利咽启闭方对脑卒中大鼠吞咽功能及皮质吞咽中枢神经细胞凋亡的影响

Effects of Lipopharyngeal Qibi Formula on swallowing function and apoptosis in central cortical swallowing neurons in rats after stroke

背景:中药复方利咽启闭方治疗脑卒中吞咽障碍取得了良好疗效。外周血清5-羟色胺及中枢孤束核神经递质与吞咽密切相关,因此该研究利用分子生物学等现代医学实验方法,探索中药利咽启闭方对外周血清及吞咽中枢孤束核神经递质的调控作用,为其机制的探索开拓新思路。目的:验证利咽启闭方对脑卒中吞咽障碍的治疗作用,并探究其作用机制。方法:将38只SD大鼠随机分为模型组14只、治疗组14只和假手术组10只,模型组和治疗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;造模后第2天开始治疗组给予中药复方利咽启闭方灌胃治疗,其余两组给予生理盐水灌胃;造模后第2,7,14,30天记录各组大鼠的体质量及24 h进食、进水量;造模后第14,30天采用生物信号采集器及张力换能器检测大鼠吞咽启动反应时间及吞咽次数;吞咽功能检测后取材,采用TTC染色测定每组大鼠的脑缺血面积,采用免疫组化法检测延髓吞咽中枢孤束核5-羟色胺表达,采用RT-PCR、Western blot法检测各组大鼠岛叶、前运动皮质、扣带皮质、丘脑处BCL-2、BAX的mRNA及蛋白表达水平。结果与结论:①与假手术组相比,治疗组和模型组在灌胃第14天时的体质量、24 h进食量、进水量均减少,吞咽启动反应时间均延长,吞咽次数均减少(P<0.05);在灌胃第30天时,与模型组相比,治疗组大鼠体质量、24 h进食量、进水量均增加(P<0.05),但仍低于假手术组(P<0.05);②与模型组相比,治疗组大鼠的吞咽启动反应时间缩短、吞咽次数增加,但吞咽次数仍较假手术组减少,差异均有显著性意义(P<0.05);③治疗组大鼠脑缺血面积较模型组减小,治疗组延髓孤束核5-羟色胺阳性表达较模型组增加,但仍低于假手术组,差异有显著性意义(P<0.05);④与模型组相比,治疗组大鼠岛叶、扣带皮质、丘脑BCL-2 mRNA及蛋白表达均升高,BAX mRNA及蛋白表达均下降,BCL-2/BAX比值均增加,差异有显著性意义(P<0.05);⑤结果表明:中药复方利咽启闭方可以改善脑卒中吞咽障碍大鼠的吞咽次数及吞咽启动反应时间以及24 h进食进水量、体质量等吞咽功能相关指标,其作用机制可能是通过改善脑缺血面积,抑制大鼠岛叶、扣带皮质、丘脑的神经细胞凋亡,进而改善高级中枢对延髓吞咽中枢的调控,以及调节孤束核内的神经递质5-羟色胺水平来实现的。

BACKGROUND:The treatment of post-stroke dysphagia with Lipopharyngeal Qibi Formula has achieved good efficacy,and 5-hydroxytryptamine in peripheral serum and neurotransmitters in the nucleus tractus solitarius are closely related to swallowing.Therefore,this study was conducted to explore the modulatory effects of peripheral serum and nucleus tractus solitarius neurotransmitters in swallowing by using modern medical experimental methods such as molecular biology,thereby developing new ideas for the exploration of their mechanisms.OBJECTIVE:To verify the therapeutic effect of Lipopharyngeal Qibi Formula on post-stroke dysphagia and to investigate its mechanism of action.METHODS:Thirty-eight Sprague-Dawley rats were randomly divided into model group(n=14),treatment group(n=14)and sham-operated group(n=10).Animals in the model and treatment groups were modeled by reperfusion after 90 minutes of transient cerebral ischemia by wire bolus method.At 6 hours after modeling,neurological function was scored,and rats with a score of 2 were selected for subsequent experiments.The treatment group was given compound Lipopharyngeal Qibi Formula by gavage starting from the 2nd day after modeling and the remaining two groups were given normal saline by gavage.Changes in body mass,24-hour food and water intake were recorded on days 2,7,14 and 30.The swallowing initiation response time and the number of swallows were detected using a biosignal collector and a tonic transducer on days 14 and 30.After the swallowing test,the ischemic area of the brain in each group was measured by TTC staining.The expression of 5-hydroxytryptamine in the nucleus tractus solitarius of the medulla oblongata was measured by immunohistochemistry.The mRNA and protein expression levels of BCL-2 and BAX in the insula,premotor cortex,cingulate cortex and thalamus of rats in each group were measured by RT-PCR and Western blot,respectively.RESULTS AND CONCLUSION:Compared with the sham-operated group,the body mass,24-hour food intake and water intake were reduced,the swallow initiation response time was prolonged,and the number of swallows was reduced in the treatment and model groups at day 14 of gavage(P<0.05).Compared with the model group,the body mass,24-hour food intake and water intake of rats were increased in the treatment group at day 30 of gavage(P<0.05),but were still lower than those in the sham-operated group.Compared with the model group,the swallow initiation reaction time was shortened and the number of swallows increased in the treatment group,but the number of swallows was still significantly lower than that in the sham-operated group(P<0.05).Cerebral ischemia area was reduced in the treatment group compared with the model group,and the number of 5-hydroxytryptamine-positive cells in the nucleus tractus solitarius of the medulla oblongata was increased in the treatment group compared with the model group,but it was still significantly lower than that in the sham-operated group(P<0.05).Compared with the model group,the expression of BCL-2 mRNA and protein in the insula,cingulate cortex and thalamus of rats in the treatment group were significantly increased,the expression of BAX mRNA and protein were significantly decreased,and the BCL-2/BAX ratio was significantly increased(P<0.05).To conclude,the Chinese herbal compound Lipopharyngeal Qibi Formula could improve the number of swallows and swallowing initiation response time,as well as 24-hour food intake,body mass and other swallowing-related indexes in rats with post-stroke dysphagia.The mechanism of action may be achieved by improving the area of cerebral ischemia,inhibiting the apoptosis of neuronal cells in the insula,cingulate cortex and thalamus of rats,thus improving the regulation of the higher centers on the medulla oblongata swallowing center,and regulating the level of 5-hydroxytryptamine in the nucleus tractus solitarius.