治呛方穴位敷贴治疗短暂性脑缺血再灌注大鼠吞咽障碍的机制。方法:70只SD大鼠随机分为正常组(n=16),缺血再灌注损伤模型组(n=18),贴敷组(n=18),假贴敷组(n=18)。模型组、贴敷组、假贴敷组采用线栓法短暂脑缺血90 min后再灌注进行造模,造模6 h后进行神经功能评分,选取评分为2分的大鼠进入后续实验;造模第2天贴敷组给予祛痰治呛方穴位敷贴治疗,假贴敷组在模型制备的基础上给予无药物贴敷,其余两组正常饲养1个月。记录各组大鼠造模后第2,7,14,30天体质量、吞咽启动反应时间及吞咽次数。治疗4周后,测定各组大鼠的脑组织含水量,采用免疫组化法、RT-PCR、Western blot法检测延髓吞咽中枢疑核处谷氨酸受体1N-甲基-D-天门冬氨酸受体(NMDAR1)、氨基丁酸A型受体α1(GABAA Rα1)和c-fos蛋白的表达。结果与结论:①与正常组相比,模型组、贴敷组和假贴敷组大鼠体质量、1 min内吞咽次数在造模后第14,30天时均减少(P<0.05),吞咽启动反应时间均延长(P<0.05);第30天时,与模型组相比,贴敷组大鼠体质量、吞咽次数增加(P<0.05),吞咽启动时间缩短(P<0.05);②治疗4周后,与模型组相比,贴敷组大鼠脑组织含水量减少(P<0.05);③与模型组相比,贴敷组延髓疑核处氨基丁酸A型受体α1阳性数量、mRNA和蛋白表达增加,但仍低于正常组,1N-甲基-D-天门冬氨酸受体和c-fos阳性数量、mRNA和蛋白表达降低,但仍高于正常组,差异有显著性意义(P<0.05);④结果表明,祛痰治呛方穴位贴敷可以改善脑卒中后吞咽障碍大鼠的吞咽功能,其作用机制可能与改善脑水肿程度,调节吞咽中枢疑核内的神经递质谷氨酸受体、γ氨基丁酸受体和c-fos蛋白等密切相关。

BACKGROUND:Previous clinical studies have found that acupoint dressing therapy using Expectorant Choking Formula is effective in improving swallowing function in patients with post-stroke swallowing disorders,but the mechanism of action is unclear.In the brainstem swallowing center,the nucleus ambiguus is the main component of the swallowing center in the ventral region,which can induce swallowing movements and swallowing time,while glutamate receptors,γ-aminobutyric acid receptors,and c-fos protein in the nucleus ambiguus are closely related to swallowing.OBJECTIVE:To investigate the effects of acupoint dressing therapy using Expectorant Choking Formula on the expression of glutamate receptors,γ-aminobutyric acid receptors,and c-fos protein in the nucleus ambiguus of rats with ischemia-reperfusion injury,and to investigate the mechanism of swallowing disorder in rats with cerebral ischemia-reperfusion treated by acupoint dressing therapy using Expectorant Choking Formula.METHODS:Seventy Sprague-Dawley rats were randomly divided into normal group(n=16),ischemia-reperfusion injury model group(n=18),patching group(n=18),and sham patching group(n=18).The model group,patching group,and sham patching group were modeled by reperfusion after 90 minutes of transient cerebral ischemia using the thread-occlusion method.After 6 hours of modeling,neurological function was scored and the rats with a score of 2 were selected for subsequent experiments.The rats were treated with acupoint dressing therapy using Expectorant Choking Formula on the second day of modeling,the sham patching group was given drug-free dressing on the basis of the model preparation,and the other two groups were kept normally for 1 month.Body mass,swallowing initiation response time and the number of swallows were recorded in each group on days 2,7,14 and 30 after modeling.After 4 weeks of treatment,the water content in brain tissue was measured;the expression of gamma-aminobutyric acid A receptorα1,N-methyl-D-aspartate receptor 1 and c-fos protein in the nucleus ambiguus of the medullary swallowing center was detected by immunohistochemistry,RT-PCR and western blot.RESULTS AND CONCLUSION:Compared with the normal group,the body mass,the number of swallows within 1 minute and swallow initiation reaction time of rats in the model,patching and sham patching groups decreased on days 14 and 30 after modeling(P<0.05),and the swallow initiation reaction time was prolonged(P<0.05);on day 30,the body mass and the number of swallows of rats increased in the patching group compared with the model group(P<0.05),while the swallowing initiation time was shortened(P<0.05).After 4 weeks of treatment,the water content of brain tissue was significantly reduced in the patching group compared with the model group(P<0.05).Compared with the model group,the positive expression and the mRNA and protein expression of gamma-aminobutyric acid A receptorα1 in the nucleus ambiguus of the medulla oblongata were increased in the patching group but still lower than those in the normal group;the positive expression and the mRNA and protein expression of N-methyl-D-aspartate receptor 1 and c-fos were decreased in the patching group but significantly higher than those in the normal group(P<0.05).These findings indicate that the acupoint dressing therapy using Expectorant Choking Formula could improve the swallowing function of rats with post-stroke swallowing disorder,and its mechanism of action might be related to improving the degree of brain edema,regulating the neurotransmitters glutamate receptors,gamma-aminobutyric acid A receptors and c-fos protein levels in the nucleus ambiguus.